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CHARACTERIZING AQP9: A REGULATOR OF
EPIPHYSEAL PLATE CHONDROCYTE PROLIFERATION,
HYPERTROPHY, AND LONG BONE GROWTH
by
Pontius Pu Tian Tang
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Institute of Medical Science
University of Toronto
© Copyright by Pontius Pu Tian Tang (2018)
ii
Abstract
Characterizing Aqp9: a regulator of epiphyseal plate
chondrocyte proliferation, hypertrophy, and long bone growth
Pontius Pu Tian Tang
Master of Science
Institute of Medical Science
University of Toronto
2018
Aquaporin-9 (AQP9) is a membrane channel protein suspected to regulate growth in the
epiphyseal plate. As long bone defects often possess limited non-surgical options, novel factors
underlying bone growth must be continuously explored to advance effective treatments. I
hypothesized that Aqp9 is an important epiphyseal plate chondrocyte channel regulating the
process of endochondral ossification. In this study, Aqp9 -/- mouse long bones compared to
wildtype mouse long bones showed a neonatal hindlimb-specific acceleration of growth followed
by reduced length in the juvenile age. Analysis of Aqp9 -/- epiphyseal plates and chondrocytes
showed an early disposition for proliferation and aversion from hypertrophy, suggesting that
Aqp9 may function similarly to genes such as Col10a1 and Mmp13. This study provides insight
into chondrocyte membrane channel proteins and their regulation of the growing epiphyseal
plate, demonstrating that Aqp9 may be a novel therapeutic target for the non-invasive
intervention of leg length discrepancies.
iii
Acknowledgements
I would like to take this opportunity to thank everyone who has helped me throughout my
degree. Firstly, I would like to express my gratitude to my supervisor, Dr. Peter Kannu, for
granting me the opportunity to dive into graduate work and explore a novel protein in a state-of-
the-art facility. Secondly, I would like to thank my Program Advisory Committee members, Dr.
Brian Ciruna and Dr. Marco Magalhaes, for their insight and constructive criticism.
I would like to thank our associate Kashif Ahmed for guiding me through the basics of cell
culture, our MSc candidate Liliana Vertel for assisting greatly with mouse management and
dissection, our previous lab technician Angela Weng for establishing preliminary findings in the
Aqp9 project, our colleague Raymond Poon for providing incredible guidance with genotyping
and mouse work, previous summer students with the Alman lab for assisting with supporting
research, our colleagues in the Alman lab for providing guidance with basic techniques, our
summer students William Xie and Lisa Vi for assistance with cell culture and sectioning,
members of the Wall lab for sharing equipment, and members of the Justice lab for sharing
helpful reagents. I would also like to thank The Centre for Phenogenomics for assistance with
cage maintenance and reminders for mouse weaning and health conditions.
On a personal note, I would like to thank my family and friends for their continuous support and
encouragement. I would also like to thank Michael Liang for guidance with data interpretation,
Nicole Park for assistance with bioinformatic and literature searches, Mushriq Al-Jazrawe for
introductory tips on laboratory work, and Neeti Vashi for guidance with qPCR analysis, and Erin
Chown for guidance with writing and thesis defense.
iv
Table of Contents
Acknowledgements........................................................................................................................ iii
Table of Contents ............................................................................................................................ iv
List of Abbreviations .....................................................................................................................vii
List of Tables ................................................................................................................................... x
List of Figures .................................................................................................................................. x
Chapter 1: Introduction .................................................................................................................... 1
1.1 An overview of endochondral ossification and epiphyseal plate regulation ................. 1
1.2 Models of differential long bone growth ..................................................................... 19
1.3 Aquaporins and chondrocytes: expression, function, and regulation .......................... 25
1.4 Characterization of AQP9 and its novel role in chondrocyte and bone activity .......... 30
1.5 Aqp9 -/-: an accessible knockout mouse model for epiphyseal chondrocyte and bone
length investigation ............................................................................................................ 43
1.6 Aqp9 has a novel function in cartilage and murine long bone growth ........................ 45
Chapter 2: Research Aims, Hypothesis, and Summary Plan ......................................................... 49
2.1 Rationale ...................................................................................................................... 49
2.2 Hypothesis ................................................................................................................... 49
2.3 Objectives .................................................................................................................... 50
2.4 Clinical significance .................................................................................................... 51
2.5 Cellular mechanisms .................................................................................................... 51
Chapter 3: Methods........................................................................................................................ 53
3.1 Mouse creation, maintenance, genotyping, and age selection ..................................... 53
3.2 In situ hybridization ..................................................................................................... 53
3.3 Skeletal staining ........................................................................................................... 54
v
3.4 Staining and immunohistochemistry of epiphyseal plates........................................... 54
3.5 Visualization and measurement of limbs and epiphyseal plates ................................. 55
3.6 Primary chondrocyte culture, qPCR, and RNA silencing ........................................... 56
3.7 Statistical analyses ....................................................................................................... 57
Chapter 4: Results .......................................................................................................................... 58
4.1 Body weight and superficial comparisons of WT and Aqp9 -/- mice.......................... 58
4.2 Histological analysis of Aqp9 expression in the developing epiphyseal plate ............ 63
4.3 Skeletal staining of P5 WT and Aqp9 -/- mice ............................................................ 65
4.4 Skeletal staining of P21 WT and Aqp9 -/- mice .......................................................... 72
4.5 Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates .................................. 79
4.6 Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates ............................ 84
4.7 Analysis of old WT and Aqp9 -/- epiphyseal plates .................................................... 87
4.8 Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates ..................... 90
4.9 Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate
chondrocytes ...................................................................................................................... 93
4.10 Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate
chondrocytes ...................................................................................................................... 95
4.11 Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes ..................................... 97
Chapter 5: Discussion .................................................................................................................... 99
5.1 Aqp9 temporally influences hindlimb length ............................................................. 100
5.2 Epiphyseal plate irregularities underscore Aqp9-mediated bone length.................... 108
5.3 Aqp9 mutant chondrocytes show a differential phenotype ........................................ 116
5.4 A model for Aqp9 function in murine endochondral ossification ............................. 122
Chapter 6: Conclusions ................................................................................................................ 126
vi
Chapter 7: Future Directions ....................................................................................................... 128
7.1 In situ hybridization of Aqp9 during mesenchymal condensation............................. 128
7.2 Histomorphometry of WT and Aqp9 -/- long bones .................................................. 129
7.3 Flow cytometry cell cycle analysis ............................................................................ 130
7.4 RNA-sequencing of WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate
chondrocytes .................................................................................................................... 131
7.5 Therapeutic strategies ................................................................................................ 132
Appendix...................................................................................................................................... 159
Statement of Contributions .............................................................................................. 159
vii
List of Abbreviations
A Adenine
Adam A disintegrin and metalloproteinase
Akt Protein kinase B
Aqp Aquaporin
AQPap Human aquaporin adipose
ARE Androgen response element
Atf AMP-dependent transcription factor
Bgp Osteocalcin
Bmp Bone morphogenetic protein
Bsp Bone sialoprotein
C Cytosine
CACNA1H Calcium Voltage-Gated Channel Subunit Alpha1 H
CD Cluster of differentiation
CDH2 Cadherin-2
Cdk Cyclin-dependent kinase
cDNA Complementary DNA
CHIP Channel-forming integral protein
CHO Chinese hamster ovary
Col10a1 Collagen, type X, alpha 1
Col1a1 Collagen, type 1, alpha 1
Col2a1 Collagen, type II, alpha 1
DAB Diaminobenzidine
DEPC Diethyl pyrocarbonate
DIG Digoxigenin
Dkk Dickkopf-related protein
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DSLR Digital single-lens reflex
E. coli Escherichia coli
ECM Extracellular matrix
EDNRB Endothelin B receptor
EDTA Ethylenediaminetetraacetic acid
EMSA Electrophoretic mobility shift assay
ERE Estrogen response element
Fgf Fibroblast growth factor
G Guanine
GDF Growth/differentiation factor
GH Growth hormone
GlpF Glycerol uptake facilitator protein
GLUT4 Glucose transporter type 4
H&E Hematoxylin and Eosin
H2O2 Hydrogen peroxide
HB Helix B
HE Helix E
HIF Hypoxia-inducible factors
Hox Homeobox
Hp1bp3 Heterochromatin protein 1, binding protein 3
HRP Horseradish peroxidase
HSC Hematopoietic stem cell
IGF-1 Insulin-like growth factor 1
Ihh Indian hedgehog
IL Interleukin
IRE Insulin response element
viii
KCNB1 Potassium voltage-gated channel, Shab-related subfamily, member 1
KOH Potassium hydroxide
Lepr Leptin receptor
LLD Leg Length Discrepancy
MAPK Mitogen-activated protein kinase
Matn Matrilin
MCDS Metaphyseal Chondrodysplasia Schmid Type
MEM Minimum Essential Medium
MKRN3 Makorin ring finger protein 3
Mmp13 Matrix metallopeptidase 13
mRNA Messenger RNA
MSC Mesenchymal stem cell
mTOR Mammalian target of rapamycin
AMPK AMP-activated protein kinase
N-CAM Neural cell adhesion molecule
Nfat Nuclear factor of activated T-cells
NPA Asn-Pro-Ala
Npr Natriuretic peptide receptor
OARSI Osteoarthritis Research Society International
Osx Osterix
Panx Pannexin
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PEPCK Phosphoenolpyruvate carboxykinase
PF4V1 Platelet factor 4 variant 1
PFA Paraformaldehyde
pH Power of hydrogen
PI3K Phosphoinositide 3-kinase
PKC Protein kinase C
Prx1 Paired related homeobox 1
PTHrP Parathyroid hormone-related protein
PTP Protein tyrosine phosphatase
qPCR Quantitative polymerase chain reaction
RANK-L Receptor activator of nuclear factor kappa-Β ligand
RNA Ribonucleic acid
ROI Region of Interest
ROS Reactive oxygen species
Rspo R-spondin
RT-PCR Reverse transcription polymerase chain reaction
Runx Runt-related transcription factor
SDS Sodium dodecyl sulfate
Shh Sonic hedgehog
Shox Short stature homeobox
siRNA Silencing RNA
Smad "small" "Mothers Against Decapentaplegic"
Sox Sry-related HMG box
Spred2 Sprouty-related, EVH1 domain-containing protein 2
SSC Saline-sodium citrate
Stat1 Signal transducer and activator of transcription 1
T Thymine
Tak1 Transforming growth factor beta-activated kinase 1
TBST Tris-buffered saline, polysorbate 20
Tbx T-box
TCP The Centre for Phenogenomics
TEA Triethylamine
Tgf Transforming growth factor
ix
TNE Tris, EDTA, NaCl
TonEBP Tonicity-responsive enhancer binding protein
TRPM7 Transient receptor potential cation channel subfamily M member 7
TRPV4 Transient receptor potential cation channel subfamily V member 4
VEGF Vascular endothelial growth factor
VILO Variable input, linear output
Wnt Wingless-related integration site
WT Wildtype
Yap Yes-associated protein
x
List of Tables
Table 1.1 Models of differential bone growth ............................................................................... 24
Table 5.1 Primary epiphyseal plate chondrocyte proliferation rates (48-96 hours) .................... 117
List of Figures
Figure 1.1 Mesenchymal condensation ........................................................................................... 5
Figure 1.2 Early chondrogenesis ..................................................................................................... 8
Figure 1.3 Chondrocyte proliferation, columnar formation, and pre-hypertrophy........................ 11
Figure 1.4 Chondrocyte hypertrophy ............................................................................................. 14
Figure 1.5 Ossification and long bone growth .............................................................................. 18
Figure 1.6 AQP9 protein structure................................................................................................. 33
Figure 1.6 cont. .............................................................................................................................. 35
Figure 1.7 Expression and regulation of AQP9 ............................................................................. 38
Figure 4.1 Body weight and superficial comparisons of WT and Aqp9 -/- mice .......................... 59
Figure 4.2 Histological analysis of Aqp9 expression in the developing epiphyseal plate ............. 65
Figure 4.3 Skeletal staining of P5 WT and Aqp9 -/- mice ............................................................. 67
Figure 4.4 Skeletal staining of P21 WT and Aqp9 -/- mice ........................................................... 74
Figure 4.5 Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates .................................. 82
Figure 4.6 Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates ............................ 86
Figure 4.7 Analysis of old WT and Aqp9 -/- epiphyseal plates ..................................................... 89
Figure 4.8 Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates ..................... 92
Figure 4.9 Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate
chondrocytes .................................................................................................................................. 95
xi
Figure 4.10 Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate
chondrocytes .................................................................................................................................. 97
Figure 4.11 Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes ..................................... 99
Figure 5.1 A model for Aqp9 function in murine endochondral ossification ............................. 125
1
1 Introduction
The epiphyseal plate is a region of growing tissue near the ends of long bones in children and
adolescents (Mirtz, Chandler, & Eyers, 2011). It is an exceptional model for understanding
genetic mechanisms that underlie long bone development because it is:
1) The foremost location where long bone grows,
2) Easily visualized and accessible,
3) Regulated by a myriad of genes, and
4) Organized in specific zones with remarkable cell function and morphology.
Numerous studies have provided information on the mechanisms governing long bone growth.
Sectioning of multispecies epiphyseal plates and histological analyses paved the way for several
of the basic structures recognized today, such as the epiphyseal plate zones. The
characterizations of simple elements—such as chondrocytes; the primary cellular unit of
epiphyseal plate cartilage—were critical towards a richer understanding of long bone
development (Brighton, Sugioka, & Hunt, 1973). These studies further led to the investigation of
spatiotemporal markers at each zone during skeletal morphogenesis, including the expression of
now-familiar genes Ihh and Bmp-6 (Iwasaki, Le, & Helms, 1997). This process has been
accelerated by the advent of mutant mouse models, which have revealed the functional role of
specific genes contributory to long bone development. Currently, the challenge is to understand
the interaction of novel genes and pathways that coordinate in the epiphyseal plate to control cell
proliferation, progression into cellular hypertrophy, and ultimately extend long bone length. In
particular, transmembrane residents of the channelome—the diverse set of ion channels and
pores on the chondrocyte membrane—are emerging as targets to further understand cartilage and
bone biology (Barrett-Jolley, Lewis, Fallman, & Mobasheri, 2010).
1.1 An overview of endochondral ossification and epiphyseal plate
regulation
Osseous tissues in the mammalian skeleton are formed through two different processes—
intramembranous ossification and endochondral ossification (Berendsen & Olsen, 2015). In the
2
former, osteoblasts derived from mesenchymal cells during embryonic skeletal development
secrete osteoid that eventually calcify and form mature bone matrix (Lefebvre & Bhattaram,
2010). This process gives rise to flat bones, the majority of cranial bones, and the clavicles. In
contrast, endochondral ossification creates the long bones of the body by replacing hyaline
cartilage (Mackie, Ahmed, Tatarczuch, Chen, & Mirams, 2008). Here, mesenchymal cells
condense and differentiate into chondrocytes that secrete matrix, proliferate, undergo
hypertrophy, and expire sequentially to allow osteoblast and vascular invasion. Bone matrix is
formed at the epiphyseal plate until the primary and secondary centres of ossification join and
prevent further growth (Ortega, Behonick, & Werb, 2004).
This process is tightly regulated and dictates the skeletal structure of the individual, impacting
their appearance, health, and quality of life. Examining the structures and regulatory markers
present during each step of murine endochondral ossification will entrench an understanding of
the known factors important to long bone growth—as well as provide insight into novel
contributory genes.
1.1.1 Mesenchymal condensation (see Figure 1.1)
Endochondral ossification commences with the migration of mesenchymal cells from the lateral
plate mesoderm and subsequent condensation at the respective limb field (Long & Ornitz, 2013).
Initiating limb outgrowth requires T-box transcription factors such as Tbx5 in the forelimb and
Tbx4 in the hindlimb to establish fibroblast growth factor (FGF) gene expression and eventual
chondrogenesis (Nishimoto & Logan, 2016). This embryonic process occurs as early as E9.5-
10.5 in the murine limb bud and sets the foundation for later chondrogenic differentiation and
production of early chondrocyte-specific extracellular matrix (ECM) products such as Col2a1
and aggrecan (Hata, Takahata, Murakami, & Nishimura, 2017). At this stage, the mesenchymal
cells are tightly packed and rely on body-plan genes such as Hox to direct their condensation
position. Expression of Hox10 guides growth of the proximal long bone—such as the femur—
and expression of Hox11 guides the distal long bone—such as the tibia (Rux & Wellik, 2017). A
member of the Hedgehog signalling pathway, Shh, is expressed in the posterior margin of the
limb bud—named the zone of polarizing activity—to guide appropriate anterior-posterior
3
patterning of the developing limb (Yang, Andre, Ye, & Yang, 2015). While the evolutionary
background of appendicular skeleton development requires further investigation, genes important
to future chondrogenic maturation also play a role in driving initial mesenchymal condensation.
Bone morphogenetic proteins (BMPs) are part of the transforming growth factor-β (TGF- β)
family and play fundamental roles in embryonic skeletal development (Beederman et al., 2013).
BMPs bind to serine/threonine kinase receptors, inducing their phosphorylation, which then
phosphorylate SMAD proteins that can complex with SMAD4 and enter the nucleus to perform
transcriptional activation of genes such as CDH2 to synthesize N-cadherin (Massagué, Seoane,
& Wotton, 2005; Wang, Zhao, & Zhang, 2017). Both N-cadherin and N-CAM are proteins
responsible for cell-to-cell adhesion during mesenchymal condensation, as driven by the BMP-
SMAD pathway (DeLise, Fischer, & Tuan, 2000). The modern consensus is that BMP signalling
is required for mesenchymal cells to form necessary small aggregates and cluster into
condensations with definite boundaries for later chondrogenic differentiation (Barna &
Niswander, 2007). BMP2 and BMP4 are two such explored genes that are required for proper
long bone formation (Bandyopadhyay et al., 2006). Later genes such as Sox9 are necessary for
chondrogenesis but rely on BMP signalling to maintain the condensation aggregate, as Sox9
alone is insufficient to rescue chondrocyte formation in Smad4-deficient embryos (Akiyama,
Chaboissier, Martin, Schedl, & de Crombrugghe, 2002; Lim et al., 2015).
Amidst the importance of BMPs, fibroblast growth factors (FGFs) are also necessary precursors
to condensation formation. FGFR1-4 in both mice and humans are cell surface tyrosine kinase
receptors that bind FGF and phosphorylate important downstream pathways involving MAPK,
PI3K, STAT1, and PKC (Turner & Grose, 2010). Primarily, FGFR1 and 2 are expressed widely
in limb bud mesenchyme prior to condensation and their deletion from the limb bud produces
diminished skeletal products (Peters, Werner, Chen, & Williams, 1992; Yu et al., 2003). FGF
signalling in the mesenchyme provides essential cell survival signals during pre-condensation, as
well as in subsequent chondrogenic differentiation (Sun, Mariani, & Martin, 2002).
Condensation may ultimately rely on mechanical factors such as ion channels, cilia, and the
cytoskeleton for mesenchymal cells to communicate (Hughes et al., 2018). The presence of
4
mechanosensitive calcium channels such as TRPM7 and potassium channels such as KCNB1 in
mesenchymal cell membranes highlight specific proteins through which condensation may be
initiated (Xiao, Chen, & Zhang, 2016; Pillozzi & Becchetti, 2012). Hence, even the earliest stage
of long bone growth—at the limb bud—may require channelome members to progress toward
chondrogenesis. At this stage, mesenchymal cells have formed the cartilage anlage necessary for
further chondrocytic differentiation and eventual ossification.
Figure 1.1. Mesenchymal condensation
Developing
embryo
Developing
Forelimb
Tbx4
Tbx5
Lateral plate
mesoderm
Developing
Hindlimb
Developing digitsHox10 Hox11
Limb bud
Zone of
polarizing
activity
Shh
Condensing
mesenchymal
cells
BMPs
SMADs
> N-cadherin
> N-CAM
FGFs
> MAPK
> PI3K
> STAT1
> PKC
TRPM7
KCNB1
Other channelome proteins…
Encourage
survival,
promote cell-to-
cell adhesion,
and guide
condensation
5
Mesenchymal cell
6
1.1.2 Early chondrogenesis (see Figure 1.2)
After condensation, mesenchymal cells in the core of the anlage can differentiate into early
chondrocytes by committing to a chondrogenic fate (Somoza, Welter, Correa, & Caplan, 2014).
These pre-chondrocytes cease production of adhesion molecules and begin secreting cartilage
matrix including collagen types II, IX, and XI into the extracellular space. As these cells migrate
towards the anlage periphery, a portion commit to the osteogenic fate and form the
perichondrium that surrounds the developing epiphyseal plate (Lefebvre & Bhattaram, 2010).
Three main proteins drive early chondrogenesis—Sox5, Sox6, and Sox9 (Smits et al., 2001;
Akiyama et al., 2002). This trio performs cooperative binding at the promoter regions of the
aforementioned matrix genes to drive transcription and protein synthesis. Sox5 and Sox6 share
similar promoter-binding capacities and act in concert, but Sox9 is expressed upstream and acts
earlier than the two in chondrogenesis. In fact, homozygous deletion of murine Sox9 in early
chondrocytes completely prevents chondrocytic differentiation, whereas deletion of Sox5 and
Sox6 only diminishes differentiation (Bi, Deng, & Zhang, 1999). Overall, early chondrogenesis
relies on the Sox triad greatly—but other factors are necessary as Sox does not function uniquely
in the chondrocytic lineage (Han & Lefebvre, 2008).
BMP signalling is necessary for the Sox trio to function (Yoon et al., 2005). Without BMP1
receptors and their downstream SMAD modules, chondrogenic cells fail to activate Col2a1 and
differentiate, resulting in severe dysplasia—a dramatic dysregulation of skeletal stature (Retting,
Song, Yoon, & Lyons, 2009). FGF is also a transcriptional enhancer of Sox9, where FGFR1-4
are capable of increasing its expression (Murakami, Kan, McKeehan, & de Crombrugghe, 2000).
However, members of the Wnt/β-catenin pathway inhibit differentiation and overexpression of
β-catenin signaling can a) reroute chondrogenic cells to soft connective tissue formation, b)
inhibit Sox9 activity, and c) result in achondrodysplasia, or dwarfism (ten Berge, Brugmann,
Helms, & Nusse, 2008; Hill, Spater, Taketo, Birchmeier, & Hartmann, 2005; Akiyama et al.,
2004). Furthermore, ectopic Notch signalling suppresses chondrogenesis through binding of the
Notch Intracellular Domain and suppression of Sox9 in the budding limb (Mead & Yutzey,
2009). Evidently, Sox9 is an important transcription factor in early chondrogenesis.
7
In relation to the channelome, cation channels such as TRPV4 can be pharmacologically
activated to artificially raise Sox9 reporter activity (Muramatsu et al., 2007). Removal of calcium
channels such as CACNA1H in cartilage is also able to attenuate Sox9 expression (Lin et al.,
2014). As pre-chondrocytes gradually differentiate into primordial chondrocytes, their membrane
proteome likely evolves in channel expression to reflect the impending need for robust
proliferation, volume expansion, and calcification.
Figure 1.2. Early chondrogenesis 8
Condensing
mesenchymal
cells
Cartilage anlage
Pre-chondrocytes Secreting collagens II, IX, XI
Perichondrium
Sox5, 6, 9
BMPs
SMADs
FGFs
Wnt/β-catenin signalling
TRPV4
CACNA1H
Other channelome proteins…
Developing
epiphyseal plate
Pre-chondrocyte
Prepare for
chondrocyte
proliferation
Developing anlage
Developing bone
Pre-chondrocytes at
the periphery
envelop the anlage
9
1.1.3 Chondrocyte proliferation, columnar formation, and pre-hypertrophy (see Figure
1.3)
By E11.5, chondrocytes have begun to proliferate and orient into a long shaft—the diaphysis—
surrounded on either end by globular masses—the epiphyses. Diaphysis growth occurs through
the epiphyseal plate, where layers of phenotypically different chondrocytes exist as a spectrum
toward eventual long bone development. While a portion of chondrocytes remain round and
randomly distributed throughout the ECM—deemed resting chondrocytes—another population
begins to proliferate, flatten, and organize into columns towards the diaphysis (Abad et al., 2002;
Long, Schipani, Asahara, Kronenberg, & Montminy, 2001). BMP1/BMP2 receptors signal here
with SMAD1, 5, and 8 to secrete type II collagen and proceed with cyclin-dependent kinase
(Cdk) activation for cell division (Sherr & Roberts, 2004; Schmidl et al., 2006).
The matrilins, such as Matn1 and Matn3, are ECM proteins expressed at the proliferating stage
(Yang et al., 2014). Matn3 is able to bind to Bmp2, suppressing its Col10a1 activation and
delaying hypertrophy. Col10a1 encodes type X collagen, a marker of chondrocyte hypertrophy
that is suppressed at the proliferating stage. Also, FGFR3 acts as a negative regulator of
proliferation and accelerates hypertrophy through mitogen-activated protein kinase
phosphorylation of Stat1 (Murakami et al., 2004). Furthermore, the Sox genes are still required:
deletion of Sox5 and Sox6 block proliferation, and Sox9 delays hypertrophy (Smits, Dy, Mitra, &
Lefebvre, 2004; Huang, Chung, Kronenberg, & de Crombrugghe, 2001). Sox9 protein
degradation is a requisite for hypertrophy, and Runx2—a gene that will become more active in
hypertrophy—is activated during pre-hypertrophy by Atf-3 to inhibit Sox9, cyclins, and Cdks
(James, Woods, Underhill, & Beier, 2006; Yamashita et al., 2009).
However, as the columnar proliferating chondrocytes enter the pre-hypertrophic stage and
prepare themselves for hypertrophy, the Ihh/PTHrP negative feedback loop plays one of the most
critical roles (Kronenberg & Chung, 2001). As Kronenberg (2003) describes, PTHrP, the
parathyroid hormone-related protein, is first secreted from the perichondrium and chondrocytes
near the epiphysis. It binds to proliferating chondrocytes and helps them retain their phenotype,
promoting division and delaying hypertrophy. As these chondrocytes divide, form columns, and
10
eventually distance themselves sufficiently from the top of the epiphyseal plate—in a paracrine
fashion—the level of circulating PTHrP is low enough for Ihh production. At this time, the
chondrocytes have become pre-hypertrophic and cease with proliferation. Ihh, a member of the
Hedgehog family, acts on Ihh receptors in the preceding resting and proliferating chondrocytes
and increases their division rate (Chau et al., 2011). Simultaneously, Ihh signals to the
chondrocytes near the epiphysis and stimulates PTHrP production, essentially retaining the
proliferative phenotype and preventing hypertrophy in a negative feedback fashion. This way,
the Ihh/PTHrP duo reduces possible fluctuations in hypertrophic maturation and promotes
stability. Modulation of this gene pair therefore controls the spatial boundaries that the
proliferative and hypertrophic zones can consume in the epiphyseal plate.
Similar to previous stages of endochondral ossification, the chondrocyte membrane induces
diverse channel proteins to support differentiation. Ihh is a mechanosensitive gene that promotes
the proliferative phenotype by receiving mechanical stress signals through membrane stretch-
activated channels (Nowlan, Prendergast, & Murphy, 2008; Wu, Zhang, & Chen, 2001).
Interestingly, aquaporin-5, an integral membrane protein and transporter of water, is expressed in
human proliferating chondrocytes as well as the surrounding mesenchyme (Shimasaki,
Kanazawa, Sato, Tsuchiya, & Ueda, 2018). As columnar chondrocytes exit the cell cycle, their
channelome must begin to reflect the demand for hypertrophic differentiation by expressing
membrane channel proteins that help increase intracellular volume. Here, water-selective pores
such as aquaporins place their stake.
Figure 1.3. Chondrocyte proliferation, columnar formation, and pre-hypertrophy 11
Diaphysis EpiphysisEpiphysis
Developing epiphyseal plate
Resting
chondrocytesResting
chondrocytes
Proliferating
chondrocytesProliferating
chondrocytes
Pre-hypertrophic
chondrocytes
Eventual direction of long bone growth
Eventual direction of long bone growth
BMPs
SMADs
Cdks
Col2a1
Matns
FGFR3
Sox 5, 6, 9
Runx2
PTHrP
AQP5
Other channelome proteins . . .
Proliferating
chondrocyte
Prepare for pre-
hypertrophy
Pre-hypertrophic
chondrocyte
Ihh Prepare for
hypertrophy
Secreting collagens II, IX, XI
12
1.1.4 Chondrocyte hypertrophy (see Figure 1.4)
By the time the developing embryo reaches E12.5-E13.5, the first wave of proliferating
chondrocytes leave the cell cycle and begin differentiating into hypertrophic chondrocytes. A
volume increase occurs prior to terminal differentiation. Chondrocyte volume increase
contributes to growth rate and the final long bone length primarily through accumulation of
water (Wilsman, Farnum, Leiferman, Fry, & Barreto, 1996; Buckwalter, Mower, Ungar,
Schaeffer, & Ginsberg, 1986). However, whether the expansion occurs through imbalanced fluid
uptake or through a matched growth of intracellular organelles was initially unclear. Cooper et
al. (2013) explain that murine tibial epiphyseal plate chondrocytes undergo three specific phases
of hypertrophy.
Using P5 mice, it was observed that chondrocytes first increase 3-fold from ~600 femtolitres (fl)
to 2000 fl in volume—the internal macromolecules also grow proportionately and the dry mass
density is therefore retained. In the second stage, a 4-fold increase in volume occurs from 2000 fl
to ~8000 fl—but without an increase in internal dry mass, effectively quartering the dry mass
density. In the third and final stage, the dry mass is permitted to stabilize before the chondrocyte
enlarges 2-fold to ~14000 fl. The dry mass grows proportionately with this increase at this stage
and the final density remains as approximately ¼ of the initial density. Cooper et al. (2013) show
in a linear regression that without disproportional growth, the chondrocyte volume would not
even reach 10000 fl if dry mass grew linearly. This process of hypertrophy, like endochondral
ossification as a whole, is henceforth sequential and tightly regulated by a variety of factors.
BMPs generally promote hypertrophy as explored in a variety of cell culture experiments, but
results can vary from stimulation to delay (Kobayashi, Lyons, McMahon, & Kronenberg, 2005).
For example, mice overexpressing Bmp4 have enhanced chondrocyte hypertrophy but BMP7
addition to ATDC5 cell-line chondrocytes suppresses their hypertrophy (Tsumaki et al., 2002;
Caron et al., 2013). As mentioned before, the Ihh/PTHrP duo tightly controls the rate of
hypertrophy. Transcription factor Mef2c binds and activates Col10a1 as a marker of hypertrophy
(Arnold et al., 2007). The hypoxia-inducible factors (HIFs) are heterodimers that can also bind
the Col10a1 promoter potently to transition chondrocytes into hypertrophy (Saito et al., 2010).
13
Not surprisingly, the most common markers of hypertrophic chondrocytes are type X collagen as
well as Mmp13, an enzyme responsible for collagen matrix restructuring for bone formation
(Nurminskaya & Linsenmayer, 1996). Runx2 and Runx3 are both expressed during hypertrophy
as requirements for chondrocyte maturation, albeit in a redundant manner (Inada et al., 1999).
Runx2 directly binds and activates Col10a1, Mmp13, and Ihh to maintain the hypertrophic
phenotype (Zheng et al., 2003; Selvamurugan, Kwok, Alliston, Reiss, & Partridge, 2004;
Yoshida et al., 2004). Proteins important for impending bone formation and blood vessel
invasion, such as osteopontin, osteocalcin, and VEGF, are also expressed during hypertrophy
(Lian, McKee, Todd, Gerstenfeld, 1993; Horner et al., 1999). As for the Sox trio genes, they are
likely turned off by Runx2 activity through reciprocal inhibition (Cheng & Genever, 2011).
As chondrocyte hypertrophy relies on water accumulation, members of the channelome capable
of water transport likely play an important role. Passive water intake through aquaporin channels
in articular chondrocytes surrounding the epiphyses have been well documented (Liang, Feng, &
Ma, 2008; Mobasheri et al., 2004). Furthermore, Aquaporin-1 is expressed throughout rat
epiphyseal plates and promotes hypertrophy (Claramunt et al., 2017). Other aquaporin isoforms
may be present to conduct water intake but have not been largely explored in the epiphyseal
zones. Channels such as calcium ion transporters can import calcium to activate calmodulin,
RUNX2, and maintain the hypertrophic phenotype (Chen, Fu, Cong, Wu, & Pei, 2015). As well,
channels that can import reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) may
generate hypoxic environments necessary to increase HIF activity and promote hypertrophy
(Lennicke, Rahn, Lichtenfels, Wessjohann, & Seliger, 2015). Members of the channelome,
cellular volume expansion, and specific gene expression work together to prepare hypertrophic
chondrocytes for terminal differentiation, ossification, and ultimately long bone growth.
Figure 1.4. Chondrocyte hypertrophy 14
Eventual direction of long bone growth
Hypertrophic
chondrocytes
1
Phases of chondrocyte
hypertrophy
2
3
Pre-hypertrophic
chondrocyte Volume 3x, dry mass 3x
Volume x2, dry mass x2
Volume 4x
Result:
Volume ~24x
Dry mass ~6x
Dry mass density = ¼ initial densityBMPs
Mef2c
Col10a1
HIFs
Mmp13
Runx2, Runx3
Prepare for
ossification
Hypertrophic
chondrocyte
Aqp1
Calcium ion transporters
Other channelome proteins . . .
Diaphysis
Epiphysis (not fully
shown)
Epiphysis (not fully
shown)
15
1.1.5 Ossification and long bone growth (see Figure 1.5)
Thus far, the entire process of endochondral ossification has been occurring in the centre of the
diaphysis; the primary centre of ossification. As Salazar, Gamer, & Rosen (2016) describe, the
centre is dominated by hypertrophic chondrocytes and chondrocytes are sequentially less
differentiated towards either of the epiphyseal ends, reflecting the spectrum of chondrocyte
phenotypes. By E14.5, hypertrophic chondrocytes in the centre core undergo apoptosis and blood
vessels invade to deliver hematopoietic stem cells (HSCs) that differentiate into osteoclasts.
Osteoclasts are cells that perform matrix resorption, whereas osteoblasts—which are required
after resorption—originate from MSCs similarly to chondrocytes and perform bone matrix
formation instead (Caetano-Lopes, Canhão, & Fonseca, 2007). The collagen matrix left behind
by the hypertrophic chondrocytes is excavated by osteoclasts and forms the bone marrow cavity.
Here, the remaining hypertrophic chondrocyte population is bisected by the vasculature and
osteoclasts, forming a longitudinal growth axis with one population of chondrocytes pointing
towards one epiphysis and the second population heading toward the other. As the populations
grow further in distance appositionally from the marrow cavity, osteoblasts arrive with the
invading blood vessels to synthesize osteoid for bone mineralization in the spaces where
hypertrophic chondrocytes have died and osteoclasts have cleared.
At postnatal days 5-7 (P5-P7), vasculature invades the globular ends of each epiphysis and
chondrocytes in their centre undergo the entire aforementioned process of differentiation towards
hypertrophy (Xing, Cheng, Wergedal, & Mohan, 2014). These secondary centres of ossification
ossify in the same manner, with the chondrocytes spreading to the ends of the bone becoming
articular cartilage, and the chondrocytes approaching either one of the primary centre
populations forming a union known as the epiphyseal plate. Eventually, ossification from either
end of the plate permeates into the chondrocyte population, causing the plate to narrow until the
diaphysis meets the epiphysis and long bone growth ceases. The primary centre of ossification
eventually becomes the bone marrow cavity, while the secondary centre of ossification is filled
with trabecular bone. Trabecular bone is a porous, spongy tissue found at the epiphyses and is
the main load-bearing bone in vertebrates (Oftadeh, Perez-Viloria, Villa-Camacho, Vaziri, &
Nazarian, 2015). The outer layer surrounding the bone is hard cortical bone, the compact tissue
16
enveloping the diaphysis (Eriksen, 2010). The process of ossification therefore requires many
steps of regulation as the entire long bone structure lengthens. Here, osteoblastogenesis is critical
to promote bone growth.
Lefebvre & Bhattaram (2010) succinctly describe the roles of osteoblasts: 1) to secrete non-
mineralized osteoid for later mineralization, 2) to secrete alkaline phosphatase and provide
inorganic phosphate for mineralization, and 3) to produce osteocalcin and other bone-specific
proteins to help mineralize the osteoid matrix. Osteoblasts primarily rely on Runx2 as a master
factor to differentiate from mesenchymal stem cells (MSCs); lack of Runx2 at the ossification
stage completely ablates bone formation (Otto et al., 1997). Osx is another transcription factor
that activates several bone genes—Col1a1 for type 1 collagen, Bgp for osteocalcin, and Bsp for
bone sialoprotein—in MSCs and transforms them into functional osteoblasts (Nakashima et al.,
2002; Sinha, Yasuda, Coombes, Dent, & de Crombrugghe, 2010). However, osteoblasts are not
only derived from MSCs. Hypertrophic chondrocytes in the epiphyseal plate are also able to
transdifferentiate into osteoblasts as an alternative to the apoptotic fate (Tsang, Chan, & Cheah,
2015). Here, Runx2 and Osx can stimulate them to re-enter the cell cycle, secreting Col1a1 and
osteocalcin as newly differentiated osteoblasts. This optional ‘chondro-osteoblastic’ lineage
highlights that osteogenic differentiation of hypertrophic chondrocytes is yet another point of
control in ossification.
In the case of rare metaphyseal chondrodysplasias—diseases that affect skeletal stature—such as
MCDS, a failure of chondro-osteoblastic differentiation is responsible for delayed ossification,
shorter long bones, and bowed legs (Ho et al., 2007). Nevertheless, general osteoblastogenesis
control still relies on many key factors from previous steps of endochondral ossification. Wnt/β-
catenin signalling is required in osteoblasts after Osx expression for osteoblasts to mature and
produce osteocalcin (Day & Yang, 2008). FGFR3 is a promoter of MSC-osteoblast
differentiation, but inhibitor of mature osteoblast mineralization (Su, Jin, & Chen, 2014). BMP2
also upregulates Runx2 and Osx expression during osteoblastogenesis (Matsubara et al., 2008).
During ossification, gene dysregulation can lead to abnormal length in specific bones (Panda,
Gamanagatti, Jana, & Gupta, 2014). Unnatural shortening of the major long bones can be
17
rhizomelic—affecting the proximal limbs; the humerus and femur—or mesomelic—affecting the
distal limbs; the radius, ulna, tibia, and fibula. Aside from mutations in the main players of
endochondral ossification, genes located on the sex chromosomes can also cause dysplasias.
Mutation of SHOX, the Short Stature Homeobox-containing gene, can cause idiopathic short
stature (Marchini, Rappold, & Schneider, 2007). Interestingly, the malformation of singular or
multiple individual bones can also occur; however, these are classified as dysostoses instead of
dysplasias (Offiah & Hall, 2003). Overall, ossification may not necessarily proceed at the same
rate in chiral limbs. Furthermore, irregular ossification and abnormal skeletal stature may arise
from mutations not necessarily related to the major genes in the epiphyseal plate.
In the channelome, aquaporin-5 enhances MSC apoptosis in the bone marrow through high water
permeability and decelerates femoral bone healing, where osteoblasts play a critical role (Yi et
al., 2012). In the osteoblast nuclear membrane, vitamin D receptors receive the 1,25D hormone
to stimulate bone-specific protein production and matrix secretion through chloride and calcium
channel activation (Wang, Zhu, & DeLuca, 2014; Zanello & Norman, 2004). Overall, a myriad
of genes, transcription factors, and protein channels dictate the cellular profile and ossification
status of the growing long bone. To further examine their specific contributions, the generation
of mutant mouse models is paramount to understanding effectors of bone elongation. The
deletion or overexpression of important aforementioned genes—such as Sox9 or Ihh—has helped
provide insight into the known and unknown processes affecting endochondral ossification.
Figure 1.5. Ossification and long bone growth 18
Developing
articular
cartilage
Developing
articular
cartilage
Secondary centre
of ossification
(developing
trabecular bone)
Secondary centre
of ossification
(developing
trabecular bone)
Epiphyseal plate Epiphyseal plate
Bone
marrow
cavity
Growing bone
tissue
(osteoblasts,
osteoclasts,
trabecular bone)
Growing bone
tissue
(osteoblasts,
osteoclasts,
trabecular bone)
Cortical bone
Direction of long bone growth
Primary centre of
ossification
Narrowing
epiphyseal
plate
Narrowing
epiphyseal
plate
Growing
secondary
centre
Growing
secondary
centre
Diaphysis
Epiphysis Epiphysis
Osx
Col1a1
Bgp
Bsp
Runx2
FGFR3
BMP2
Prepare for
apoptosis or
transdifferentiation
Terminal hypertrophic
chondrocyte
Aqp5
Vitamin D receptors &
chloride/calcium channels
Other channelome proteins . . .
Osteoblast (forms bone matrix) Osteoclast (resorbs bone matrix)
MSCsHSCs
19
1.2 Models of differential long bone growth
The house mouse, Mus musculus, is an invaluable model organism to understanding human
biology due to strikingly similar genomes and physiology (Perlman, 2016). Using homologous
recombination or Cre-Lox recombination, genes implicated in development and disease can be
knocked out globally or in specified sites throughout the mutant. However, both knockout and
overexpression models are useful in determining the effect of a gene in absence or abundance.
Here, murine mutants of the most critical transcription factors in endochondral ossification and
their skeletal phenotypes are described in detail. Their human mutation counterparts, if known,
are also described (see Table 1.1).
1.2.1 Sox mutants
As previously mentioned, the Sox protein trio is important towards chondrogenesis and early
differentiation, but delay hypertrophy. Sox9flox/flox; Prx1-Cre embryos, which have targeted limb
bud deletion of Sox9 prior to mesenchymal condensation, are unable to form either cartilage or
bone (Akiyama et al., 2002). In the same study, Sox9flox/flox; Col2a1-Cre embryos, which have
Sox9 deletion after condensation, present with severe chondrodysplasia. Akiyama et al. (2004)
also examined Sox9 overexpression with Col2a1/Sox9 knock-in embryos, observing decreased
chondrocyte proliferation, delayed hypertrophy, and diminished long bone formation.
Interestingly, Smits et al. (2001) found that Sox5 and Sox6 single-null mice presented with mild
skeletal abnormalities. However, Sox5; Sox6 double-null fetuses present with severe
chondrodysplasia, suggesting that Sox5 and Sox6 are redundant but essential together for bone
growth. Sox5 and Sox6 help secure Sox9 to the Col2a1 enhancer for type II collagen production
(Han & Lefebvre, 2008).
Overall, the Sox trio carefully regulates endochondral ossification and fluctuations in
expression—up or down—can severely dysregulate the final skeletal structure. In humans, any
mutations in the SOX9 coding region can lead to campomelic dysplasia: a typically lethal
disorder resulting in long bone bowing and other skeletal defects (Mansour et al., 2002).
20
1.2.2 Bmp mutants
Bmp signalling is present in all stages of endochondral ossification and primarily functions
through Smad pathways (Retting, Song, Yoon, & Lyons, 2009). Limb bud deletion of Bmp2 and
Bmp4 from MSCs severely impairs osteogenesis (Bandyopadhyay et al., 2006). However,
Bmp4flox/flox; Col2a1-Cre embryos alone do not display a striking cartilage phenotype—only
when Bmp2 is deleted alongside do severe chondrodysplasias occur (Shu et al., 2011). In the
study, it was concluded that Bmp4 alone is insufficient to affect chondrocyte differentiation but
Bmp2 alone is. Also, Bmp6 -/- mice at 10 weeks of age have smaller long bones and reduced
longitudinal growth rate following estrogen treatment (Perry, McDougall, Hou, & Tobias, 2008).
The BMP6 promoter is transcriptionally regulated by estrogen receptor α, suggesting that Bmp6
may be hormonally stimulated (Ong, Colley, Norman, Kitazawa, & Tobias, 2004). Yet, not all
Bmp mutants with bone phenotypes have diminished growth. GDF-7 is the murine homologue of
human BMP12 and GDF-7 -/- mice have accelerated endochondral bone growth, due to a shorter
hypertrophic phase duration (Mikic, Ferreira, Battaglia, & Hunziker, 2008). Mutations in specific
Bmp isoforms do not all result in long bone defects, but many known isoforms are important in
embryogenesis. In humans, dysregulation of BMP signalling can lead to osteogenesis
imperfecta—through BMP1—and osteoarthritis—through BMP5 and BMP14 (Wang et al.,
2014).
1.2.3 Fgfr mutants
The Fgfs mediate many endochondral cellular responses by binding to the four unique Fgfrs (Su,
Jin, & Chen, 2014. Since activated Fgfrs then phosphorylate and activate pathways downstream,
modulating Fgfr expression greatly influences the efficacy of Fgf signalling (Ornitz, 2005).
Fgfr1flox/flox; Col2-Cre embryos have osteo-chondroprogenitor cells with delayed osteoblast
differentiation, but show increased bone mass by adulthood (Jacob, Smith, Partenen, & Ornitz,
2006). In humans, activating mutations of FGFR1 leads to osteoglophonic dysplasia and
rhizomelic dwarfism, where proximal limbs are shortened (White et al., 2005). Fgfr2 mutants
have bone defects which are mainly cranial rather than endochondral. However, deletion of the
3c alternative of Fgfr2 results in premature loss of growth and diminished long bones
21
(Eswarakumar et al., 2002). Human gain-of-function mutations of FGFR2 cause a variety of
craniosynostes, which are premature fusions of skull sutures (Wilkie, 2005). In Fgfr3 mutants,
activating mutations result in shortened long bones, disorganized proliferating zone columns, and
smaller body size overall (Ornitz & Marie, 2002). Deletion of the 3c isoform of Fgfr3 results in
overstimulation of the proliferating zone and dramatic skeletal overgrowth with reduced bone
mineral density (Eswarakumar & Schlessinger, 2007). In humans, Ornitz & Marie (2002) also
review that a variety of different FGFR3 point mutations result in many dysplasias, such as
achondroplasia and hypochondroplasia. On the contrary, Fgfr4 -/- mutants are conversely normal
in development—but when deleted alongside Fgfr3 in a double knockout, they are dwarfed in
overall size (Weinstein, Xu, Ohyama, & Deng, 1998). Overall, Fgfrs control balance among the
cellular players of skeletal growth.
1.2.4 Ihh & PTHrP mutants
Indian hedgehog and PTHrP are responsible for controlling the rate at which chondrocytes
undergo hypertrophy, meaning that their modulations should critically alter the long bone
phenotype in mutants. Indeed, Ihh -/- mutants show severe shortening of limbs at all
endochondral ossification embryonic timepoints with almost completely reduced appendicular
bones by the newborn stage (St-Jacques, Hammerschmidt, & McMahon, 1999). These mutants
have drastically reduced chondrocyte proliferation, as well as delayed chondrocyte maturation
into hypertrophy. In humans, homozygous mutations of IHH result in acrocapitofemoral
dysplasia: a rare autosomal recessive disorder featuring short limbs and irregular-shaped hands
and hips (Hellemans et al., 2003). As the partner of Ihh, PTHrP mutations display similar
phenotypes. The epiphyseal plates of PTHrP -/- mutants have markedly shorter proliferating
zones as well as advanced hypertrophy, apoptosis, and terminal mineralization (Lee et al., 1996).
Without surprise, human mutations of PTHrP result in chondrodysplasia (Nissenson, 1998).
1.2.5 Runx2 mutants
Runx2 plays a pivotal role in the hypertrophic and mineralization stages of endochondral
ossification. Runx2 -/- mutants die shortly after birth due to a complete lack of ossification
22
(Komori et al., 1997). Takarada et al. (2013) show that even Runx2flox/flox; Col2-Cre mutants are
unable to prevent this perinatal lethality, suggesting that Runx2 is a requirement for proper
endochondral ossification in both embryonic and postnatal stages. However, they also show that
Runx2flox/flox; Col1-Cre present with no observable skeletal abnormalities. Runx2 may therefore
be important but redundant by the time committed osteoblasts arrive to build bone, where Col1a1
is expressed. Using the same type 1 collagen promoter, Runx2 was overexpressed in osteoblasts
(Liu et al., 2001). Surprisingly, these transgenic mutants are stunted in growth, prone to
fractures, and have diminished osteoblast mineralization capacities. Runx2 is likely a negative
regulator of bone growth in late osteoblast development to control bone mass, but normally
promotes mineralization at physiological levels of expression. In humans, the importance of
RUNX2 is also evident. RUNX2 haploinsufficiency causes cleidocranial dysplasia, where the
clavicles, teeth, and overall stature are underdeveloped (Xu et al., 2017).
1.2.6 Mutant models with membrane protein and channelome modulation
Of the aforementioned critical players of endochondral ossification, proteins such as Fgf, Bmp,
and Ihh bind their receptors on the plasm membrane for downstream signalling. However, the
chondrocyte membrane is not static; its protein expression must change to reflect every stage of
its transformation during the long bone growth process. The channelome is home to a variety of
membrane proteins that are gaining explorative value as more mutant mice are generated to
investigate their skeletal outcomes.
Pannexin-3, or Panx3, is one such transmembrane gap junction channel with robust expression in
skin and cartilage (Penuela et al., 2007). As Panx3 is also expressed in pre-hypertrophic
chondrocytes, hypertrophic chondrocytes, and osteoblasts, Oh et al. (2015) generated Panx3 -/-
mice to observe their skeletal phenotypes. Panx3 -/- embryo epiphyseal plates showed delayed
hypertrophy, delayed osteoblast differentiation, delayed mineralization, and ultimately shortened
long bones that persisted till adulthood. Indeed, the Panx3 promoter region contains binding sites
where Runx2 may act and promote bone growth (Bond et al., 2011). Adam17 is a membrane
protein that functions as a metalloproteinase, releasing factors critical to hypertrophy such as
tumor necrosis factor alpha (Hall & Blobel, 2012). Hall & Blobel (2012) generated
23
Adam17flox/flox; Col2a1-Cre mutants and showed that newborn mice have expanded hypertrophic
zones with retarded long bone growth. Concerning hypertrophy, it should be reiterated that fluid
uptake—primarily water—is the root mechanism for chondrocyte enlargement. Investigating the
channelome members that transport water, the aquaporins, may be valuable to deciphering how
ubiquitous membrane proteins contribute to bone growth. However, mutant mice with modulated
aquaporin isoform genes are not well explored, and neither are the examination of their chondro-
osseous physiologies. Previously, Wu et al. (2007) ventured to measure the femoral bone density
of aquaporin-1-null mice and found that 2-month old mutants had reduced density, calcium, and
phosphorous in the bones. A focus on the precursors to ossification—the different chondrocyte
phenotypes—will characterize how these bone irregularities might arise. Specifically, the
aquaporins show promise as effectors of chondrocyte gene expression, differentiation, and
potentially long bone growth.
24
Table 1.1. Models of differential bone growth
Mutation / Affected gene(s) Hallmarks Disorder (if described)
BMP1 Fragile bones Osteogenesis imperfecta
BMP14 Articular cartilage damage Osteoarthritis
Bmp2C/C ; Bmp4C/C Severe chondrodysplasia -
Bmp4flox/flox; Col2a1-Cre Mild cartilage abnormalities -
BMP5 Articular cartilage damage Osteoarthritis
Bmp6 -/- (w/ estrogen) Smaller long bones, reduced growth rate -
Col2a1/Sox9 knock-in Decreased chondrocyte proliferation, delayed
hypertrophy, decreased long bone formation -
FGFR1 (activating) Craniofacial abnormalities, dwarfism; proximal
limb shortening
Osteoglophonic dysplasia;
rhizomelic dwarfism
Fgfr1flox/flox; Col2-Cre Delayed osteoblast differentiation, increased
adult bone mass -
FGFR2 Premature fusions of skull sutures Craniosynostis
Fgfr2 (3c) Chondrodysplasia -
Fgfr3 (3c) -/- Skeletal overgrowth, reduced bone mineral
density -
Fgfr3 (activating) Dwarfism, disorganized epiphyseal plate
proliferating zone columns -
GDF-7 -/- (murine homologue
of BMP12) Accelerated endochondral bone growth -
IHH Shortened long bones, irregular hands and hips Acrocapitofemoral dysplasia
Ihh -/- Severe chondrodysplasia -
PTHrP Chondrodysplasia -
PTHrP -/- Shorter epiphyseal plate proliferating zones,
advanced hypertrophy and mineralization
RUNX2 (haploinsufficiency) Short stature, underdeveloped clavicles and
teeth Cleidocranial dysplasia
Runx2 (transgenic) Fragile bones, diminished mineralization,
stunted growth -
Runx2 -/- Absence of ossification, perinatal lethality -
Runx2flox/flox; Col2-Cre Perinatal lethality -
Sox5 -/- Mild skeletal abnormalities -
Sox5; Sox6 -/- Severe chondrodysplasia -
Sox6 -/- Mild skeletal abnormalities -
SOX9 Lethality, long bone bowing, generalized
skeletal defects Campomelic dysplasia
Sox9flox/flox; Col2a1-Cre Severe chondrodysplasia -
Sox9flox/flox; Prx1-Cre Absence of cartilage and bone -
25
1.3 Aquaporins and chondrocytes: expression, function, and regulation
Aquaporins (AQPs) are a subfamily of integral membrane proteins that facilitate water and
solute conductance in a wide variety of physiological structures (Agre, 2006). AQPs are
distinguished from other integral transporters by a series of conserved hydrophobic residues and
a signature ‘NPA’ motif (Kruse, Uehlein, & Kaldenhoff, 2006). Functionally, AQPs are defined
as the isoforms that display selectivity in water and small solute transport across a membrane
(Takata, Matsuzaki, & Tajika, 2004). Thirteen mammalian isoforms (AQP0—AQP12) exist
throughout the human body with tissue-specific patterns and a higher concentration in organs
that constantly process water, such as the kidneys. However, homeostatic fluid transport is an
integral function to all healthy cells in maintaining basic tissue upkeep and controlled growth. In
particular, chondrocytes undergo notable water transport during a) volume changes in resting and
loaded cartilage tissue at the articular region, and b) phased volume changes for hypertrophy in
the epiphyseal plate that underlie skeletal growth (Oswald, Chao, Bulinkski, Ateshian, & Hung,
2008; Cooper et al., 2013). The chondrocyte-aquaporin relationship has been explored among a
variety of isoforms and substantiates the importance of the channelome in chondrocyte
proliferation, growth, and apoptosis.
1.3.1 AQP1
Aquaporin-1 (AQP1) was initially discovered as a tetrameric 28-kDa integral membrane protein
in human red blood cells when co-purified with the 32-kDa subunit of the Rh polypeptides
(Agre, Saboori, Asimos, & Smith, 1987). Originally named CHIP28 (channel-forming integral
protein of 28 kDa), expression of the gene in the X. laevis oocyte expression system exhibited
swollen oocytes with higher osmotic water permeability coefficients than their control
counterparts when placed in osmolarity-tempered solution (Agre et al., 1993). This demonstrated
that an abundant, archetypal membrane channel was capable of specific water transport and
confirmed its existence in a variety of mammalian organs as the newly termed ‘aquaporin’. The
wide distribution of AQP1 in ordinary human tissue was first illustrated using tissue microarray
analysis and visualized with immunohistochemistry, where the protein was found to be strongly
localized in chondrocytes within the deep zone of articular cartilage (Mobasheri & Marples,
26
2004). This presence supported a role of AQP1-mediated water transport across the chondrocyte
membrane, suggesting that compressive and osmotic forces finely balanced by the protein
channel may be necessary in the maintenance of healthy cartilage. Water content and type II
collagen collectively contribute to articular cartilage load bearing, where the loss of either
component can strain the other with a greater weight burden (Mow, Kuei, Lai, & Armstrong,
1980; Fox, Bedi, & Rodeo, 2009). As dysregulated water content in articular chondrocytes may
promote pathophysiological degradation of cartilage collagen fibers, these findings were
extrapolated to investigations in the possible contribution of AQP1 to osteoarthritis—a
degenerative disease featuring breakdown of cartilage in articular regions.
Meng, Ma, Li, & Wu (2007) simultaneously measured Aqp1 and Aqp3 mRNA expression in
adult rats after inducing osteoarthritis in their temporomandibular joints, observing that Aqp3—
but not Aqp1—mRNA was highly upregulated in the dissected cartilage groups. Gao et al.
(2011) then focused on determining a relationship between Aqp1 expression and osteoarthritis,
albeit in the long bone joints. After amputation of knee ligaments and partial damage to the
medial menisci in different test groups, aged Sprague-Dawley rats presented with significantly
elevated Aqp1 mRNA, Caspase-3 mRNA, and Caspase-3 protease activity several weeks post-
operation. As caspases execute cell apoptosis, the concomitant rise of Aqp1 expression suggested
a positive co-regulatory relationship between the two genes in chondrocyte death and henceforth
the onset of osteoarthritis.
Musumeci et al. (2013) further clarified the relationship between Aqp1 and osteoarthritis by
performing medial and lateral meniscectomies on thirty-six rats, observing that Aqp1 expression
was increased in the test groups via immunochemistry and Western blot analysis. In human
chondrocytes, AQP1 mRNA is also significantly higher in osteoarthritic cartilage and is further
elevated through IL-1B treatment, hinting at an inflammation-controlled regulation of AQP1 in
osteoarthritis progression (Haneda et al., 2018). Thus, the breadth of AQP1-osteoarthritis
investigations suggests that the aquaporin may exacerbate osteoarthritic swelling in the articular
regions and compromise the cellular morphology and metabolism of chondrocytes—although a
purported mechanism has not been elucidated thus far. Overall, AQP1 plays a functional role in
27
chondrocyte water uptake and its expression is implicated as a contributor to the onset of
osteoarthritis.
1.3.2 AQP2
The concrete ability of aquaporins to perform bidirectional transport of water in cartilage further
led to investigations of their presence in chondrocyte-like cells. Aquaporin-2 (AQP2) expression
was initially measured in the nucleus pulposus and annulus fibrosus of human intervertebral
discs, both consisting of hyperosmotic fibrocartilage responsible for absorbing daily compressive
loads in bodily movement (Richardson, Knowles, Marples, Hoyland, & Mobasheri, 2008). The
study sought to discover if AQP1, AQP2, and AQP3 were localized in discs and controlled cell
volume and extracellular matrix metabolism. While their immunohistochemistry was negative
for AQP2 protein in all fibrocartilage region cells, Gajghate et al. (2009) found AQP2 protein
expression in vivo in both rat and humans nucleus pulposus tissue, regulated by the tonicity-
enhancer binding protein TonEBP. Using mouse embryonic fibroblasts—a precursor of the
murine chondrocytic lineage—they demonstrated that TonEBP/NFAT5-null mutants experience
~50% decreases in Aqp2 promoter activity. AQP2 is largely unexplored in chondroskeletal
studies; yet its capacity to control water balance in chondrocyte-like resident cells by the
osmosensitive transcription factor TonEBP suggests that other aquaporin isoforms are also
transcriptionally regulated for cellular volume control. In chondrocytes, the diverse membrane
channels and porins rely on these signalling molecules to control not only disease—such as
osteoarthritis in the articular region—but also their natural differentiation in strictly-regulated
processes such as endochondral ossification. In chondrocyte hypertrophy, water accumulation is
a defining characteristic (Buckwalter et al., 1986). Therefore, aquaporins—the only known
membrane proteins that directly transport water—likely contribute to hypertrophy during
endochondral ossification (Kozono, Yasui, King, & Agre, 2002).
1.3.3 AQP3
Several aquaporin family members—deemed ‘aquaglyceroporins’—are also capable of glycerol
and uncharged solute transport in addition to water. Aquaporin-3 (AQP3) was the first
28
mammalian aquaglyceroporin to be cloned and was explored in chondrocytes to determine if
equine articular cartilage expressed aquaporins as a means of volume regulatory behavior
(Mobasheri & Marples, 2004). AQP3 and AQP1 were two isoforms expressed in the articular
region, suggesting their roles in the transport of metabolites and water respectively across the
chondrocyte membrane. Indeed, AQP3 was later found to be expressed in human articular
chondrocytes through human tissue microarrays (Mobasheri et al., 2005). Due to the presence of
both AQP3 and AQP1 in the articular region, both members were investigated during the
chondrogenic differentiation of human mesenchymal stem cells derived from adipose tissue
(Graziano et al., 2018). Chondrogenic markers including SOX9, aggrecan, and type II collagen
were measured during a four-week period alongside AQP3 and AQP1. Graziano, Avola,
Pannuzzo, & Cardile (2018) discovered that while AQP1 protein levels decreased after 21 days,
AQP3 protein persisted at high levels by 28 days. This suggests that while AQP1 may play
important roles at the earlier stages of differentiation, AQP3 is necessary throughout the process
and its alteration may lead to chondrogenic death and cartilage damage. As AQP3 is also
expressed in other immature cell types such as murine bone-marrow derived dendritic cells and
bovine blastomeres, aquaporins may be responsible for important developmental aspects in the
embryonic stages in addition to postnatal conditions such as osteoarthritis (Song et al., 2011;
Zhao et al., 2015). In particular, the importance of AQP3 expression during chondrogenesis
highlights a critical role of aquaglyceroporins in chondrocyte generation. Other members of the
aquaglyceroporin family—AQP7, AQP9, and AQP10—may play special functional roles during
prenatal processes, although their chondrogenic significance has yet to be delineated.
1.3.4 AQP4
Aquaporin-4 (AQP4) is largely explored in brain disorders, with a particular focus on its
involvement in astrocyte activity and the central nervous system (Verkman, Smith, Phuan,
Tradtrantip, & Anderson, 2017). However, Aqp4 is expressed in rat articular chondrocytes and
its siRNA inhibition during IL-1B-induced apoptosis reduces chondrogenic death by reducing
p38 MAPK activity (Cai et al., 2017). Similar to the AQP1 findings of Haneda et al. (2018),
Aqp4 likely contributes to chondrocyte apoptosis in joint diseases such as osteoarthritis and
rheumatoid arthritis. The presence of all aquaporins isoforms in varying tissues has not yet been
29
clearly characterized, and Aqp4 appears to have therapeutic value as a knockdown target in
organs aside from the brain. Due to the ubiquitous nature of aquaporins in a wide range of
mammalian cells, even uninvestigated isoforms may emerge as promising candidates as meta-
analyses continue revealing relationships between the channelome and diseases. The rising
recognition of aquaglyceroporins in metabolic syndrome and obesity-related pathologies
substantiates this phenomenon (da Silva, Rodrigues, Rebelo, Miranda, & Soveral, 2018).
1.3.5 Other aquaporins in chondrocyte activity
The remaining mammalian aquaporin isoforms in relation to chondrocytes have been largely
uninvestigated. Aquaporin-0 (AQP0) expression has been discovered in osteoarthritic articular
cartilage, but is mostly explored in its essential homeostatic role in the lens of the eye (Haneda et
al., 2018; Schey, Petrova, Gletten, & Donaldson, 2017). Aquaporin-5 (AQP5) is regulated by
BMP6—an inducer of cartilage and bone growth—in the salivary gland of Sjögren's syndrome-
like mice, yet its relationship to chondrocyte function is tangential (Lai et al., 2016). Aquaporin-
6 (AQP6) is expressed in Meckel’s cartilage in human orofacial tissues, but there are no studies
in its function in articular and epiphyseal plate chondrocytes (Wang et al., 2003). Aquaporin-7
(AQP7) is expressed in adipose tissue, and its deficiency results in increased glycerol kinase
activity, triglyceride accumulation, and ultimately obesity and a Type-2 diabetic phenotype (Iena
& Lebeck, 2018). Interestingly, these phenotypes are reminiscent of metabolic syndrome
symptoms, which include inflammation and osteoarthritis. Beyond cartilage damage from
obesity-added compressive forces, hyperglycemia from insulin resistance triggers production of
mitochondrial reactive oxygen species, inflammatory mediators, and metalloproteinases in
chondrocytes (Courties, Sellam, & Berenbaum, 2017). It is possible that Aqp7-null mice
chondrocytes are abnormally prone to degradation in the articular region and hypertrophy in the
epiphyseal plate due to these factors, although a definitive study has not been made.
The other aquaporin isoforms (8, 10, 11, and 12), aside from Aquaporin-9 (AQP9), have not
been explored in fields related to cartilage, bone, or pathologies that influence cartilage or bone
development. AQP11 and 12 are ‘super-aquaporins’ that are more recent discoveries and have
been briefly explored in major murine organs without attention to chondrocytes or skeletal
30
phenotypes (Ishibashi, Tanaka, & Morishita, 2014). As a whole, the majority of aquaporin
investigations focus on isoforms in physiological structures that unambiguously require water
transport due to the nature of their functions. The chondrocyte-aquaporin relationship is
delineated in several isoforms, but characterization of previously unexplored aquaporins in
chondrocytes will highlight the importance of the channelome in both the articular and
epiphyseal zones. In particular, AQP9 is a strong candidate for investigation in chondrocyte
function due to its indiscriminate conductance nature and supporting literature for its relationship
with hypertrophy-inducing mediators. It has previously been explored in bone, osteoclasts, and
synoviocytes. A definitive study of AQP9 in chondrocytes may unveil an aquaporin isoform with
the capacity to control chondrocyte proliferation, hypertrophy, death, and even biogenesis.
1.4 Characterization of AQP9 and its novel role in chondrocyte and
bone activity
AQP9 was initially discovered in a systematic gene analysis of human adipocytes which revealed
a unique sequence encoding for a 342-amino-acid membrane protein (Kuriyama et al., 1997).
This channel was named ‘Aquaporin-9’ and was tested via expression in X. laevis oocytes,
demonstrating a potent 7-fold increase in water permeability and the ability to conduct glycerol.
Over a decade of literature has revealed its capacity to facilitate transport of urea, purines,
pyrimidines, arsenic, hydrogen peroxide, and a variety of other small uncharged solutes (Viadiu,
Gonen, & Walz, 2007; Liu et al., 2002; Watanabe, Moniaga, Nielsen, & Hara-Chikuma, 2016).
The indiscriminate substrate specificity of AQP9 makes it unique among other aquaporin
isoforms, where it may conduct a wider range of solutes that regulate cellular metabolism. Given
that endochondral ossification relies on harmony between transcription factors, gene expression,
and channelome activity, the liberal solute conductance of AQP9 shows promise as an important
aquaporin in chondrocytes as they differentiate and extend bone length.
1.4.1 General aquaporin protein structure
All aquaporin isoforms are tetramers and emit a positive electrostatic field that prohibits cations
from passing through (Rothert, Rönfeldt, & Beitz, 2017). Each monomer forms an independent
31
pore, and all aquaporins share a common intramembranous protein fold comprised of two amino
acid tandem repeats in opposing directions. These repeats largely define aquaporin function and
ultimately the identity of solutes that chondrocytes may receive during regulated processes, such
as differentiation in the epiphyseal plate. In the first repeat, 3 helices aptly named ‘helices 1-3’
are present alongside a reentrant loop named ‘B’. Yan & Luo (2010) characterized that reentrant
loops are important structural motifs in alpha-helical transmembrane proteins that penetrate
halfway into the membrane and then return to their side of origin. These loops feature low
hydrophobicity and may serve to limit the amount of water permeated through aquaporins, as
more hydrophilic residues in the inner pore constriction can attract water molecules to the
channel walls and raise the energy cost of passage (Murata et al., 2000). In the second repeat, 3
more helices named ‘helices 4-6’ are present alongside a reentrant loop named ‘E’. Loops B and
E form the short pore-lining alpha-helices HB and HE respectively, which contain important
residues located midway into the channel. These loops also contain the hallmark motifs of
aquaporins, the Asn-Pro-Ala (NPA) motifs. These two motifs are co-localized and attract passing
water molecules to the same side, where the helices HB and HE are nestled midway into the
membrane. Murata et al. (2000) discovered that as water molecules pass through the pore in
single-file due to efficient hydrogen-bond arrangement, Asn residues 76 and 192 from HB and
HE respectively will use their amido groups to force every incoming water molecule to switch
their hydrogen-bonding partners to themselves instead. Having abandoned the hydrogen bonds
formed with the molecule ahead and the molecule above, the targeted water molecule hydrogen-
bonds to the Asn residues and is contorted perpendicularly to the direction of water flow. With
the target’s hydrogen atoms facing 90° from the single-file water chain, the efficient stream is
broken. However, Murata et al. (2000) further explained that the pore walls feature exclusively
hydrophobic residues at the Asn constriction site, promoting exit and total permeation of water
molecules through the channel with an ultimately low energy barrier. For cations such as protons
that rely on a stable ‘proton wire’ built on the hydrogen bonds of the single-file water chain, the
chain breakage prohibits them from passage through the channel (Pomès & Roux, 1996). The
relatively tight intramembranous constrictions and wider membranous openings of aquaporins
creates a high dielectric barrier to the majority of ions. Hence, neutral solutes are favored for
passage among all aquaporin isoforms.
32
1.4.2 AQP9 protein structure (see Figure 1.6)
Despite their shared structural features and resulting permeation capacities, AQP9 is
distinguished by having the widest range of substrate specificity among the aquaporins. Viadiu et
al. (2007) created an AQP9 projection map by reconstituting glycosylated rat AQP9 into two-
dimensional crystals with aid from magnesium and calcium ions. The crystals were then
embedded in glucose, frozen, and processed at 7 angstrom resolution to view the protein
structure. Interestingly, the projected AQP9 tetramer resembled the E. coli-derived glycerol
facilitator protein GlpF with greater fidelity than pure water-specific aquaporins, such as AQP0
and AQP1. AQP9 and GlpF monomer projections presented with a more square-shaped
phenotype than those of AQP0, which are more wedge-shaped in characteristic. Furthermore,
GlpF boasts a round pore diameter of 7 angstroms while AQP9 has a more oval-shaped pore of
approximately 7x12 angstroms in dimension. Viadiu et al. (2007) identified a region of lower
electron density existing in both GlpF and AQP9 specifically, which may explain their capacities
to conduct larger solutes. AQP9 not only boasts a larger pore area, but also has ‘tripathic pores’
where each channel has a hydrophobic corner, a hydrogen-bond donor corner, and a hydrogen-
bond acceptor corner (Stroud et al., 2003). These designated areas allow for substrates to be
oriented by region, increasing their likelihood of reaching an optimal spatial configuration and
passage. Viadiu et al. (2007) analyzed this characteristic and identified seven amino acid
residues conserved among all AQP9 homologues, but different from other aquaporins and GlpF.
These residues are Phe 64, Gly 81, Met 91, Val 176, Phe 180, Leu 209 and Cys 213. As most of
these substitutions occur at the hydrophobic corner near the entrance of the pore, the residues
themselves may be responsible for the lower density and easier substrate access. Overall, AQP9
is an isoform that features larger pores, a lower density barrier, and unique residues compared to
other aquaporin members. Describing substances that the channel conducts will provide insight
into AQP9 function in chondrocyte differentiation and long bone growth.
Figure 1.6. AQP9 protein structure
Conducts:
Water
Urea
Purines
Pyrimidines
Arsenic
Hydrogen peroxide
Glycerol
Lactate
Selenite
Pore size
(~7Å x 12Å)
Projection map of AQP9 tetramer
Aqp9
monomer
7Å
12Å
Tripathic pore
components and
unique residues
33
34
1.4.3 AQP9 protein function (see Figure 1.6. cont.)
The elucidated pore size and residue composition of AQP9 grants the ability to transport both
orthodox and unorthodox molecules. In the chondrocyte channelome, many membrane proteins
are multifunctional and can be ion channels, receptors, and signalling elements all in one
(Mobasheri et al., 2018). The diversity of substrates that a channel can tolerate may parallel the
amount of control it has over highly regulated and sequential processes, such as endochondral
ossification. Aside from water, AQP9 is permeable to glycerol, urea and smaller molecules
including purines and pyrimidines (Tsukaguchi, Weremowicz, Morton, & Hediger, 1999).
Lactate is also transported by AQP9 (Badaut & Regli, 2004). Chondrocytes rely heavily on
glycolysis in culture and glycerol may be imported by AQP9 as gluconeogenic fuel (Jackson,
Huang, & Gu, 2012). Conversely, lactate is a by-product of glycolysis and is produced heavily
during chondrocyte growth (Hossain, Bergstrom, & Chen, 2015). AQP9 may be important in
exporting excess lactate to maintain physiological pH and cell growth rate. As a neutral solute
conductor, AQP9 is also permeable to arsenic and even selenite (Liu et al., 2002; Geng et al.,
2017). Interestingly, arsenic contributes to telomere attrition and apoptosis by creating ROS,
which is an inducer of chondrocyte hypertrophy (Liu, Trimarchi, Navarro, Blasco, & Keefe,
2003; Morita et al., 2007). It is sensible to question if AQP9 in the chondrocyte channelome may
be an important modulator of chondrocyte differentiation under arsenic exposure. Interestingly,
arsenic exposure has been shown to decrease hypertrophic zone height in rats during
endochondral ossification (Aybar Odstrcil, Carino, Ricci, & Mandalunis, 2010).
In relation to ROS transport, one of the most striking permeants of AQP9 is H2O2 (Watanabe et
al., 2016). It was previously discussed that the HIF family of transcription factors induce
hypertrophic differentiation in the epiphyseal plate under hypoxic conditions. As H2O2
accumulates in eukaryotes in response to hypoxia, AQP9 H2O2 transport may therefore be critical
toward differentiation as well (Vergara, Parada, Rubio, & Pérez, 2012). Overall, AQP9 channels
conduct a broad spectrum of substrates—some of which have clear connections to chondrocyte
growth and possibly ossification. Their distribution and transcriptional control should be further
described to support a role in chondrocyte activity.
Figure 1.6. cont. 35
AQP9 monomer side view
MembraneHE
HB
Hallmark
NPA
motif
Other helices (H1-H6)
Residues
unique to
AQP9
Gly
81
Met
91
Val
176
Phe
180
Leu
209
Cys
213
Asn
Pro
Ala
Hydrophobic
corner
Phe
64
H-bond
corner
H-bond
corner
Selective
passage
through
interaction
with residues
WaterUrea
Purines
Pyrimidines
Arsenic
Hydrogen peroxide
Glycerol
Lactate
Selenite
36
1.4.4 Expression and regulation of AQP9 (see Figure 1.7)
Aqp9 protein is expressed in a variety of tissues, such as rat Leydig cell membranes, spleens,
brains, and murine spinal cords (Nicchia, Frigeri, Nico, Ribatti, & Svelto, 2001; Elkjaer et al.,
2000; Oshio et al., 2004). In mice, Aqp9 is expressed developmentally in the trophoblast
(Barcroft, Offenberg, Thomsen, & Watson, 2003). Human placentas and peripheral leukocytes
from the blood also express AQP9, although the function has not been fully explored (Damiano,
Zotta, Goldstein, Reisin, & Ibarra, 2001; Ishibashi et al., 1998). AQP9 is also expressed in liver
hepatocytes, where hepatic gluconeogenesis occurs (Rodríguez et al., 2014). In fact, their study
found that AQP9 was the most abundantly expressed aquaporin isoform in the human liver. This
coincides with the function of AQP9 as a glycerol transporter useful for energy metabolism.
Aqp9 is also localized within mitochondrial membranes, where lactate import can influence the
mitochondrial matrix pH and regulate the formation of ROS which may be critical to
chondrocyte hypertrophy (Amiry-Moghaddam et al., 2005).
Concerning regulation, the promoter sequence of Aqp9 features the insulin response element
(IRE) sequence TGTTTTC (-496/-502), sharing homology with core negative IREs found in the
promoters of genes including PEPCK and AQPap/7 (Kuriyama et al., 2002). In their study,
insulin downregulated Aqp9 mRNA expression in cultured hepatocytes and it is expected that
Aqp9 in other organs is transcriptionally suppressed by insulin at the same putative site. Insulin is
a known inducer of chondrogenic differentiation, although conflicting results have been found
(Phornphutkul, Wu, & Gruppuso, 2006; Torres, Andrade, Foncesa, Mello, & Duarte, 2003).
Nevertheless, transcriptional regulation of Aqp9 in varying tissues has been largely unexplored.
Aqp9 expression in hepatocytes is decreased by estrogen in a concentration-dependent manner,
as well by estrogen receptor agonists (Lebeck et al., 2012). As well, Vitamin-D receptor deficient
mice present with lower estrogen levels, higher Aqp9 mRNA expression, and higher estrogen
receptor alpha expression—likely due to compensation for the hormone deficiency (Zanatta et
al., 2017). Estrogen is a gonadal steroid that can promote epiphyseal growth and slow it as well,
and it may use AQP9 as a signalling intermediate to regulate growth during puberty (Nilsson et
al., 2014). Testosterone is also able to rescue Aqp9 expression after estrogen treatment in the
developing rat epididymis, although the effect of steroid hormones on Aqp9 may depend strongly
37
on the cell type Aqp9 is expressed in (Pastor-Soler et al., 2010). Hence, the Aqp9 promoter is
known to contain androgen response elements (AREs) and estrogen response elements (EREs)
where hormones can bind to regulate transcription (Joseph, Shur, & Hess, 2011).
A natural phenol, phloretin, is a known inhibitor of AQP9 and has been used as a channel
blocker in a variety of studies (Tsukaguchi et al., 1998; Dibas, Yang, Bobich, & Yorio, 2007;
Calamita et al., 2012). However, phloretin is not AQP9-specific and also affects other proteins
like anion channels (Sabirov, Kurbannazarova, Melanova, & Okada, 2013). AQP9 is a viable
candidate for control of endochondral ossification; however, it needs to be further explored in
both chondrocytes and bone. Here, the expression and function of AQP9 in chondro-osseous
tissues and pathways is described in detail.
Figure 1.7. Expression and regulation of AQP9 38
AQP9 protein expression has been shown in the mammalian…
Testicles (Leydig cells) Spleen Brain
Spinal cord Trophoblast Placenta
Liver (hepatocytes) Leukocyte Mitochondria
Known AQP9 gene regulation
Regulatory region
Insulin
Downregulates Aqp9 in
hepatocytes through the
insulin response element (IRE)
Estrogen / estrogen receptor agonists
Downregulates Aqp9 in hepatocytes
through the estrogen response
element (ERE)
Testosterone
May upregulate Aqp9 in the
epididymis through the androgen
response element (ARE)
Phloretin
Downregulates Aqp9 activity
AREEREIRE
Known AQP9 protein regulation
Introns and exons
Transcribed region
Inhibitory effect
39
1.4.5 H2O2, an inducer of chondrocyte hypertrophy, is transported by AQP9
AQP8 was among the first aquaporin isoforms to be implicated in general ROS transport
(Bienert et al., 2007). It was demonstrated that expression of AQP8 in yeast cells increased their
sensitivity to exogenously supplied H2O2, through observing increased fluorescence in AQP8-
transformed yeast cells supplied with fluorescent-dyed H2O2. Then, AQP3 was shown to import
H2O2 in keratinocytes when stimulated with TNF-α in psoriasis development (Hara-Chikuma et
al., 2015). Watanabe et al. (2016) eventually demonstrated that AQP9 expression in CHO-K1
cells increased H2O2 import with exogenously added H2O2, and that AQP9 silencing in HepG2
cells reduced extracellular import. Their study further used Aqp9 -/- mice to observe suppressed
uptake of H2O2 in knockout erythrocytes and mast cells compared to their WT (wildtype)
counterparts, highlighting a third aquaporin isoform capable of ROS conductance.
The developing epiphyseal plate is typically regarded as hypoxic, where ROS expression has
been shown to increase as well as decrease in separate studies (Schipani et al., 2001; Bell,
Klimova, Eisenbart, Schumacker, & Chandel, 2007; Frandrey, Frede, & Jelkmann, 1994). To
complicate matters, HIF-1α, an oxygen-sensitive component of the transcription factor HIF-1,
does not appear to be expressed linearly with ROS formation (Qutub & Popel, 2008).
Nevertheless, the HIF homologue HIF-2α is important for chondrocyte hypertrophy and its
encoding gene, Epas1, increases in expression alongside other important genes such as Col10a1
and Mmp13 (Saito et al., 2010). As well, AQP9 may be responsible for controlling intracellular
H2O2 levels in the epiphyseal plate chondrocyte mitochondria, where HIF-1α can accumulate in
response to ROS generation (Chandel et al., 2000). Describing the function of AQP9 in
chondrocyte-oriented processes such as endochondral ossification or cartilage degradation would
further clarify its role in chondrocyte activity, possibly through controlled H2O2 uptake.
1.4.6 AQP9 expression is upregulated in osteoarthritis
To suggest that AQP9 affects chondrocyte activity, solely highlighting its conductance of a
known chondrocyte hypertrophy modulator is insufficient—evidence of its gene expression and
functional protein activity in physiological or disease processes is necessary. In patients with
40
osteoarthritis and rheumatoid arthritis, AQP9 was detected in their synovial tissues at the RNA
and protein levels (Nagahara et al., 2010). More importantly, osteoarthritic tissues with
hydrarthrosis—irregular fluid accumulation in the knee—featured significantly higher AQP9
mRNA expression than in tissues without. This suggests that in chondrocyte diseases such as
osteoarthritis, AQP9 may not only contribute to abnormal water homeostasis but may also be a
genetic marker of the disorder. Osteoarthritis involves elevated chondrocyte production of
proteolytic enzymes such as MMP13 which subsequently cause cartilage damage and diminished
joint function (van der Kraan & van den Berg, 2012). Given that AQP9 expression parallels the
expression of degradative genes in chondrocyte disease—and that these same genes are
necessary in physiological hypertrophy of chondrocytes during endochondral ossification—the
function of AQP9 in chondrocytes appears to mimic a hypertrophic or inflammatory phenotype.
Another study investigating upregulated genes in human osteoarthritic synovial tissues found
that AQP9 expression was significantly higher among other angiogenic-classified genes when
compared to normal tissues, such as PF4V1 and EDNRB (Lambert et al., 2014). Angiogenesis
and osteogenesis in endochondral ossification are post-hypertrophic processes, suggesting that
AQP9 may favor a non-proliferative chondrocyte phenotype. Whether AQP9 contributes to
hypertrophy in the articular cartilage or epiphyseal plate chondrocytes through water
conductance, H2O2 uptake, an uninvestigated metabolic pathway, or a combination of the
aforementioned is currently unknown. Nevertheless, AQP9 is associated with hypertrophic
chondrocyte gene expression. It may underlie chondrocyte disorders that induce a hypertrophic
phenotype, such as osteoarthritis where type X collagen expression is increased (Walker,
Fischer, Gannon, Thompson, & Oegema, 1995). If AQP9 plays a role in dysregulating
hypertrophy at the articular cartilage, it may also function in chondrocyte regions that undergo
natural hypertrophy—such as the epiphyseal plate. However, a functional study of AQP9 in the
epiphyseal zones has yet to be performed. The identification of AQP9 in the terminal products of
endochondral ossification may highlight a gap in the literature for the channel’s function in the
epiphyseal plate. In particular, it would support that AQP9 is important in the exterior of bone—
at the articular cartilage—as well as in bone tissue itself—in cells or matrix—but is unexplored
in between the two regions. Investigating AQP9 in the epiphyseal plate may describe a novel
gene potentially important to long bone formation.
41
1.4.7 Aqp9 expression rises during osteoclast biogenesis
To further support a role of AQP9 in regulating chondrocyte activity and prospective bone
growth, a function of the channel in bone cells would connect these two phases of endochondral
ossification and suggest its importance in the process.
Aharon & Bar-Shavit (2006) hypothesized that Aqp9 would be critical in osteoclast
differentiation due to a significant volume change—like chondrocyte hypertrophy—from their
murine bone marrow macrophage precursors. When differentiated via RANK-L, the subsequent
osteoclasts presented with higher Aqp9 and Mmp9 expression. Mmp9 is a well-known matrix
metallopeptidase that is elevated in rheumatoid arthritis just as Aqp9 is, suggesting the two genes
are active in concert during both chondrocyte dysregulation and osteoclast differentiation. The
RANK-L-stimulated macrophages were then treated with phloretin to investigate if an Aqp9
inhibitor would diminish the differentiation process. The macrophages were unable to survive
the differentiation process, suggesting that Aqp9 plays a critical role in bone cell formation.
However, Liu et al. (2009) compared osteoclasts between Aqp9 WT and -/- mice, discovering
that -/- osteoclasts did not differ in morphology and resorption ability in comparison to their WT
counterparts. Due to phloretin’s capacity to block other channels aside from Aqp9, the -/- mouse
model is more representative of the importance of Aqp9 in osteoclast differentiation.
Nevertheless, they also observed increased Aqp9 expression during differentiation of the pre-
osteoclast cell line, RAW264.7. While Aqp9 -/- chondrocytes have not been explored, the
presence and rise of Aqp9 expression in osteoclasts suggests that Aqp9 may be important at the
culminating steps of endochondral ossification. The two main osteoclast resorption enzymes,
MMP9 and MMP13, are present during osteoarthritic cartilage damage as well as Aqp9
expression. If Aqp9 and Aqp9-affiliated genes play functional roles at the articular and bone
matrix regions respectively, Aqp9 is likely to act in the structure adjoining the two as well—the
epiphyseal plate. Describing roles of Aqp9 in macroscopic bone metabolism would further
clarify its importance throughout the entire endochondral ossification process and resultant bone
tissue formation.
42
1.4.8 AQP9 is a possible target for bone loss attenuation and bone length modulation
AQP9 has been described as a target for the prevention of bone loss, suggesting its activity may
span anywhere from the cartilage anlage to post-endochondral ossification structures. Since
endochondral ossification underlies long bone development, it is likely that Aqp9 functions in the
epiphyseal plate and affects bone health. In postmenopausal women, a single nucleotide
polymorphism in intron 1 of AQP9 is associated with increased bone mineral density at the
femoral neck (Chanprasertyothin, Saetung, Rajatanavin, & Ongphiphadhanakul, 2010). Subjects
with a thymine nucleotide rather than an adenine nucleotide at the rs2414539 A/T position had
the highest estimated odds ratio for differential density, suggesting the role of AQP9 in human
bone metabolism. Although the polymorphism is located in an intronic region, it may affect
splice variance and result in AQP9 protein isoforms that behave according to their host cell type
(Wang & Cooper, 2007). Therefore, AQP9 may play an important role in the cells upstream of
bone formation. Interestingly, Aqp9 -/- mice are able to retain more bone tissue after
microgravity-induced bone loss when compared to their WT counterparts (Bu, Shuang, Wu, Ren,
& Hou, 2012). Femoral Aqp9 expression was also elevated during the simulated microgravity,
suggesting that Aqp9 is at least partly responsible for the bone loss observed. The study also
captured postmenopausal effects on Aqp9 expression by checking for femoral bone mineral
density in ovariectomized mice; the lack of estrogen did not change expression and hints that
Aqp9 may be reactive to stressful bone conditions. Aqp9 may therefore play important roles
throughout endochondral ossification and mature bone remodeling.
Previously, the osteoclast findings of Liu et al. (2009) had included comparisons of femur and
tibia lengths between one-year old female Aqp9 WT and -/- C57BL/6 mice; they revealed no
significant difference in bone length between the two genotypes. If Aqp9 is important in bone
development, its functional role may not be apparent in mature bone tissue but rather in the
earlier murine stages before longitudinal growth has slowed. However, a definitive study of
Aqp9 in early postnatal chondrocytes and bone has not yet been made. Examining the long bone
lengths of Aqp9 WT and -/- mice at a time before the juvenile P21 age may determine if
chondrocytes active in endochondral ossification are functionally different in the knockout
43
mutation. The use of a mutant model with inactivated Aqp9 will clarify the role of Aqp9 in
epiphyseal chondrocyte activity and long bone growth.
1.5 Aqp9 -/-: an accessible knockout mouse model for epiphyseal
chondrocyte and bone length investigation
Rojek et al. (2007) initially created the Aqp9 -/- mouse to investigate the role of Aqp9 in type 2
diabetes mellitus. In obese type 2 diabetic patients, hepatic glucose production is elevated in part
from the gluconeogenic properties of glycerol. Given that Aqp9 is a glycerol conductor, the
expectation was that inactivation of Aqp9 might reduce the import of the gluconeogenic substrate
and decrease blood glucose levels. Aqp9 -/- mutants feature different metabolic characteristics in
comparison to their heterozygous counterparts that may support the role of Aqp9 in chondrocyte
differentiation. Furthermore, the mutants do not experience any embryonic or early postnatal
mortality, easing dissection and examination of bone and cartilage phenotypes.
1.5.1 Aqp9 -/- mutants have abnormal glycerol metabolism
In comparison to +/- mutants, Aqp9 -/- mutant mice present with increased plasma glycerol and
triglyceride levels after 24 hours of fasting (Rojek et al., 2007). This seemingly contradictory
observation may be credited to compensatory glycerol uptake by the other aquaglyceroporin
isoforms to stabilize glycerol blood levels. Glycerol is a gluconeogenic precursor vital for
phospholipid synthesis, entry into the glycolytic pathway, and conversion to glucose for
metabolic homeostasis and skeletal growth. While the knockdown of Aqp9 would then appear to
dysregulate metabolism, Rojek et al. (2007) further demonstrated that administration of
exogenous glycerol in both genotypes did not result in significantly differential glucose levels
between the two. Hence, the capacity for -/- mutants to generate glucose despite the blockage of
a gluconeogenic precursor conductor suggests they are sufficient in maintaining a relatively
normal whole-body metabolic phenotype. However, the plasma measurements were implicitly
performed in mature mice several weeks past the young pup/neonatal age; the early postnatal
phenotypes were not characterized. The availability of glucose and gluconeogenic precursors
greatly influence the capacity for early bone growth. Maor & Karnieli (1999) demonstrated that
GLUT4, IGF-1, and insulin receptors play critical roles in preventing retardation of skeletal
44
growth in P6-P8 mice. Indeed, glucose transporters and insulin-related receptors are critical
components of anabolism in bone (Klein, 2014). Furthermore, epiphyseal plate chondrocyte
proliferation is dependent on the presence of glucose intake, as insulin and IGF-1 are both
capable of promoting chondrogenic differentiation (Zhang et al., 2014). Given that the building
blocks of anabolism in skeletal growth—energy-generating precursors such as glycerol—were
unexplored in the early age Aqp9 -/- mutants where they would have the greatest influence on
bone development, the mouse model presents as a prime candidate for discovering the effects of
Aqp9 in the epiphyseal plate. The uninvestigated glycerol defect in early postnatal mutants
suggests that Aqp9 deletion may induce a possible unique skeletal phenotype.
1.5.2 Mature Aqp9 -/- mutants show superficially normal physical characteristics
In comparison to age-matched WT and +/- littermate controls, the Aqp9 -/- mutants presented
with no apparent differences in body weight and physical appearance. Rojek et al. (2007)
implicitly performed all observations at mature timepoints, as referenced by immunostaining for
Aqp9 protein in 6-week-old littermates, 15-week periods of waiting for development of type 2
diabetes in Leprdb and Aqp9 knockout mice, and 2-week periods of waiting post-shave to prepare
for skin regeneration experiments. Indeed, the body weight measurements that were presented
were reported from 6-14 weeks of age, and only in obese (Leprdb/Leprdb) Aqp9 +/- and -/-
mutants. If physical measurements were indeed made at early postnatal timepoints for Aqp9 -/-
mutants—before the P21 timepoint—it is unlikely they were statistically analyzed due to their
absence of presentation. However, it cannot be excluded that measurements may have been
statistically analyzed but omitted from presentation due to a lack of significant differences. In the
case of epiphyseal plate and long bone length differences, dissection and extraction of the
structures would be necessary for accurate measurement.
Liu et al. (2009) investigated osteoclast function in Aqp9 -/-mutants and extensively
characterized Aqp9 WT femurs and tibias in comparison to their -/- mutant counterparts. They
show that in one-year-old female mice, both genotypes do not differ in femur length, tibia length,
whole-body and long bone density, and osteoclast cell density. In femur and tibia length, Aqp9 -
/- mutants show minor decreases in comparison but without statistical significance. Bone density
45
was also measured at 4, 6, and 10 weeks of age in both genotypes and showed no significant
differences, suggesting that mature WT and -/- mice share physically similar bones phenotypes.
However, if Aqp9 plays an important functional role in bone development, it may be apparent
only during timepoints where epiphyseal plate chondrocytes are actively differentiating to
lengthen bone—before approaching maturity, partial epiphyseal plate fusion, and dramatic
retardation of longitudinal growth. To best capture the influence of Aqp9 in epiphyseal plate
chondrocyte activity, experiments should be performed at postnatal timepoints close to the onset
of bone development at E14.0 as endochondral ossification begins. Timepoints ranging between
P0 and P21 would serve as candidates for long bone measurement, where Aqp9 may be
functioning without apparent influence from pubertal skeletal mediators including sex steroids
and growth hormone (Courtland et al., 2011). There are currently no characterizations of early
postnatal Aqp9 -/- mutant mouse epiphyseal plate chondrocytes and long bone length
measurements. Observed differences in long bone length between -/- mutants and their WT or
heterozygous littermates at an appropriate postnatal timepoint—such as P5—would support
Aqp9 as a regulator of chondrocyte activity in the epiphyseal plate.
1.6 Aqp9 has a novel function in cartilage and murine long bone growth
In the developing cartilage anlage, joint-site associated MSCs cease expression of early
chondrocyte markers such as Col2a1 to form the joint menisci (Hyde, Boot-Handford, & Wallis,
2008). MSCs that retain Col2a1 expression in the center of the premature joint—the interzone—
form articular cartilage (Jiang & Tuan, 2015). Although articular cartilage is hyaline, simple, and
generally non-proliferative, these interzonal cells reside where chondrocytes once occupied and
are henceforth undifferentiated chondrocytic descendants (Nalin, Greenlee, & Sandell, 1995).
Articular and epiphyseal plate chondrocytes are then discriminated by their location, function,
and microenvironment despite their common origin. These cell types share conducting pathways
that affect their development and dysregulation. In particular, the Wnt/β-catenin signaling
pathway has been implicated in osteoarthritis development where genes such as Wnt7b and Dkk1
expression levels correlate with osteoarthritic severity (Nakamura, Nawata, & Wakitani, 2005;
Honsawek et al., 2010). This canonical pathway is complex and not only also regulates skeletal
phenotypes—such as Wnt3a and Wnt5a +/- mutants which exhibit lower bone mineral density—
46
but may also target lesser known genes and pseudogenes such as those from the aquaporin
families (Okamoto et al., 2014). Here, the Kannu lab has demonstrated the merit of investigating
aquaporin/β-catenin interactions in osteoarthritis and also skeletal bone length.
1.6.1 Inhibition of Aqp9 protects against cartilage damage in humans and mice
Previous work in the Kannu lab identified AQP7P1 as a β-catenin target gene in resected femoral
condyle articular cartilage from patients undergoing total knee replacement for osteoarthritis
(Ma, Vi, Whetstone, Kannu, & Alman, 2014). AQP7 presented with a -2 fold change through
ChIP-sequencing and was not known to be functional in osteoarthritis or a known target of β-
catenin at the time. Dkk1, a target of the β-catenin pathway and ameliorator of osteoarthritis, was
then used to treat articular chondrocyte samples in culture from 5 patients undergoing knee
replacement surgery (Kannu, Weng, Poon, Ali, & Ma, 2015). Interestingly, a microarray showed
that AQP9 was differentially regulated by Dkk1 with a greater than 2-fold change; this supported
that aquaporins from the aquaglyceroporin family appeared to be critical in osteoarthritis
pathogenesis. Aqp9 -/- mice aged to 18 months were then sacrificed and measured for knee
cartilage damage, revealing less osteoarthritis severity in the mutants in comparison to the WT
mice, as determined through the recommended method of OARSI scoring (Glasson, Chambers,
Van den berg, & Little, 2010). Kannu et al. (2015) also demonstrated that 18 month Aqp9 -/-
mice were protected against age-induced cartilage damage in the knee, as measured by decreased
Col10a1 immunohistochemistry staining. With the previous literature regarding Aqp9 in
osteoarthritis, osteoclast biogenesis, and bone mineral density, Ma et al. (2014) and Kannu et al.
(2015) were able to further highlight the importance of aquaporins such as Aqp9 in the articular
region and possibly the growing epiphyseal plate as well. However, preliminary evidence of
Aqp9 function in a mouse model would be required to substantiate this postulation.
1.6.2 Early-aged Aqp9 -/- pups appear to be larger than their WT littermates
During examination of P5 WT and Aqp9 -/- pups, the Kannu lab also noticed that the -/- pups
appeared to be slightly larger than their WT counterparts. These differences were occasionally
observable by eye but complicated by the skin appearance and arrangement of the limbs which
47
may have biased interpretations. Initial weight measurements of both genotypes did not reveal
any significant differences, as relayed by colleagues of the Kannu lab. However, even mice that
exhibit skeletal overgrowth defects—such as Fgfr3c -/- mutants—have lower mean body
weights, suggesting that any long bone differences in Aqp9 -/- mice may not necessarily correlate
with their weight measurements (Eswarakumar & Schlessinger, 2007). Instead, performing
skeletal preparations and epiphyseal plate measurements would be necessary in delineating any
particular long bone phenotype. Previous work with the Kannu lab performed preliminary
measurements of Aqp9 -/- Col10a1-stained epiphyseal plate regions at unidentified timepoints
but did not return any significant differences (Yang et al., 2014). Preliminary measurements of
Col2a1-staining in P14 Aqp9 -/- epiphyseal plates also returned no significant differences,
although the proliferative and hypertrophic zones appeared to be partially shorter in height (Shao
et al., 2016). Overall, appropriate replicates of epiphyseal regions would be required alongside
skeletal preparations at various timepoints to identify Aqp9 as a regulator of epiphyseal
chondrocyte function. Furthermore, characterizing any remarkable transport function of Aqp9 -/-
chondrocytes at the in vitro or in vivo level would support its role in modulating the epiphyseal
plate and long bone growth.
1.6.3 Aqp9 may be a critical regulator of chondrocyte proliferation, hypertrophy, and
long bone growth
With the Kannu lab, Shao et al. (2016) also demonstrated that Aqp9 -/- knee epiphyseal
chondrocytes treated with 1 mM H2O2 were more viable than their WT counterparts after 24
hours. This result substantiates that H2O2 transport in Aqp9 -/- chondrocytes is reduced and is
able to elicit an anti-apoptotic effect. Furthermore, examination of intracellular H2O2
concentrations after treatment showed a >0.01 mM reduction in the Aqp9 -/- chondrocytes. The
proportion of apoptotic cells was then measured after treatment through Annexin V and
propidium iodide gating in flow cytometry, showing a >20% reduction in the Aqp9 -/- group. If
these epiphyseal plate chondrocytes are capable of resisting apoptosis induced by exogenous
means, they may also be resistant in physiological environments where a reduction of
intracellular H2O2 may affect gene expression, chondrocytic phenotype, and ultimately long bone
growth. Indeed, Shao et al. (2016) found that Mmp13 expression in Aqp9 -/- chondrocytes was
48
>6-fold less in comparison to WT chondrocytes post-treatment. The expression of Sox9 was
conversely >1.5 fold greater, suggesting that Aqp9 deletion supports chondrocytic retention of
the proliferative phenotype. Taken together, the Kannu lab has demonstrated that Aqp9 may be
important in regulating epiphyseal plate chondrocyte gene expression and directing their fate
toward long bone growth. H2O2 may also be the main substrate blocked to resist the hypertrophic
phenotype. Nevertheless, scrutiny of the long bones, epiphyseal plates, and chondrocyte gene
expression of Aqp9 -/- mutants at pup, juvenile, and old age timepoints are required to test this
hypothesis. My Master’s thesis project is to further characterize any purported long bone
phenotypes in Aqp9 -/- mice and study their essential structures: epiphyseal plate zones and
chondrocyte gene profiles.
49
2 Research Aims, Hypothesis, and Summary Plan
Investigating the epiphyseal plate and chondrocyte behavior can provide insight into new
mechanisms by which long bones grow. Specific experiments must be planned to measure the
long bones, epiphyseal plate zones, and chondrocyte activity in both control and test groups.
Aqp9 is a transporter of H2O2 and expressed in articular cartilage and osseous tissues, but
unexplored in the epiphyseal plate. I hypothesized that Aqp9 is a regulator of epiphyseal plate
chondrocyte proliferation, hypertrophy, and long bone growth. Here, the significance and
structure of the investigation are described.
2.1 Rationale
Chondrocyte hypertrophy, where massive fluid uptake is performed, is the largest contributor to
long bone growth during endochondral ossification (Cooper et al., 2013). Given that the
channelome comprises the set of membrane ion channels and porins that dictate chondrocyte
function, aquaporins may be responsible for governing how chondrocytes differentiate in the
epiphyseal plate (Mobasheri et al., 2018). Aqp9 is an isoform capable of transporting diverse
substrates across the membrane and potentially influencing chondrocyte gene expression
(Tsukaguchi et al., 1999). Aqp9 is important in articular cartilage and in the bone shaft where
osteoclasts function (Kannu et al., 2015; Liu et al., 2009). However, the function of Aqp9 at their
structural intermediary—the epiphyseal plate—is unclear.
2.2 Hypothesis
Preliminary findings in the Kannu lab have shown that Aqp9 deletion is protective against
chondrocyte hypertrophy, but analysis of the Aqp9 -/- skeletal and epiphyseal plate phenotype
has not been completed. I hypothesized that Aqp9 is a regulator of epiphyseal plate chondrocyte
proliferation, hypertrophy, and long bone growth. As Aqp9 is a transporter of H2O2, an inducer
of chondrocyte hypertrophy, Aqp9 -/- mice epiphyseal plates may present with dysregulated
hypertrophy.
50
2.3 Objectives
2.3.1 Objective 1: Long bone measurements of WT and Aqp9 -/- mice
To continue the preliminary findings of the Kannu lab, the first aim of this study was to
determine if long bone differences existed between WT and Aqp9 -/- littermate mice. Full-body
dissections of mice at the pup age (P5) and male mice at the juvenile age (P21) were performed
to liberate whole skeletons for skeletal preparation staining (Mitchell, Gould, Smolik, Koek, &
Daws, 2013; Rigueur & Lyons, 2014). Finished skeletons were photographed, then further
dissected to isolate long bones for manual and digital measurement. Measurements were
analyzed to observe for any statistical significance.
2.3.2 Objective 2: Epiphyseal plate analysis of WT and Aqp9 -/- mice
The second aim of the study was to examine the long bone epiphyseal plate zones of WT and
Aqp9 -/- mice to determine if differences in zone heights existed. In situ hybridization of Aqp9 to
the WT epiphyseal plate was performed to confirm gene expression. H&E staining of P5
epiphyseal plates was performed to observe any differences in zone heights. Col10a1
immunohistochemistry of P21 epiphyseal plates was performed to observe if Col10a1, a marker
of hypertrophy, stained differently between the WT and Aqp9 -/- mice. Toluidine blue staining of
18 month old mice was performed to observe any differences in epiphyseal line fusion. To
observe if Aqp9 deletion affected the epiphyseal plate at the developmental stage, E16.5 embryo
epiphyseal plates were stained with Safranin-O to visualize the chondrocyte distribution. All
epiphyseal plate zone heights were quantified and analyzed for statistical significance.
2.3.3 Objective 3: Primary chondrocyte analysis of WT and Aqp9 -/- mice
The third aim of the study was to determine the proliferation rate and gene profile of WT and
Aqp9 -/- epiphyseal plate chondrocytes. P5 WT and Aqp9 -/- primary chondrocytes were seeded
at 100,000 cells and counted daily over 96 hours. Cell counts were analyzed for statistical
significance to determine if Aqp9 -/- chondrocytes had a proliferation defect. Whole RNA from
P5 WT and Aqp9 -/- primary chondrocytes was also extracted to synthesize cDNA for qPCR
analysis with probes for Aqp9 and other markers of proliferation and hypertrophy. P5 WT
51
primary chondrocytes were also cultured with an Aqp9-siRNA, harvested for whole RNA
extraction, and used for cDNA synthesis for qPCR analysis with the aforementioned probes.
2.4 Clinical significance
Leg length discrepancies (LLDs) are inequalities in the length of the lower limbs that can arise
from anatomical or functional means (Murray & Azari, 2015). Anatomical LLD is characterized
by actual bone asymmetry in the lower limbs, whereas functional LLD is characterized by
abnormalities in other structures—such as the lower spine or pelvis—that result in apparent
shortening of one leg (Gurney, 2002; Subotnick, 1981). LLDs are universal and affect up to 90%
of the population with a mean length inequality of 5.2mm between the lower limbs (Knutson,
2005). However, deviations from this discrepancy can alter the weight-bearing capacities of the
lower extremities and contribute to osteoarthritis, scoliosis, lower back pain, and gait disruption
(Golightly, Allen, Helmick, Renner, & Jordan, 2009; Raczkowski, Daniszewska, & Zolynski,
2010; Kaufman, Miller, & Sutherland, 1996). Currently, the only interventions are physical
methods—such as shoe lifts for mild cases—or surgery—such as bone resection, mechanical
lengthening, or guided growth in severe cases (Brady, Dean, Skinner, & Gross, 2003; Hasler,
2000). These procedures are complex, invasive, and often depend on extensive rehabilitation.
Hence, there is a continuous need to determine the mechanisms underlying long bone growth in
order to identify therapeutic targets and create effective treatments.
2.5 Cellular mechanisms
LLDs can be congenital—through disorders such as dysplasias—or acquired through trauma, and
the root mechanism lies in dysregulated long bone growth (Murray & Azari, 2015). Long bone
growth occurs at the epiphyseal plate through the process of endochondral ossification. Here,
chondrocytes proliferate, undergo hypertrophy, and undergo apoptosis or transdifferentiate into
osteoblasts to allow for bone formation (Mackie et al., 2008). Each of these stages are highly
regulated by expression of key marker proteins including SOX9, BMPs, RUNX2, and FGFs
(Long & Ornitz, 2013). Among others, these transcription factors dictate chondrocyte division
capacity, shape, and expression of specific cartilage matrix collagens. Deletion of these critical
genes often results in dysregulation of chondrocyte activity, malformed epiphyseal plates during
52
development, and ultimately shortened and disfigured long bones. However, the contribution of
each protein isoform is variant: in FGFs, Fgfr3 -/- mice have longer bones than their WT
counterparts (Su et al., 2010). This suggests that Fgfr3 negatively regulates long bone growth.
Overall, investigating the expression of unique marker genes at each stage of epiphyseal plate
chondrocyte differentiation may provide insight into factors which may be controlled to
ameliorate LLDs.
53
3 Methods
3.1 Mouse creation, maintenance, genotyping, and age selection
The Aqp9 mutant mouse model was generated by substituting 55 nucleotides of exon 2 in Aqp9
with a neomycin phosphotransferase expression cassette, resulting in correct translation of the
first 47 amino acids, then 100 randomized amino acids, and then a stop codon. RT-PCR with
customized primer pairs confirmed the mutated Aqp9 gene and western blotting with the
AQP9A-1 primary antibody confirmed the absence of Aqp9 protein in the mutants. Aqp9 +/-
mutants were crossed with a C57BL/6 mouse background and initial heterozygote crosses
produced offspring expected of Mendelian ratios, suggesting that -/- mutants can be bred without
difficulty. No embryonic or early postnatal mortality is present in the mutants. The Aqp9 mutants
were a generous gift imported by Dr. Soren Nielsen from Aarhus University, Denmark. Mice
used in this study were housed in standardized cages at The Centre for Phenogenomics (TCP) in
Toronto, ON according to their guidelines. General mouse maintenance was performed by
employees at TCP. For the genotyping of embryonic and pup stage mice, yolk sacs and tail clips
were harvested respectively and digested in 50ul of QuantaBio Extraction Reagent for 30
minutes. Then, 50ul of QuantaBio Stabilization Buffer was added to neutralize the mixture. 2uL
of DNA was used for PCR. For genotyping of juvenile and old mice, ear notches were made by
employees at TCP and sent to Transnetyx, Inc. for DNA analysis.
For specific experiments, the P5 age was selected to investigate endochondral ossification at the
pup stage where pubertal growth has not yet occurred. The P14 age was selected to best visualize
the discrete zones of the epiphyseal plate. The P21 age was selected to investigate endochondral
ossification at the juvenile stage where pubertal growth and early adolescence has begun. The 18
month age was selected to investigate a timepoint where the epiphyseal plate is senescent.
3.2 In situ hybridization
RNA digoxigenin-dUTP-labeled riboprobes were created from linearized template DNA
plasmids for Aqp9. Riboprobes were synthesized using a DIG-labeling mix (Roche) according to
54
the manufacturer’s instructions and precipitated in 5M LiCl DEPC, 100% ethanol at -80°C for 2
hours. The riboprobes were resuspended in 75ul sterile DEPC-water, 75ul formamide and stored
at -80°C until usage. In brief, mouse lower limbs were harvested in cold DEPC-PBS, embedded
in paraffin blocks, and sectioned. Slides were prepared through a xylene/DEPC-ethanol/DEPC-
PBS/PFA/TEA solution series and then air dried. Hybridization buffer (distilled formamide, 20x
DEPC-SSC pH 4.5, 10% DEPC-SDS, and 20mg/ml heparin) was warmed at 85°C prior to
addition of the riboprobes. Riboprobes were then added and hybridization was performed
overnight at 55°C. After a post-hybridization SSC/formamide/TNE/TBST wash series, slides
were blocked with blocking reagent (Roche) for 1 hour. Anti-DIG antibody (1:2000, Roche) was
then added for room temperature incubation, for 1 hour. BM purple colour substrate (Roche) was
used for colour development over 8-20 hours. The colour reaction was stopped with a distilled
water/100% ethanol/xylene rinse series. Slides were then mounted in Permount and covered in
coverslips for air drying.
3.3 Skeletal staining
For staining of bone and cartilage, whole skeletons were dissected from mice and fixed in 95%
ethanol for 48 hours in room temperature. Then, skeletons were submerged in Alcian blue
staining solution (2.5ml 0.3% Alcian Blue SGS (Sigma), 10ml glacial acetic acid, and 40ml
ethanol) for 48 hours at 37°C. Alcian blue staining solution was replaced with 95% ethanol daily
over 3 days. Skeletons were then submerged in Alizarin red staining solution (0.5ml 0.2%
Alizarin Red S (Sigma), 5ml 10% KOH, and 45ml distilled water) for 24 hours in room
temperature. Alizarin red staining solution was then replaced with 20% glycerol, 1% KOH for 3
days in room temperature and then 50% glycerol, 1% KOH until muscle and fat tissue was
dissolved. The dissolving step varied in time depending on the mouse size. Skeletons were then
stored in 80% glycerol for 24 hours and then 100% glycerol for long-term storage.
3.4 Staining and immunohistochemistry of epiphyseal plates
Mouse limbs were harvested in PBS and stored in 70% ethanol prior to paraffin embedding and
sectioning. Limbs were then paraffin embedded and sectioned. Slides were rehydrated in a
100%/90%/70%/50% ethanol series, then washed twice in distilled water for 3 minutes. For
55
H&E staining, slides were then immersed in 0.3% ammonium hydroxide for 20 dips and rinsed
twice in water for 1 minute, followed by immersion in Eosin Y certified biological stain (Fisher
Scientific) for 10 dips. For Toluidine blue staining, slides were then immersed in 1% Toluidine
Blue O (Sigma) for 10 minutes. For Safranin-O staining, slides were then immersed in Weigert’s
iron hematoxylin solution (Sigma) for 10 minutes and rinsed in distilled water followed by
immersion in 1% Safranin O (Sigma) for 5 minutes. After all staining procedures, slides were
washed in water thrice for 1 minute, dehydrated in a 95%/100% ethanol and xylene series, then
mounted and covered. For immunohistochemistry, slides were then bleached in 6% H2O2 for 6
hours, followed by rehydration in 1% Tween-20 in PBS. Blocking was performed with sheep
serum:DMSO 4:1 and then incubated in antibody solution containing 1:100 anti-phosphorylated
H3 antibody (Cell Signaling Technology) for 24 hours at 4°C. After a wash series of 1% Tween-
20 in PBS, slides were incubated in an antibody solution containing 1:200 HRP-conjugated
secondary antibody. Slides were then covered in DAB and incubated for 20 minutes for the
colour reaction to occur. Then, slides were mounted and covered.
3.5 Visualization and measurement of limbs and epiphyseal plates
Skeletal staining preparations of mice were photographed with a Canon DSLR over a backlight
and self-made cardboard aperture to maintain the same magnification. A ruler was placed in each
shot for measurement references, and images were rotated in ImageJ and PowerPoint.
Measurements of limbs were performed using a Mastercraft digital caliper. The measurements
were performed blinded and each bone was measured twice with its averaged value reported for
statistical analyses.
For P5 mice, humerus bones were measured from the most proximal staining point (at the
articulation with the distal glenoid cavity) to the most distal staining point (at the articulation
with the proximal ulnar aspect). Femur bones were measured from the most proximal staining
point (at the articulation with the distal acetabulum) to the most distal staining point (preceding
the knee femoral cartilage). Tibia bones were measured from the most proximal staining point
(proceeding the knee tibial cartilage) to the most distal staining point (at the articulation with the
talus bone). For P21 mice, humerus bones were measured from the most proximal staining point
56
(at the head of the humerus) to the most distal staining point (at the condyle). Femur bones were
measured from the most proximal staining point (the femoral head and greater trochanter) to the
most distal staining point (at the femoral condyles). Tibia bones were measured from the most
proximal staining point (the head and tuberosity) to the most distal staining point (at the
malleoli). Skull condylo-basal lengths were measured from the most anterior aspect of the nasal
bone to the most posterior aspect of the braincase.
Epiphyseal plates were visualized and photographed microscopically, then transferred to ImageJ
and PowerPoint for cropping. Measurements of epiphyseal plate zones and cellularity were
performed using ImageJ, then recorded using the Region of Interest (ROI) Manager. The
measurements were also performed blinded and each replicate was measured twice with its
averaged value reported for statistical analyses.
3.6 Primary chondrocyte culture, qPCR, and RNA silencing
Epiphyseal cartilage tissue was excised from mouse limb knee joints in PBS and digested in
1mg/ml Pronase powder (Sigma) for 1 hour at 37°C. The tissue contained all cartilage normally
stained by Alcian blue in the knee region. The tissue was digested in 0.5mg/ml Collagenase P
powder (Sigma) for 24 hours at 37°C. Chondrocytes were re-suspended in PBS and plated in
Dulbecco’s Modified Eagle Medium F/12 (Gibco) with 5% anti-anti and 10% fetal bovine
serum. Cell counting was performed using a manual counter and ThermoFisher Scientific
Countess® Automated Cell Counter. RNA was extracted using the RNeasy Mini Kit (Qiagen)
according to manufacturer’s instructions. RNA was then reverse transcribed into cDNA using a
SuperScriptTM VILOTM cDNA Synthesis Kit (Invitrogen) according to manufacturer’s
instructions. Gene probe primers were designed using Primer3 Version 4.0.0 to target the exon-
exon junctions of murine mRNA. Design parameters included primer sizes of 18-30bp, Tm of 58-
60°C, and GC content of 40-60%. Reactions were performed using Fast Evagreen® qPCR
Master Mix (Biotium) according to manufacturer’s instructions, and run on the ViiA 7 Real-
Time PCR System (Agilent Technologies) with actin and GAPDH controls. RNA silencing was
performed using the Lipofectamine® 2000 Reagent (Invitrogen) kit according to manufacturer’s
instructions. Primary chondrocytes were cultured until ~50% confluency in 6-well plates for
57
transfection. 100pmol Aqp9 siRNA oligomer was diluted in 250ul Opti-MEM® Medium. 5ul
Lipofectamine® 2000 was diluted in 250ul Opti-MEM® Medium and incubated for 5 minutes at
room temperature. The diluted mixture was incubated for 20 minutes at room temperature. The
mixture was made for each chondrocyte sample and added to each well for 48 hours of
incubation at 37°C. An equimolar diluted control siRNA (Santa Cruz Biotechnology) was added
to control wells. RNA extraction, cDNA synthesis, and qPCR were then performed.
3.7 Statistical analyses
Data (mouse weights, bone measurements, epiphyseal plate measurements, chondrocyte
experiments, etc.) were analyzed using the Student’s two-tailed heteroscedastic t test by
comparing all test groups (heterozygous, knockout, silenced, etc.) to corresponding control
groups (wildtype, wildtype with control, etc.). The ANOVA test was not utilized as multiple
group comparisons were not performed in any analyses. Significance was defined as the p-value
(P), with *P <0.05, **P <0.01, ***P <0.001. Means and error bars were graphed using
Microsoft Excel chart and error bar formatting tools.
58
4 Results
4.1 Body weight and superficial comparisons of WT and Aqp9 -/- mice
Mice were selected at the pup/neonatal age (P5), juvenile age (P21), and old age (18 months old)
for imaging and mean body weight measurements. This would serve to determine if any
superficial body size differences existed across the Aqp9 -/- mouse lifespan. At the P5 timepoint,
the mice were not sexed due to sex determination unreliability. Whole body differences due to
sex are typically unobservable at this age (Schlomer et al., 2013; Bouleftour et al., 2014). At the
P21 and 18 month timepoints, male mice were used to prevent any sex-specific body weight
differences.
At P5, decapitated WT and Aqp9 -/- pups do not appear to differ significantly in body size or
average body weight (Figure 4.1A, 4.1D). At P21, WT and Aqp9 -/- juvenile mice also appear
superficially similar and do not differ significantly in average body weight (Figure 4.1B, 4.1E).
18 month old WT and Aqp9 -/- mice display the same characteristics (Figure 4.1C, 4.1F). The
measurements at the three timepoints charted over time show consistent body weights between
WT and Aqp9 -/- mice.
59
Figure 4.1. Body weight and superficial comparisons of WT and Aqp9 -/- mice
Side-by-side physical appearance comparison of WT and Aqp9 -/- mice at P5 to observe any
superficial differences at the pup stage (A). n=6.
A
WT Aqp9 -/- 5cm
60
Figure 4.1. cont.
Side-by-side physical appearance comparison of WT and Aqp9 -/- mice at P21 to observe any
superficial differences at the juvenile stage (B). n=6.
WT Aqp9 -/- 10cm
B
61
Figure 4.1. cont.
Side-by-side physical appearance comparison of WT and Aqp9 -/- mice at 18-months of age to
observe any superficial differences at the mature stage (C). n=3.
WT Aqp9 -/- 10cm
C
62
Figure 4.1. cont.
Body weight measurements of WT and Aqp9 -/- mice at P5 (D), P21 (E), and 18 months of age
(F). n=6, 6, and 3 respectively. Average body weights of WT and Aqp9 -/- plotted over time (G).
Student’s two-tailed t test was performed for statistical analysis, with the level of significance set
at *P < 0.05.
0
5
10
15
20
25
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35
40
P5 P21 5 mos.
Avera
ge w
eig
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(g)
Mouse age
WT
Aqp9 -/-
0.00
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2.00
3.00
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6.00
8.00
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12.00
14.00
WT Aqp9 -/-
Ave
rag
e w
eig
ht (g
) N.S
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
WT Aqp9 -/-
Ave
rag
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) N.S D E F
G
P5 P21 18 months
63
4.2 Histological analysis of Aqp9 expression in the juvenile epiphyseal
plate
Aqp9 expression was checked in the P14 WT mouse epiphyseal plate to confirm that Aqp9 is
normally present in the area where endochondral ossification occurs. In situ hybridization of
Aqp9 in the proximal tibia epiphyseal plate from dissected lower limbs was performed using an
Aqp9 DIG-labeled RNA probe (Figure 4.2A).
The transcript was detected in specific regions of the epiphyseal plate using the antisense probe.
Aqp9 is expressed in chondrocytes in the proliferating zone, as observed by the violet columns of
stacked cells. Furthermore, Aqp9 is strongly expressed in chondrocytes in the pre-hypertrophic
zone, as observed in the dark violet lower portions of the columns as chondrocytes begin to
deviate from their columnar organization. In the hypertrophic zone, Aqp9 staining is not robust
and suggests that Aqp9 is not expressed there. The sense probe produced less prominent staining
of Aqp9 in the epiphyseal plate, suggesting that the antisense probe is transcript specific (Figure
4.2B).
64
Figure 4.2. Histological analysis of Aqp9 expression in the juvenile epiphyseal plate
In situ hybridization of Aqp9 using the Aqp9 antisense probe in the WT P14 proximal tibia (A)
and with its corresponding sense probe (B). The gene can be detected in the proliferating and
pre-hypertrophic zones of the epiphyseal plate. R = resting zone, P = proliferating zone, PH =
pre-hypertrophic zone, H = hypertrophic zone, OC = ossification centre.
65
4.3 Skeletal staining of P5 WT and Aqp9 -/- mice
P5 mice were sacrificed and dissected for whole-mount skeletal staining. This experiment served
to identify if any long bone phenotype existed in neonatal mice with Aqp9 deletion. The staining
protocol was used to digest excess muscle and fat tissue, stain cartilage with Alcian blue, and
stain bone with Alizarin red to identify skeletal regions for accurate measurement. No gross body
differences are observable between WT and Aqp9 -/- mice (Figure 4.3A). Isolation of the upper
limb and scapula show no significant difference between the average Aqp9 -/- humerus bone
length in comparison to the WT humerus bone (Figure 4.3B, 4.3F, 4.3G). Isolation of the lower
limb shows that the average Aqp9 -/- femur bones also show no significant difference compared
to their WT counterparts, but the tibia bones are significantly different (Figure 4.3C, 4.3H, 4.3I,
4.3J, 4.3K).
To determine the overall percent differences between P5 WT and Aqp9 -/- long bones, the right
and left humerus, femur, and tibia bones from each biological replicate were grouped according
to bone type. The average Aqp9 -/- long bone length was divided by the average WT long bone
length to determine the percent change. The Aqp9 -/- humerus (Figure 4.3L) and femur (Figure
4.3M) bones do not show any significant percent differences compared to their WT counterparts
on average. The Aqp9 -/- tibia bones (Figure 4.3N) are significantly longer compared to their WT
counterparts on average. To test if the Aqp9 -/- long bone observation was specific to bone
formation and not affecting other organs, excised spleens from P5 WT and Aqp9 -/- mice were
weighed (Figure 4.3E). No gross differences in appearance or weight were observed, suggesting
that the mutation does not cause full body overgrowth (Figure 4.3O).
Furthermore, to test if the Aqp9 -/- long bone observation was specific to endochondral
ossification, P5 WT and Aqp9 -/- skulls were measured for their condylo-basal lengths (Figure
4.3D). The condylo-basal length is the length of the skull. No gross differences in skull length or
size were apparent, suggesting that the rate of intramembranous ossification—how skulls form—
may not be affected by the mutation (Figure 4.3P).
66
A
B
50mm
WT Aqp9 -/-
WT Aqp9 -/-
10mm
67
Aqp9 -/-WT
25mm
D
C WT Aqp9 -/-
10mm
68
Figure 4.3. Skeletal staining of P5 WT and Aqp9 -/- mice
Images of WT and Aqp9 -/- whole body lengths (A), humerus bones (B), femur and tibia bones
(C), and condylo-basal lengths (D) stained with Alcian blue and Alizarin red for cartilage and
bone identification. Green lines indicate the observable ossified regions. Excised spleens are
shown as well (E).
WT Aqp9 -/-
10mm
E
69Right Humerus Left Humerus
Right Femur Left Femur
Right Tibia Left Tibia
0
1
2
3
4
5
6
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
1
2
3
4
5
6
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
1
2
3
4
5
6
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
1
2
3
4
5
6
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
1
2
3
4
5
6
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
*
0
1
2
3
4
5
6
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
*
F G
H I
J K
70
Humerus Femur
Tibia
Figure 4.3. cont.
Measurements of WT and Aqp9 -/- right humerus bones (F), left humerus bones (G), right femur
bones (H), left femur bones (I), right tibia bones (J), and left tibia bones (K). Average percent
differences of Aqp9 -/- humerus bones (L), femur bones (M), and tibia bones (N) compared to
their WT counterparts. n=5. Student’s two-tailed t test was performed for statistical analysis
comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05.
75%
80%
85%
90%
95%
100%
105%
110%
115%
120%
125%
WT Aqp9 -/-
Avera
ge p
erc
ent
diffe
rence
Mouse genotype
N.S
75%
80%
85%
90%
95%
100%
105%
110%
115%
120%
125%
WT Aqp9 -/-
Avera
ge p
erc
ent
diffe
rence
Mouse genotype
N.S
75%
80%
85%
90%
95%
100%
105%
110%
115%
120%
125%
WT Aqp9 -/-
Avera
ge p
erc
ent
diffe
rence
Mouse genotype
*
L M
N
71
Figure 4.3. cont.
Measurements of WT and Aqp9 -/- excised spleen weights (O). n=3. Measurements of WT and
Aqp9 -/- condylo-basal lengths (P). n=5. Student’s two-tailed t test was performed for statistical
analysis comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05.
0
1
2
3
4
5
6
7
8
9
10
WT Aqp9 -/-
Avera
ge s
ple
en w
eig
ht
(mg)
Mouse genotype
N.S
0
5
10
15
20
WT Aqp9 -/-
Avera
ge c
ondyl
o-b
asal
length
(m
m)
Mouse genotype
N.S
O
P
72
4.4 Skeletal staining of P21 WT and Aqp9 -/- mice
P21 male mice were sacrificed and dissected for whole-mount skeletal staining. This experiment
served to identify if the P5 bone phenotype persisted from the neonatal stage to the juvenile
stage. The same Alcian blue-Alizarin red staining protocol was used to identify the skeleton
elements for accurate measurement. Gross body differences are not apparent between the WT
and Aqp9 -/- mice (Figure 4.4A). Isolation of the upper limb and scapula show no significant
difference between the average Aqp9 -/- humerus bone length in comparison to the WT humerus
bone (Figure 4.4B, 4.4F, 4.4G). Isolation of the lower limb shows that the average Aqp9 -/- tibia
bones also show no significant difference compared to their WT counterparts, but the femur
bones are significantly different (Figure 4.4C, 4.4H, 4.4I, 4.4J, 4.4K).
To determine the overall percent differences between P21 WT and Aqp9 -/- long bones, the right
and left humerus, femur, and tibia bones from each biological replicate were grouped according
to bone type. The average Aqp9 -/- long bone length was divided by the average WT long bone
length to determine the percent change. The Aqp9 -/- humerus (Figure 4.4L) and tibia (Figure
4.4N) bones do not show any significant percent differences compared to their WT counterparts
on average. The Aqp9 -/- femur bones (Figure 4.4M) are significantly shorter compared to their
WT counterparts on average.
To test if the P21 Aqp9 -/- bone observation was specific to bone formation and not affecting
other organs, excised spleens from P21 WT and Aqp9 -/- mice were weighed (Figure 4.4E). No
gross differences in appearance or weight were observed, suggesting that the mutation does not
cause full body dwarfism (Figure 4.4O). Furthermore, to test if the Aqp9 -/- short bone
observation was specific to endochondral ossification, P5 WT and Aqp9 -/- skulls were measured
for their condylo-basal lengths (Figure 4.4D). No gross differences in skull length or size were
apparent, similar to the P5 mice. This suggests that the rate of intramembranous ossification—
how skulls form—may not be affected by the mutation at this timepoint either (Figure 4.4P).
73
Aqp9 -/-WT
100mm
A
B
74
C
D
75
Figure 4.4. Skeletal staining of P21 WT and Aqp9 -/- mice
Images of WT and Aqp9 -/- whole body lengths (A), humerus bones (B), femur and tibia bones
(C), and condylo-basal lengths (D) stained with Alcian blue and Alizarin red for cartilage and
bone identification. Green lines indicate the observable ossified regions. Excised spleens are
shown as well (E).
E
76
Right Humerus Left Humerus
Right Femur Left Femur
Right Tibia Left Tibia
0
2
4
6
8
10
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
2
4
6
8
10
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
2
4
6
8
10
12
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
2
4
6
8
10
12
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
*
0
2
4
6
8
10
12
14
16
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
0
2
4
6
8
10
12
14
16
WT Aqp9 -/-
Avera
ge length
(m
m)
Mouse genotype
N.S
F G
H I
J K
77
Humerus Femur
Tibia
Figure 4.4. cont.
Measurements of WT and Aqp9 -/- right humerus bones (F), left humerus bones (G), right femur
bones (H), left femur bones (I), right tibia bones (J), and left tibia bones (K). Average percent
differences of Aqp9 -/- humerus bones (L), femur bones (M), and tibia bones (N) compared to
their WT counterparts. n=6. Student’s two-tailed t test was performed for statistical analysis
comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05.
75%
80%
85%
90%
95%
100%
105%
110%
115%
120%
125%
WT Aqp9 -/-
Avera
ge p
erc
ent
diffe
rence
Mouse genotype
N.S
75%
80%
85%
90%
95%
100%
105%
110%
115%
120%
125%
WT Aqp9 -/-
Avera
ge p
erc
ent
diffe
rence
Mouse genotype
*
75%
80%
85%
90%
95%
100%
105%
110%
115%
120%
125%
WT Aqp9 -/-
Avera
ge p
erc
ent
diffe
rence
Mouse genotype
N.S
L M
N
78
Figure 4.4. cont.
Measurements of WT and Aqp9 -/- excised spleen weights (O). n=2. Measurements of WT and
Aqp9 -/- condylo-basal lengths (P). n=6. Student’s two-tailed t test was performed for statistical
analysis comparing WT to Aqp9 -/- measurements, with the level of significance set at *P < 0.05.
10
11
12
13
14
15
16
17
18
19
20
WT Aqp9 -/-
Avera
ge s
ple
en w
eig
ht
(mg)
Mouse genotype
N.S
0
5
10
15
20
25
WT Aqp9 -/-
Avera
ge c
ondyl
o-b
asal
length
(m
m)
Mouse genotype
N.S
O
P
79
4.5 Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates
P5 WT and Aqp9 -/- littermate distal femur epiphyseal plates were sectioned and stained with
H&E to visualize their chondrocyte distributions. This would help support if dysregulation in the
epiphyseal plate influenced the P5 bone phenotype. The femur was selected rather than the tibia
due to the availability of sections. Furthermore, all mouse models previously described with long
bone phenotypes—including those with phenotypes in specific bones only—have spatial
irregularities in those epiphyseal plate zones. Investigating the tibia epiphyseal plate was
determined to be repetitious. As the long bone phenotypes observed at both P5 and P21 were
isolated to the hindlimb only, the hindlimb remained of interest. Hence, the adjoining epiphyseal
plate at the distal femur was selected for histological analysis.
An overview of the epiphyseal plates show subtle spatial differences near the top of the
proliferating zone in Aqp9 -/- mice in comparison to WT mice (Figure 4.5A, 4.5B). Image
enlargement shows the chondrocyte distribution ranging from the resting zone to the
hypertrophic zone (Figure 4.5C, 4.5D). In the WT epiphyseal plate, the proliferating zone is
separated from the resting zone by a clear boundary where chondrocytes are different in shape
and distribution. The proliferating chondrocytes are flat and columnar, while resting
chondrocytes are rounder and randomly dispersed with less organization. In the Aqp9 -/-
epiphyseal plate, chondrocytes at the top of the proliferating zone become gradually rounder but
retain columnar organization transitioning into the resting zone.
The Aqp9 -/- proliferating zone appears expanded in comparison to the WT proliferating zone.
The pre-hypertrophic zones of WT and Aqp9 -/- epiphyseal plates are demarcated by where
chondrocytes at the bottom of the proliferating zones begin to lose their columnar organization
and have enlarged cytoplasmic regions. No differences in pre-hypertrophic zone height are
apparent. The hypertrophic zones are demarcated by where the chondrocytes are engulfed in
round and pale cytoplasm and lose their H&E staining, up until the heavily stained bone matrix
at the bottom of the epiphyseal plate.
80
Proliferating and hypertrophic zone heights were measured by drawing five vertical lines across
each zone per biological replicate and averaging their heights. Statistical analysis of the
proliferating and hypertrophic zone heights show that proliferating zones in P5 Aqp9 -/-
epiphyseal plates are significantly taller than their WT counterparts (Figure 4.5E). No significant
differences in hypertrophic zone height were observed.
The average number of chondrocytes per column in proliferating and hypertrophic zones were
quantified by counting the number of chondrocytes that passed through each vertical line and
averaging them. Aqp9 -/- proliferating zones have significantly more chondrocytes per column in
comparison to their WT counterparts (Figure 4.5F). No significant differences were observed in
the hypertrophic zone.
The cellular density of proliferating and hypertrophic zones, in cells/um2, was also measured by
counting the individual chondrocytes in each zone and dividing the number by the total area of
the zone. Neither Aqp9 -/- proliferating or hypertrophic zones were significantly denser than their
WT counterparts (Figure 4.5G).
81
Figure 4.5. Histological analysis of P5 WT and Aqp9 -/- epiphyseal plates
H&E staining of P5 WT and Aqp9 -/- distal femurs (A, B) with their respective image
enlargements (C, D). Dotted black lines demarcate the observable zone boundaries. Yellow and
red lines indicate the observable proliferating and hypertrophic zones respectively (RZ = resting
zone, PZ = proliferating zone, PH = pre-hypertrophic zone, HZ = hypertrophic zone). n=3.
200um 200um
Aqp9 -/- WT
100um 100um
HZ HZ
PZ
PZ
Aqp9 -/- WT
A B
C D
RZ
RZ RZ
PH PH
82
Figure 4.5. cont.
Quantification of the proliferating and hypertrophic zone heights (E) and average cells/column
(F) of the WT and Aqp9 -/- distal femurs (PZ = proliferating zone, HZ = hypertrophic zone).
n=3. Student’s two-tailed t test was performed for statistical analysis, with the level of
significance set at *P < 0.05.
0
50
100
150
200
250
300
350
400
450
500
PZ HZ Total
Heig
ht
(um
)
Epiphyseal plate zone
WT
Aqp9 -/-
*
N.S
N.S
0
5
10
15
20
25
30
PZ HZ
Avera
ge c
ells
/colu
mn
Epiphyseal plate zone
WT
Aqp9 -/-
*
N.S
E
F
83
Figure 4.5. cont.
Quantification of the cellular density of the proliferating and hypertrophic zones (G) of the WT
and Aqp9 -/- distal femurs (PZ = proliferating zone, HZ = hypertrophic zone). n=3. Student’s
two-tailed t test was performed for statistical analysis, with the level of significance set at *P <
0.05.
0
1
2
3
4
PZ HZ
Cells
/um
2(1
0-3
)
Epiphyseal plate zone
WT
Aqp9 -/-
N.S
N.S
G
84
4.6 Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates
P21 WT and Aqp9 -/- littermate proximal tibia epiphyseal plates were sectioned and probed with
a primary antibody against Col10a1 to qualitatively visualize their zone arrangements and
chondrocyte distributions, with a focus on the hypertrophic zone. The tibia was selected rather
than the femur due to the availability of sections. As mentioned previously, all mouse models
described with long bone phenotypes—including those with phenotypes in specific bones only—
have spatial irregularities in those epiphyseal plate zones. Therefore, investigating the femur
epiphyseal plate was determined to be repetitious. As the long bone phenotypes observed at both
P5 and P21 were isolated to the hindlimb only, the hindlimb remained of interest. The adjoining
epiphyseal plate at the proximal tibia was selected for histological analysis.
An overview of the epiphyseal plates do not show any apparent differences in zonal height
between WT and Aqp9 -/- mice (Figure 4.6A, 4.6B). The image enlargements clearly indicate the
height of the entire epiphyseal plate, as well as their zonal compartments (Figure 4.6C, 4.6D). In
the upper portions, the resting-proliferating zones were measured due to difficulty in
distinguishing between the chondrocyte types. The resting zone regions, where chondrocytes are
more randomly dispersed, are narrow in both WT and Aqp9 -/- mice. The proliferating zone
regions clearly end where the dark staining for Col10a1 begins, indicating the upper portion of
the hypertrophic zone. The hypertrophic zone is dense with chondrocytes outlined in the dark
stain, where Col10a1 protein is robustly expressed.
Resting-proliferating and hypertrophic zone heights were measured by drawing five vertical lines
across each zone per biological replicate and averaging their heights. Hypertrophic zone heights
were divided by resting-proliferative zone heights to measure the hypertrophic zone – resting-
proliferative zone ratio. Statistical analysis of the ratios show no significant difference between
the P21 WT and Aqp9 -/- mice (Figure 4.6E). Total epiphyseal plate heights were also measured
and show no significant difference (Figure 4.6F).
85
Figure 4.6. Immunohistochemistry of P21 WT and Aqp9 -/- epiphyseal plates
Col10a1 staining of P21 WT and P21 proximal tibias (A and B) with their respective image
enlargements (C and D). Blue bar sections indicate the observable epiphyseal plate, green
sections indicate the observable resting and proliferating zones combined, and red sections
indicate the observable hypertrophic zones (C and D) (R-PZ = resting-proliferative zone, H =
hypertrophic zone).
400um 400um
400um 400um
R-PZ
HZ
R-PZ
HZ
86
Figure 4.6. cont.
Quantification of WT and Aqp9 -/- hypertrophic zones as a ratio to their respective resting-
proliferating zones (E). Measurement of the total epiphyseal plate heights of WT and Aqp9 -/-
mice (F). n=2. Student’s 2-tailed t test was performed for statistical analysis, with the level of
significance set at *P < 0.05.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
WT Aqp9 -/-
Pro
xim
al tib
ia H
Z /
R-P
Z r
atio
Mouse genotype
N.S
E
0
200
400
600
800
1000
WT Aqp9 -/-
Pro
xm
inal tib
ia e
pip
hyseal pla
te h
eig
ht (u
m)
Mouse genotype
N.S F
87
4.7 Analysis of old WT and Aqp9 -/- epiphyseal plates
18 month old male WT and Aqp9 -/- femoral heads were stained with Toluidine blue to highlight
the cartilaginous epiphyseal plates. This would help determine if Aqp9 affects epiphyseal plate
narrowing into an old age timepoint. An overview shows the epiphyseal plates stained a deep
blue as a thin line traversing across the diameter of the femoral head (Figure 4.7A, 4.7B). Image
enlargement shows that in the WT head, the epiphyseal plate reaches the right side toward the
greater trochanter and makes contact with the cortical bone (Figure 4.7C). However, in the Aqp9
-/- head, the epiphyseal plate does not reach the cortical bone and is instead ablated (Figure
4.7D). The observation was initially thought to be due to sectioning inconsistencies that caught
the Aqp9 -/- epiphyseal plates at a particular depth, but statistical analysis shows the same trend
across all Aqp9 -/- biological replicates and significantly reduced width compared to the WT
replicates (Figure 4.7E).
The femoral heads were also stained with H&E to highlight the bone marrow, presence of
hematopoietic cells, and the overall trabecular bone. The epiphyseal plate is visible as a line
running in between the bone marrow and trabecular bone. In the WT femoral head, the bone
marrow is white in colour (Figure 4.7F). In the Aqp9 -/- femoral head, the bone marrow is not
white but rather stained with the H&E stain (Figure 4.7G). Furthermore, the presence of
hematopoietic cells were more visible in the Aqp9 -/- bone marrow than in WT bone marrow.
The epiphyseal plate in the Aqp9 -/- femoral head does not appear to fully contact the cortical
bone. The region of trabecular bone in the Aqp9 -/- femoral head also appears smaller than the
one in the WT femoral head.
88
Figure 4.7. Analysis of adult WT and Aqp9 -/- epiphyseal plates
Toluidine blue staining of 18 month old WT and Aqp9 -/- femoral heads (A and B) with their
respective image enlargements (C and D). Red-dotted lines (C and D) indicate how far the
epiphyseal line traverses the entire femoral head diameter. Yellow-dotted line (D) indicates the
area where the epiphyseal plate ceases to exist. EP = epiphyseal plate, CB = cortical bone.
CB
CB
EP EP
500um 500um
250um 250um
89
Figure 4.7. cont.
Quantification of each epiphyseal plate length of the WT and Aqp9 -/- mice as a percentage of
the entire femoral head diameter (E). n=3. Student’s two-tailed t test was performed for statistical
analysis, with the level of significance set at *P < 0.05.
Figure 4.7. cont.
H&E staining of 18 month old WT and Aqp9 -/- mouse femoral heads (F & G). Black arrows
indicate the region of observable bone marrow and its respective colour. Yellow arrows indicate
locations where observable hematopoietic cells reside. CB = cortical bone, EP = epiphyseal
plate, BM = bone marrow, TB = trabecular bone.
Aqp9 -/- G WT F
0.00
20.00
40.00
60.00
80.00
100.00
WT Aqp9 -/-
Epip
hys
eal
line length
/ fem
ora
l head d
iam
ete
r (%
)
Mouse genotype
E *
500um 500um
90
4.8 Histological analysis of embryonic WT and Aqp9 -/- epiphyseal
plates
E16.5 WT and Aqp9 -/- littermate proximal tibia epiphyseal plates were sectioned and stained
with Safranin-O to visualize their zone arrangements and chondrocyte distributions. This would
help determine if Aqp9 affects endochondral ossification during development. An overview of
the epiphyseal plates show similar zonal heights and total height between WT and Aqp9 -/- mice
(Figure 4.8A, 4.8B). Image enlargement shows that the Aqp9 -/- resting zone appears to cave in
towards the top of the epiphyseal plate, whereas the WT resting zone forms a straight boundary
with the proliferating zone perpendicular to the long bone (Figure 4.8C, 4.8D). The Aqp9 -/-
proliferating zone chondrocytes appear to retain columnar structures further up the epiphyseal
plate than their WT counterparts.
Resting, proliferating, and hypertrophic zone heights were measured by drawing five vertical
lines across each zone per biological replicate and averaging their heights. The hypertrophic zone
included the pre-hypertrophic zone to aid measurement boundaries. The heights were summed
and averaged to determine the total epiphyseal plate height. Then, the ratio of each zone
compared to the total height was determined by dividing each zone height by the total height.
Statistical analysis of the ratios show that the E16.5 Aqp9 -/- resting zones are significantly
shorter than those of their WT counterparts (Figure 4.8E). No significant differences were
observed in the proliferating and hypertrophic zones (Figure 4.8F, 4.8G). The sum of the
proliferating and hypertrophic zone heights divided by the total plate height are also shown, to
demonstrate that the non-resting portions of Aqp9 -/- epiphyseal plates are taller than those of
WT mice (Figure 4.8H).
91
Figure 4.8. Histological analysis of embryonic WT and Aqp9 -/- epiphyseal plates
Safranin-O staining of E16.5 WT and Aqp9 -/- proximal tibias (A and B) with their respective
image enlargements (C and D). Dotted-line regions indicate the observable resting zone of the
epiphyseal plate (RZ = resting zone, PZ = proliferating zone, HZ = hypertrophic zone).
100um 100um WT Aqp9 -/-
RZ
RZ
RZ
PZ
RZ
PZ
HZ HZ
PZ
PZ
100um 100umWT Aqp9 -/-
A B
C D
92
Figure 4.8. cont.
Quantification of each epiphyseal plate zone height (E, F, G) of the WT and Aqp9 -/- embryos as
a ratio of the entire epiphyseal plate height (EP = epiphyseal plate). Ratio of the proliferating
zone and hypertrophic zone combined to the entire epiphyseal plate height (H). n=3 (2 WT
biological replicates and 1 technical replicate, 3 Aqp9 -/- biological replicates). Student’s two-
tailed t test was performed for statistical analysis, with the level of significance set at *P < 0.05.
0
0.1
0.2
0.3
0.4
0.5
WT Aqp9 -/-
RZ
he
igh
t (u
m)
/ E
P h
eig
ht (u
m)
Mouse genotype
*
0
0.1
0.2
0.3
0.4
0.5
WT Aqp9 -/-
PZ
he
igh
t (u
m)
/ E
P h
eig
ht (u
m)
Mouse genotype
N.S
0
0.1
0.2
0.3
0.4
0.5
WT Aqp9 -/-
HZ
he
igh
t (u
m)
/ E
P h
eig
ht (u
m)
Mouse genotype
N.S
0.5
0.6
0.7
0.8
WT Aqp9 -/-
PZ
+ H
Z h
eig
ht (u
m)
/ E
P h
eig
ht (u
m)
Mouse genotype
*
E F
G H
93
4.9 Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/-
epiphyseal plate chondrocytes
P5 WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate chondrocytes were subjected to a cell
proliferation experiment to determine if deletion of Aqp9 resulted in any proliferation defect.
Any proliferation difference observed would help support the epiphyseal plate zone
dysregulation observed. Epiphyseal plate chondrocytes from P5 WT, Aqp9 +/-, and Aqp9 -/-
mice were excised and digested in a pronase and collagenase protocol, then seeded at 1 x 105
primary chondrocytes per well in a 6-well plate. Chondrocyte numbers per well were counted
every 24 hours over a 96 hour period (Figure 4.9).
WT, Aqp9 +/-, and Aqp9 -/- chondrocytes reach confluency by the 96 hour timepoint. A lag in
growth is observed in Aqp9 +/- and Aqp9 -/- chondrocytes at the 48 hour timepoint, followed by
a jump in the cell number at the 72 hour timepoint. Overall, Aqp9 +/- and Aqp9 -/- chondrocytes
appear to recover from an abnormally low cell number to match the WT cell number at the 96
hour timepoint.
94
Figure 4.9. Cell proliferation analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate
chondrocytes
Cell count of WT (black), Aqp9 +/- (orange), and Aqp9 -/- (red) epiphyseal plate chondrocytes
seeded at 100,000 cells from 0-96 hours. n=3. Student’s 2-tailed t test was performed for
statistical analysis comparing Aqp9 +/- and Aqp9 -/- to WT at each timepoint, with the level of
significance set at *P < 0.05, **P < 0.01, ***P < 0.001.
0
2
4
6
8
10
12
0 24 48 72 96
Cell
num
ber
(1e
5)
Time (hours)
WT Aqp9 +/- Aqp9 -/-
N.S
N.S
***
***
N.S
N.S
N.S
N.S
95
4.10 Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/-
epiphyseal plate chondrocytes
P5 WT, Aqp9 +/-, and Aqp9 -/- primary epiphyseal plate chondrocytes were subjected to gene
expression analysis to observe if their individual gene profiles would reflect the P5 tibia bone
phenotype and femur epiphyseal plates. The experiment would also help explain the irregular
proliferation pattern observed with the cell proliferation experiment. Epiphyseal plate
chondrocytes from P5 WT, Aqp9 +/-, and Aqp9 -/- mice were excised and digested. RNA was
extracted and then reverse transcribed to cDNA for qPCR gene expression analysis. Five gene
probes were used: Col10a1, Mmp13, Runx2, Sox9, and Aqp9. Col10a1, Mmp13, and Runx2 are
known aforementioned markers of chondrocyte hypertrophy. Sox9 is a known aforementioned
marker of early chondrogenesis, proliferation, and hypertrophy delay. Aqp9 was used to observe
if its expression levels would parallel the expression of the other probes in a remarkable manner
or trend.
Charting of the gene probe fold changes presented with significant differences in Mmp13, Sox9,
and Aqp9 (Figure 4.10). For the Mmp13 probe, Aqp9 +/- chondrocytes presented with a
significant fold change less than 1. Here, Aqp9 -/- chondrocytes did not present with a significant
change. For the Sox9 probe, Aqp9 -/- chondrocytes presented with a significant fold change
greater than 1. Here, Aqp9 +/- chondrocytes did not present with a significant change. For the
Aqp9 probe, Aqp9 -/- chondrocytes presented with a significant decrease while Aqp9 +/-
chondrocytes did not. Significant fold changes were not observed in either Aqp9 +/- or Aqp9 -/-
chondrocytes for the Col10a1 and Runx2 probes.
Overall, the fold change levels of hypertrophic markers in the Aqp9 -/- chondrocytes were not
significantly different relative to the WT control. The fold change of the proliferation marker
Sox9 was increased. Aqp9 expression was significantly decreased. In Aqp9 +/- chondrocytes, the
fold change level of the hypertrophic marker Mmp13 was significantly decreased relative to the
WT control.
96
Figure 4.10. Gene expression analysis of P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate
chondrocytes
qPCR of WT (white), Aqp9 +/- (blue) and Aqp9 -/- (orange) epiphyseal plate chondrocytes with
gene probes for markers of proliferation and hypertrophy in chondrocyte differentiation. n=3.
Student’s 2-tailed t test was performed for statistical analysis comparing Aqp9 +/- and Aqp9 -/- to
WT for each probe, with the level of significance set at *P < 0.05, **P < 0.01, ***P < 0.001.
0
1
2
3
4
5
Col10a1 Mmp13 Runx2 Sox9 Aqp9
Fo
ld c
ha
ng
e
qPCR gene
WT
Aqp9 +/-
Aqp9 -/-
N.S
N.S N.S
**
N.S
N.S
**
N.S
***
N.S
97
4.11 Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes
P5 WT primary epiphyseal plate chondrocytes were subjected to an Aqp9-siRNA silencing
protocol to observe if the Aqp9 +/- or Aqp9 -/- gene profile could be simulated. The experiment
would also help determine if the Aqp9 +/- or Aqp9 -/- gene profile was cell autonomous to the
chondrocytes. Epiphyseal plate chondrocytes were excised from P5 WT mice, digested, and
plated for transfection with the Aqp9-siRNA. qPCR using the five aforementioned probes
Col10a1, Mmp13, Runx2, Sox9, and Aqp9 was performed after 24 hours of incubation time.
Charting of the gene probe fold changes did not present with any significant differences (Figure
4.11).
98
Figure 4.11. Silencing of Aqp9 in P5 WT epiphyseal plate chondrocytes
qPCR of WT epiphyseal plate chondrocytes with an Aqp9-siRNA (yellow) and with a control
siRNA (white), with gene probes for markers of proliferation and hypertrophy in chondrocyte
differentiation. n=3. Student’s 2-tailed t test was performed for statistical analysis comparing
WT-siRNA to WT-control, with the level of significance set at *P < 0.05.
0
0.5
1
1.5
2
2.5
3
Col10a1 Mmp13 Runx2 Sox9 Aqp9
Fo
ld c
hange
qPCR gene
WT-control
WT-siRNA
N.S N.S
N.S
N.S
N.S
99
5 Discussion
The process of long bone growth is dependent on endochondral ossification, where chondrocytes
in the epiphyseal plates proliferate, undergo hypertrophy, and eventually ossify (Mackie,
Tatarczuch, & Mirams, 2011). The biology underscoring this process is complex and the clinical
management of LLDs relies on continuous endeavours in deciphering its unknown factors
(Killion, Mitchell, Duke, & Serra, 2017). Hence, identifying any novel regulators of
endochondral ossification is valuable for non-invasive therapies that can influence long bone
growth. The chondrocyte channelome is a popular area of focus that investigates the ion channels
and porins on the chondrocyte membrane that affect cartilage activity and pathogenesis
(Mobasheri et al., 2018). In particular, Aqp9 is an aquaporin isoform that indiscriminately
conducts solutes through a larger-than-usual pore (Viadiu et al., 2007). Interestingly, Aqp9 is one
of the few aquaporin species that is capable of transporting H2O2, a known inducer of
chondrocyte hypertrophy (Watanabe et al., 2016; Morita et al., 2007). Kannu et al. (2015)
initially identified that in human articular cartilage samples, those cultured with the WNT
pathway antagonist DKK1 had a greater than 2-fold change in expression of AQP9. As Dkk1
transcripts regulate embryogenesis and are restricted to the murine forelimb and hindlimb buds at
E10.5, Aqp9 may also play a role in guiding limb development (Grotewold, Theil, & Rüther,
1999; Lieven, Knobloch, & Rüther, 2010). However, the study of Aqp9 in chondrocytes and
bone has been limited to articular cartilage, osteoclasts, and bone density (Nagahara et al., 2010;
Liu et al., 2009; Bu et al., 2012). To study Aqp9 in endochondral ossification, the use of the
Aqp9 -/- mutant model developed by Rojek et al. (2007) allowed for a specific focus on its
function in the epiphyseal plate.
In this study, the Aqp9 -/- mutation was shown to be involved in differential long bone length,
epiphyseal plate zone dysregulation, and chondrocyte gene expression. Aqp9 is expressed in the
murine epiphyseal plate at the critical proliferating and pre-hypertrophic zones. The Aqp9 -/-
mice have longer tibia bones at the P5 age and shorter femur bones at the P21 age, suggesting
that Aqp9 may be important in hindlimb development. Analysis of the P5 Aqp9 -/- femur
epiphyseal plates show irregular expansion of the proliferating zone, suggesting that even long
bones not significantly different may have dysregulated endochondral ossification. Analysis of
the P21 Aqp9 -/- tibia epiphyseal plates do not show any significant differences in epiphyseal
100
zone height or hypertrophy marker staining. At the old age stage, Aqp9 -/- femoral heads show
unnatural remission and narrowing of the epiphyseal plate, suggesting that the mutants may
experience faster partial epiphyseal closure. In development, E16.5 Aqp9 -/- tibial heads show
diminished epiphyseal plate resting zone lengths, suggesting that their chondrocytes may attain a
proliferative and differentiated state faster than WT counterparts. Both Aqp9 +/- and Aqp9 -/-
chondrocytes have different proliferation rates than WT chondrocytes. Aqp9 -/- chondrocytes
also presented significantly increased expression of the proliferation marker Sox9. RNA
silencing of Aqp9 in WT chondrocytes did not present with any significant differences in
expression of the gene probes used. In this study, I hypothesized that Aqp9 is a regulator of
epiphyseal plate chondrocyte proliferation, hypertrophy, and long bone growth. The data provide
characterization of the Aqp9 -/- mutation at the anatomical, histological, cellular, and gene
expression levels. In future studies, analysis of Aqp9 -/- chondrocytes through transcriptome
sequencing may provide a richer understanding of aquaporins in endochondral ossification.
5.1 Aqp9 temporally influences hindlimb length
5.1.1 Aqp9 -/- mutants do not differ in appearance and weight
To determine if WT and Aqp9 -/- mice were comparable in terms of physical appearance and
body weight, mice at P5, P21, and 18 months of age were culled for imaging and body weight
measurements. These timepoints served to examine gross phenotypes before pubertal growth,
during pubertal growth, and during old age respectively (Mitchell et al., 2013; Jackson et al.,
2017). WT and Aqp9 -/- mice at each timepoint were comparable in terms of overall appearance
and weight, as determined by photography and weighing with a precision scale. As mentioned
previously, male mice were used at P21 and 18 months to prevent any sex-specific body weight
differences. P5 mice were not sexed due to difficulty in determining anogenital differences.
Rojek et al. (2007) reported that no detectable differences in physical appearance or body weight
were observed between age-matched WT and Aqp9 -/- littermate mice. This was confirmed
across the murine lifespan at the pup, juvenile, and old age stages. The average body weights
charted over time appear to match lifelong body weight measurements of WT male mice (List,
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Berryman, Wright-Piekarski, Jara, & Kopchick, 2013). Rojek et al. (2007) also reported
increased plasma glycerol in Aqp9 -/- mice after starvation, suggesting that Aqp9 may play a role
in repressing gluconeogenesis without significantly affecting weight. It is also possible that
deletion of a functional aquaporin isoform is compensated by the activity of other isoforms, as
shown in human pancreatic duct cell function and even plant water regulation (Burghardt et al.,
2003; Cohen et al., 2013). Hence, aquaporin redundancy may rescue any superficially observable
defect that Aqp9 deletion would have caused. Specifically, Aqp9 -/- mice may be expected to
have defected H2O2 transport, but AQP3 and AQP8 are also H2O2 transporters (Watanabe et al.,
2016). The role of these aquaporins have not been explored in epiphyseal plate chondrocytes or
bone and their contributions are unknown. Nevertheless, deletion of a channel capable of
transporting a known hypertrophy inducer may relay an effect on endochondral ossification.
In light of the literature and previous data from the Kannu lab, Aqp9 may affect long bone
growth. These differences may not be observable without skeletal dissection and analysis of the
long bones and their histology.
5.1.2 Aqp9 is expressed in the P14 mouse epiphyseal plate
Prior to long bone analysis, Aqp9 expression in the murine epiphyseal plate was checked to
confirm a possible spatiotemporal role in endochondral ossification. P14 WT mice were selected
as endochondral ossification is prominent at this timepoint (Williams, 2014). Furthermore, the
discrete zones of the murine epiphyseal plate are best visualized by in situ hybridization at P14
due to their visibility at this timepoint (Belluoccio, Bernardo, Rowley, & Bateman, 2008). In situ
hybridization of the proximal tibia epiphyseal plate with an Aqp9 riboprobe showed robust
expression of the Aqp9 transcript in the proliferating and pre-hypertrophic zone chondrocytes.
The presence of Aqp9 in the maturing epiphyseal plate suggests that it may be involved in
endochondral ossification. The use of in situ hybridization to confirm the expression of Col2a1,
Sox9, Mmp13, Col10a1, and other currently well-known chondrocytic markers is well reported
(Hall et al., 2013; Hattori et al., 2010). However, the demonstration of aquaporin expression in
the epiphyseal plate is limited with the exception of Aqp1 in the rat hypertrophic zone
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(Claramunt et al., 2017). Here, staining of Aqp9 to the proliferating and pre-hypertrophic zones
suggests that Aqp9 regulates chondrocyte activity prior to hypertrophy. The absence of Aqp9
expression in the hypertrophic zone supports that Aqp9 may function to maintain the
proliferative state and prepare chondrocytes for hypertrophy. Then, chondrocytes that attain
hypertrophy cease expression of Aqp9 and may rely on other markers to maintain hypertrophy
and prepare for ossification.
In the epiphyseal plate, Aqp9 appears to mimic the expression gradient of Col2a1 and Sox9, both
of which are known markers of the proliferating and pre-hypertrophic zones (Gómez-Picos &
Eames, 2015). However, specific Aqp9 expression in these zones does not necessarily mean
Aqp9 functions to maintain those states. The murine epiphyseal plate is a host to extremely
diverse factors that can promote certain chondrocytic phenotypes without being expressed where
those chondrocytes would be. Ihh is expressed at the pre-hypertrophic junction but serves to
maintain the proliferative state through PTHrP (Kronenberg, 2003). Wnt4, a Wnt family member
that signals through β-catenin, is also expressed in the pre-hypertrophic zone but instead
accelerates hypertrophy (Später et al., 2006; Hartmann & Tabin, 2000; Lee & Behringer, 2007).
Rspo2, an activator of Wnt/ β-catenin signalling, is expressed in the cartilage primordium and
embryonic long bone at the proliferating and pre-hypertrophic zones; yet it suppresses Col2a1
and Sox9 to promote hypertrophy (Nam, Turcotte, & Yoon, 2007; Takegami et al., 2016). It is
possible that Aqp9 imports H2O2 in proliferating chondrocytes to induce hypertrophy and then
ceases in expression, fulfilling its moonlighting role as a channelome member. However, Aqp9 is
a bidirectional transporter and may instead export H2O2 to delay hypertrophic differentiation.
Regardless, Aqp9 may function under the spatiotemporal guidance of unexplored transcription
factors within the epiphyseal plate.
Overall, Aqp9 expression in the epiphyseal plate suggests that Aqp9 may be important in
endochondral ossification and long bone growth. In situ hybridization at the proximal tibia
suggests that Aqp9 regulates hindlimb growth, although it is likely that Aqp9 is expressed in
other appendicular epiphyseal plates as well.
103
5.1.3 P5 Aqp9 -/- mice have longer tibia bones
To determine if Aqp9 -/- mice have differential long bones at the pup stage, P5 WT and Aqp9 -/-
littermates were dissected for whole-mount skeletal staining. There were no apparent gross body
differences between the two genotypes. Long bone measurements revealed that Aqp9 -/- mice
had significantly longer tibia bones than their WT counterparts. All humerus and femur bone
measurements did not present with significant differences. Excised spleens did not appear to
differ in weight, nor did the condylo-basal lengths of their skulls.
A long bone phenotype is observed in Fgfr3c -/- mice, where deletion of the Ffgf3 isoform
results in dramatic skeletal overgrowth of all long bones (Eswarakumar et al., 2007). Fgfr3 is a
known negative regulator of bone growth, as Aqp9 may also be as both their knockouts result in
bone overgrowth (Deng, Wynshaw-Boris, Zhou, Kuo, & Leder, 1996). Other knockout
mutations are reported to result in skeletal overgrowth. Deletion of a natriuretic peptide gene
through Npr3 -/- mice result in skeletal overgrowth in 2 month old mutants (Matsuwaka et al.,
1999). Conditional deletion of the cell proliferation activator Yap1 through Yap1c/c; Col2a1-Cre
mutants result in skeletal overgrowth at E16.5 and E18.5 (Deng et al., 2016). However, the
observations suggest that Aqp9 deletion is tibia-specific at P5 in bone length modulation. There
are also certain knockout mutations that are bone-specific in their phenotype. In skeletal
development, the highly regulated ECM contains proteoglycans of which the chondroitin sulfate
proteoglycans are a large proportion (Wilson et al., 2012). In mice lacking chondroitin sulfate
synthase-2, femur and tibia bone lengths are significantly reduced whereas humerus and ulnar
lengths are unchanged (Ogawa et al., 2012). Nevertheless, tibial-specific overgrowth defects are
mostly reported in physiological compensatory responses instead of mutations, such as in human
fractures and canine femoral shortening (Taylor, 1963; Schaefer, Johnson, & O’Brien, 1995). At
development, Aqp9 may normally signal with hindlimb-specific genes such as Tbx4 albeit in a
mesomelic fashion at the tibia (Rodriguez-Esteban et al., 1999; Isidor et al., 2010). Interestingly,
gene expression profiling of muscle invasive bladder cancer samples identified AQP9 and TBX4
as upregulated genes together in connective tissue disorders (Hussain et al., 2017). Hence, Aqp9
and Tbx4 may interact in the developing hindlimb. In mesomelic syndromes such as Langer
mesomelic dysplasia, shortening of the distal limbs involves inactivation of the short-stature
104
gene Shox2, Runx2, and Ihh (Cobb, Dierich, Huss-Garcia, & Duboule, 2006). Interestingly, use
of the ISMARA (Integrated System for Motif Activity Response Analysis) public dataset
‘Illumina Body Map 2’ shows that the AQP9 promoter is a top ten target for SHOX (Balwierz et
al., 2014). It is possible that the Shox transcription factor regulates endochondral ossification
partially through Aqp9. Deletion of Aqp9 may therefore repress a natural repressor of tibial
growth and resolve in abnormally lengthened tibia bones at P5. It is also possible that Aqp9 is
expressed differently throughout all long bone epiphyses and is under unexplored transcriptional
control. Conversely, the use of more biological replicates may reveal that the tibial overgrowth is
actually total long bone overgrowth. Overall, Aqp9 deletion does not severely affect skeletal
stature at the pup stage and may be dispensable or redundant in effect. However, the lack of a
difference in spleen and skull measurements between WT and Aqp9 -/- pups suggest that the
mutation targets endochondral ossification—and does not affect intramembranous ossification or
general organ development.
The early tibial overgrowth in P5 Aqp9 -/- mice appears reminiscent of precocious puberty.
Human precocious puberty is characterized by the early development of sexual characteristics
and is often idiopathic (Bourayou, Giabicani, Pouillot, Brailly-Tabard, & Brauner, 2015).
However, the cause of precocious puberty has been linked to deficiencies in MKRN3, a zinc
finger motif gene that normally helps inhibit activation of the hypothalamic-pituitary-gonadal
axis (Shin, 2016). Defects in MKRN3 lead to accelerated growth, early bone maturation, early
height gain, but ultimately reduced stature (Carel, Lahlou, Roger, & Chaussain, 2004). At P5,
Aqp9 deletion appears to mimic the early growth though tibial elongation. Interestingly, absence
of MKRN3 in Prader-Willi syndrome—a genetic disorder that includes short stature—is
accompanied by a 1.52 fold change decrease of AQP1 expression and 2100.84 fold change
decrease in AQP3 expression in tissue mitochondria (Yazdi et al., 2013). Since AQP1 and AQP3
are also aquaglyceroporins and can transport H2O2 like AQP9, it is possible that AQP9 plays a
role here to influence skeletal stature but was not detected (Plourde et al., 2015; Laforenza,
Bottino, & Gastaldi, 2016; Almasalmeh, Krenc, Wu, & Beitz, 2014). If Aqp9 deletion is involved
in precocious puberty, a shorter skeletal stature relative to WT littermates closer to adulthood
would support its role.
105
The role of estrogen in Aqp9 regulation should also be considered. Estrogen receptors are
expressed in newborn mice, where basal levels of estrogen bind and gradually stimulate long
bone growth through growth hormone activation (Zuloaga, Zuloaga, Hinds, Carbone, & Handa,
2014; Cutler, 1997; Avtanski et al., 2014). During postnatal development, estrogen promotes
chondrocyte proliferation and type II collagen expression in the epiphyseal plate (Shi, Zheng, Li,
& Liu, 2017). Aqp9 is upregulated by estrogen in rat epididymal ductules but downregulated in
rat hepatocytes (Oliveira, Carnes, França, Hermo, & Hess, 2005; Lebeck et al., 2012). The role
of estrogen in chondrocyte Aqp9 expression has not been explored. Normally, estrogen binds
alpha and beta estrogen receptors expressed in epiphyseal plate chondrocytes to promote
proliferation (Li, Wang, Jiang, & Dai, 2012). Epiphyseal plate chondrocytes can endogenously
produce 17β-estradiol to stimulate proliferation, protect against apoptosis, and promote
longitudinal growth (Chagin, Chrysis, Takigawa, Ritzen, & Sävendahl, 2006). In Aqp9 -/- mice,
Aqp9 is not available as a target and may free available estrogen to bind growth-promoting
receptors, such as growth hormone receptors (Slootweg, Swolin, Netelenbos, Isaksson, &
Ohlsson, 1997). It is also possible that if Aqp9 is a negative regulator of bone growth, estrogen
regularly binds to its enhancer to prevent rampant bone growth. Without Aqp9 as a limiter, the
P5 long bone phenotype may occur. This is similar to what is observed with insulin, which
promotes chondrocyte proliferation and long bone growth (Zhang et al., 2014). Kuryiama et al.
(2002) have shown that Aqp9 is downregulated by insulin addition via the negative insulin
response element. If Aqp9 limits bone growth, insulin may bind to the element and instead
promote bone growth. Estrogen and insulin may work together with Aqp9 as a downstream
target, as combined estradiol and growth hormone treatment has been shown to elevate IGF
levels in primates (Wilson, 1998).
Overall, Aqp9 may be a negative regulator of bone growth in early murine endochondral
ossification. It is a possible intermediate in hormonal signalling that subtly impacts tibial growth,
and its deletion in the Aqp9 -/- mutation may involve an estrogenic spurt that induces a
precocious overgrowth phenotype. This hypothesis would be supported by hastened epiphyseal
closure and shortened long bone lengths closer to the adult stage (Nilsson et al., 2014).
106
5.1.4 P21 Aqp9 -/- mice have shorter femur bones
To determine if long bone abnormalities persisted between WT and Aqp9 -/- littermates at the
juvenile stage, P21 male mice were dissected for whole-mount skeletal staining. There were no
observable gross body differences between the two genotypes. Measurement of the long bones
revealed that Aqp9 -/- mice had significantly shorter femur bones than their WT counterparts. All
humerus and tibia bone measurements did not present with significant differences. Similarly to
the P5 mice, excised spleens did not appear to differ in weight and condylo-basal skull lengths
did not differ either. This reiterates that Aqp9 deletion targets endochondral ossification and may
be independent of other growth processes.
A short bone phenotype observed in knockout mouse models is common. Sox9 +/- mice die
perinatally with premature mineralization and shorter, distorted bones (Bi et al., 2001). Runx2 -/-
mice also die shortly after birth without proper mineralization and subsequently shorter, nearly
nonexistent limbs (Otto et al., 1997). Ihh -/- mice that survive till birth display severe dwarfism
of the limbs (St-Jacques et al., 1999). Although these genes play different proliferative-
hypertrophic roles in endochondral ossification, it is evident that they are all indispensable to
long bone growth: their deletion, regardless of which chondrocytic phenotype they promote,
severely diminishes the final bone length. However, certain gene mutations can induce shortened
limbs without lethality. In Hp1bp3 -/- mice that lack the binding protein and IGF-1 modulator
Hp1bp3, mutants display reduced femoral length and overall dwarfism (Garfinkel et al., 2015).
Cobb et al. (2006) previously explored Shox2C/- mice with conditional Shox2 deletion via the
limb-specific Prx1-Cre transgene. The mutants present with virtually nonexistent humerus
bones, severely shortened femurs, and shorter tibias with bowing. At P21, Aqp9 deletion also
appears to shorten long bones without inducing lethality. However, the observations suggest that
the mutation is femur-specific at P21. There are few reported mouse mutants to model this
phenotype. In Mmp13 -/- mutants, where Mmp13 is a critical enzyme for chondrocyte
hypertrophy, a significant ~8% reduction in long bone length occurs only at the juvenile
timepoint and only in femur bones (Inada et al., 2004). Murine deletion of Spred2, an
FGFR3/MAPK intermediate, also results in bone-specific shortening in the hindlimb but at the
tibia instead (Bundschu et al., 2005). Femoral length defects are mostly reported in human
107
conditions such as Down’s syndrome and skeletal dysplasia (Morales-Roselló & Llorens, 2012).
In early puberty, Aqp9 may continue to normally signal with Tbx4 to affect hindlimb growth.
Interestingly, the short bone phenotype here appears to be rhizomelic (Panda et al., 2014). In
rhizomelic disorders such as rhizomelic chondrodysplasia punctata, shortening of the proximal
limbs involves mutation of FGFR1 (White et al., 2005). Interestingly, Col2a1-Cre-driven
deletion of Shox2 also results in rhizomelia due to precocious chondrocyte maturation and
hypertrophy (Bobick & Cobb, 2012). As mentioned previously, AQP9 is a target of SHOX
regulation and the two may function in concert at P21 to drive normal bone growth. Here,
deletion of Aqp9 appears to reverse the long bone phenotype seen at the P5 timepoint. Aqp9
expression may naturally vary throughout the hindlimb epiphyseal plates temporally. Repeating
the skeletal staining with more biological replicates may reveal that the femoral shortening
actually affects the entire hindlimb significantly, or possibly all limbs. Overall, the Aqp9 -/-
mutation does not drastically affect skeletal stature at the juvenile stage.
In comparison to the P5 bone phenotype, a striking observation is the reduced femur bone length
at P21. In skeletal overgrowth models such as Fgfr3 -/-, the long bone defect persists into early
adulthood without any apparent change in growth rate (Xie et al., 2017). Here, an early, specific
long bone phenotype has transformed into a juvenile, specific short bone phenotype within the
hindlimb. This observation is similar to precocious puberty, where mice can exhibit its
characteristics as early as 7 days of age (McGee & Narayan, 2013). As mentioned previously,
Aqp9 is also a target of estrogen and its deletion in the Aqp9 -/- mutation at P5 may promote
estrogenic binding to growth receptors. By P21, excess estrogen may lead to earlier narrowing of
the epiphyseal plate, slower growth, and shorter femurs (Weise et al., 2001). Deletion of Aqp9
appears to encourage growth through accelerated endochondral ossification but later discourages
growth through a mechanism involving dysregulated hormonal and sexual development. Aqp9
may function alongside transcription factors that affect secondary sexual characteristics at P21,
where a significant portion of sexual differentiation has occurred (Schlomer et al., 2013). In
comparison to male mice, female mice have higher plasma estrogen levels at the juvenile stage
(Saito et al., 2009). If female mice were used, the femoral shortening may be more pronounced.
108
The effects of chondrocyte senescence in the epiphyseal plate should also be considered. Lui,
Nilsson, & Baron (2011) describe that as the number of cell divisions accumulate, resting
chondrocytes may gradually lose their replicative capacity. This would translate into
proliferating chondrocytes with diminished proliferation and then growth deceleration. It is
possible that every proliferating chondrocyte is affected by an intracellular or extracellular ‘cell
cycle counter’ factor that progressively changes with each cell division. Interestingly, estrogen
has been suggested to increase the loss of proliferative capacity per cell cycle (Schrier et al.,
2006). P5 Aqp9 -/- mice likely exhibit accelerated proliferation that underscores their elongated
tibias, whereas P21 mutants likely exhibit subsequent decelerated proliferation that captures their
shortened femurs. Both observations would be supported by epiphyseal plate examination and
chondrocyte gene profiling.
Overall, Aqp9 -/- mice appear to experience subtle hindlimb defects at P5 and P21. Aqp9 may
have differential distribution on the chondrocyte membrane depending on the specific bone and
stage of endochondral ossification. Aqp9 may also be under the control of factors such as
estrogen, and its deletion may elicit the early bone elongation and later bone shortening through
accelerated but ultimately impeded growth. The observations suggest that Aqp9 may be a highly
plastic channelome gene that plays inconsistent roles in regulating long bone growth when
deleted in a mouse model.
5.2 Epiphyseal plate irregularities underscore Aqp9-mediated bone
length
5.2.1 P5 Aqp9 -/- mice have expanded distal femur proliferating zones
To determine the cause of the P5 bone phenotype, WT and Aqp9 -/- littermate distal femur
epiphyseal plates were examined with H&E. Although the overgrowth phenotype occurred
significantly in the tibias only, femurs were selected to investigate if any epiphyseal plate
dysregulation could be observed in the adjoining hindlimb long bone. In mouse models with long
bone phenotypes, spatial irregularities in those epiphyseal plate zones are predominantly
guaranteed. Hence, investigating the tibia histologically was determined to be repetitious as
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epiphyseal plate growth is predominantly coupled with bone formation (Jones et al., 2010).
Additionally, the P5 knee sections used in this study provided the best visual clarity at the distal
femur. The femur was therefore investigated; any defect observed in the Aqp9 -/- femur
epiphyseal plate would support not only how the tibial overgrowth occurs, but also the
possibility of the mutation affecting the whole hindlimb.
In the P5 Aqp9 -/- distal femur epiphyseal plate, the average height of the proliferating zone is
significantly expanded compared to the WT epiphyseal plate. No significant differences are
observed in the hypertrophic zone. The proliferating zone also features a significantly higher
number of chondrocytes per column. The cellular density in Aqp9 -/- proliferating and
hypertrophic zones did not significantly differ from that of WT littermates.
Based on the observed long bone phenotype and literature supporting accelerated proliferation,
the proliferating and hypertrophic zones were focused upon to determine if their morphology
would reflect the overgrowth. This was also supported by the fact that Aqp9 is unavailable to
likely transport H2O2 into the chondrocyte and induce hypertrophy, which may allow
chondrocytes to retain a proliferative phenotype for longer than usual in the epiphyseal plate.
The resting zones were more dispersed and difficult to border, and were not measured in this
figure. The Aqp9 -/- proliferating zone appears to have more acellular matrix surrounding the
chondrocytes, possibly due to more synthesis of type II collagen and aggrecan than in the WT
epiphyseal plate. However, a greater area of matrix and chondrocytes may have leveled off the
cellular density in comparison to the WT proliferating zone. The hypertrophic zones in WT and
Aqp9 -/- mice feature chondrocytes of varying shape and size that may influence the number of
cells permitted in the zone. Increasing the sample size of epiphyseal plates may provide a more
accurate representation. Importantly, the higher number of chondrocytes per column in the Aqp9
-/- epiphyseal plate is a hallmark of accelerated proliferation (Lui et al., 2011).
The expansion of the proliferating zone in P5 Aqp9 -/- mice supports the longer bone phenotype
observed. Many knockout mouse models that feature long bone phenotypes, such as
Fgfr3floxneo/floxneo mice, have increased expansion of proliferating and hypertrophic zones (Su et
al., 2010). However, knockout mouse models such as Stat1 -/- present with early longer bones
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and a proliferating zone-specific expansion, as observed with the P5 Aqp9 -/- mice (Sahni, Raz,
Coffin, Levy, & Basilico, 2001). Interestingly, STAT1 is a negative regulator of chondrocyte
proliferation and promoter of chondrocyte apoptosis through upstream FGFR3 signalling (Sahni
et al., 1999). This coincides with the observation that chondrocyte deletion of Aqp9 results in
improved cell viability after H2O2 treatment (Shao et al., 2016). As STAT1 is also activated
through H2O2 induction, the two genes may normally share a hypoxic method of promoting
physiological apoptosis in the epiphyseal plate (Burova, Grudinkin, Bardin, & Gamaleĭ, 2001).
Their deletion, however, may lead to expansion of the proliferating zone and subsequently longer
bones. Aqp9 may function in a similar proliferation-specific manner as Stat1 in the epiphyseal
plate to regulate bone growth.
Histological examination of the P5 Aqp9 -/- distal femur epiphyseal plate indicates that an
irregular enlargement of the proliferating zone underscores not only the tibial long bone
phenotype, but the possible enlargement of other hindlimb bones as well. Deletion of Aqp9 may
promote a proliferation-leaning, anti-apoptotic phenotype that accelerates early growth and is
highlighted by expansion of the epiphyseal plate proliferating zone.
5.2.2 P21 Aqp9 -/- mice present with typical epiphyseal plate zones
To determine the cause of the P21 bone phenotype, WT and Aqp9 -/- littermate proximal tibia
epiphyseal plates were examined with a primary antibody against Col10a1. Col10a1 is a marker
of chondrocyte hypertrophy and was expected to stain robustly in the hypertrophic zones. In
particular, the hypertrophic zone was of interest as a shorter bone phenotype is often rooted in
defected chondrocyte hypertrophy (Shu et al., 2011). Using a marker of hypertrophy served to
investigate if any hypertrophy defect would be apparent by P21 to explain the Aqp9 -/- short
bone phenotype. Although the short bone phenotype occurred significantly in the femurs only,
proximal tibias were selected for the aforementioned reason of redundancy in epiphyseal plate-
bone growth coupling. Furthermore, only tibial sections were available. The tibia was therefore
investigated histologically as an alternative. Any irregularity in Col10a1 staining of the Aqp9 -/-
proximal tibia epiphyseal plate would support not only how the femoral shortening occurs, but
also the possibility of defected hypertrophy throughout the hindlimb at the juvenile stage.
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In the P21 Aqp9 -/- proximal tibia epiphyseal plate, the ratio of the hypertrophic zone to the
resting-proliferative zone is not significantly different from that of WT littermates. Similarly, the
observable epiphyseal plate heights did not significantly differ either. As mice age, the rate of
longitudinal growth also slows and is reflected by an increasing ratio of hypertrophic zone height
to proliferating zone height (Lee, Song, Pai, Chen, & Chen, 2017). It was postulated that in mice
with comparatively lower rates of growth and shorter bones—as expected in P21 Aqp9 -/-
mutants—this ratio would be higher.
Overall, the P21 Aqp9 -/- proximal tibia epiphyseal plate is comparable to that of P21 WT mice.
Interestingly, the knockout of genes contributory to endochondral ossification do not always
result in drastic adult skeletal phenotypes. MATN3 is an aforementioned gene that encodes non-
collagenous ECM and is mutated in many epiphyseal dysplasias (Klatt et al., 2000). Embryonic
Matn3 -/- mice have hypertrophic zones 47% larger than that of embryonic WT mice, but no
long bone phenotype is observed during development or adulthood in the mutants (van der
Weyden, 2006). In the case of Aqp9 -/-, a juvenile short bone phenotype is observed but may not
necessarily be due to irregularities of the epiphyseal plate. In P15 Stat1 -/- mice, the epiphyseal
plate differences underlying the early long bone phenotype are attenuated (Sahni et al., 2001).
Hence, it possible that temporary expansions of epiphyseal plate zones do not persist into later
development. However, it is more probable that due to the small sample size of biological
replicates, the analysis is not statistically powerful. Repetition of the experiment with a greater
sample size may find that the P21 Aqp9 -/- hypertrophic zone consumes more area in the
epiphyseal plate, and that the entire epiphyseal plate is significantly shorter.
5.2.3 18 month old Aqp9 -/- mice show prominent epiphyseal plate narrowing and
reduction of primary bone marrow adipose tissue
Previously, Liu et al. (2007) had isolated femurs and tibias from one year old female WT and
Aqp9 -/- mice and found no significant difference in length between the WT and Aqp9 -/- bones.
This was not consistent with the short bone phenotype observed in the male P21 Aqp9 -/- mice.
The Aqp9 -/- mice were derived from C57BL/6 mice in both studies, suggesting that strain-
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specific differences cannot be implicated. This suggests that the bone phenotype may not persist
into adulthood, or that sex differences complicate measurement consistency. Indeed, female
C57BL/6J mice experience greater age-related declines in femoral bone volume and length than
males over time (Glatt, Canalis, Stadmeyer, & Bouxsein, 2007). The absence of Aqp9 in old
male mutants was therefore unexplored, and may present with different long bone observations
than those of female mice.
Observing the old epiphyseal plate is a strong indicator of whether or not long bone growth has
been arrested. Epiphyseal plate narrowing and closure into the epiphyseal line indicates that
chondrocyte proliferation has stopped and that bone growth can no longer proceed (Shim, 2015).
However, longitudinal bone growth does not cease in mice at sexual maturity like humans; the
rate of growth dramatically slows instead (Jilka, 2013). In mice, total epiphyseal plate fusion
does not occur but narrows as the growth velocity approaches zero (Kilborn, Trudel, & Uhthoff,
2002; Emons, Chagin, Sävendahl, Karperien, & Wit, 2011). Unusually narrow old epiphyseal
plates or striking abnormalities would then indicate a defect in bone growth and provide insight
into the final long bone length. Hence, male WT and Aqp9 -/- mice were aged to 18 months for
epiphyseal plate analysis to observe if they would reflect a shorter bone phenotype, as observed
in the male P21 mutants.
18 month old male WT and Aqp9 -/- femoral heads were examined with Toluidine blue to
analyze their cartilaginous epiphyseal plates. Femoral heads were selected due to their
availability from previous experiments in the Kannu lab. In the Aqp9 -/- mice, the epiphyseal
plate does not appear to reach the cortical bone on the right-side and is instead ablated. The
observation was initially thought to be due to sectioning inconsistencies that caught the Aqp9 -/-
epiphyseal plates at a particular depth, but statistical analysis shows the same trend and
significantly reduced width across all Aqp9 -/- biological replicates compared to WT replicates.
Overall, analysis of old male WT and Aqp9 -/- mice suggest that 18 month old male Aqp9 -/-
mice not only experience a more rapid disappearance of the epiphyseal plate, but that it may
translate into subtle long bone defects. A quicker narrowing of the epiphyseal plate suggests that
the long bone growth rate is reduced, and that the final bone length may be shortened in
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comparison. This is consistent with precocious puberty, where an accelerated period of growth—
as observed at P5—ultimately results in diminished growth by adulthood. Although this
phenotype is observed as early as in P21, the 18 month old observations here suggest that this
irregular narrowing may occur as early as the juvenile stage and into the geriatric stage.
However, epiphyseal plate analyses at each of the hindlimb bones would be required to identify
if Aqp9 deletion continues to affect specific long bones. Nevertheless, the irregular epiphyseal
plate narrowing observed here in the old age Aqp9 -/- femur is consistent with the femoral short
bone phenotype in the P21 mice. In other mutant mouse models—such as deletion of Ahsg, an
inhibitor of skeletal mineralization—discontinuities along the old epiphyseal plate also
underscore accelerated ossification and shortened long bones (Seto et al., 2012).
Interestingly, the Aqp9 -/- primary bone marrow also presented with a drastic reduction of white
adipose tissue in comparison to WT littermates. This reduction of fat was accompanied by an
increase of visible HSCs, as shown in the small, stained spots distributed throughout the bone
marrow. Typically, fat content in the femoral bone marrow increases with age (Tuljapurkar et al.,
2011). However, a lack of adipose tissue is not necessarily suggestive that the bone is immature.
When MSCs decide on a cellular lineage, they can differentiate into chondrocytes and
fibroblasts, pre-osteoblasts, or pre-adipocytes (Rosen, Ackert-Bicknell, Rodriguez, & Pino,
2009). However, the presence of marrow adipocytes may be self-promotive and induce the
differentiation of MSCs into adipocytes, preventing other cellular lineages from being attained
(Duque, 2007). In the Aqp9 -/- bone marrow, a severely diminished amount of adipose tissue
suggests that the majority of MSCs have likely committed to other lineages instead—such as
chondrogenesis or osteoblastogenesis—without coercion by adipocytes. Without fatty bone
marrow, it is possible that old Aqp9 -/- mutants proceeded with endochondral ossification at a
faster rate than WT mice because the available MSCs were condensed for long bone growth
instead. This would support the hypothesis that Aqp9 -/- mutants experienced accelerated growth
at a younger age. Furthermore, bone marrow adipocytes function to release fatty acids and
provide energy for bone growth, remodelling, and hematopoiesis (Veldhuis‐Vlug & Rosen,
2017). A reduction of adipose tissue may also suggest that during early endochondral
ossification, Aqp9 -/- chondrocytes rely on fatty reserves for accelerated growth. Examination of
the bone marrow at various timepoints would be required to verify this possibility.
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It is also possible that the reduced bone marrow adipose tissue is reflective of protection against
bone resorption through Aqp9 deletion. Bone quality declines with aging and subcutaneous fat
redistributes to sites such as the bone marrow, heart, and liver (Tchkonia et al., 2010; Rodríguez,
Catalán, Gómez-Ambrosi, & Frühbeck, 2006). A reduction of bone marrow adipose suggests that
the 18 month Aqp9 -/- mice may be protected against age-related biasing of adipogenesis and
bone loss. This suggests that Aqp9 may continue to be a negative regulator of bone growth in
adulthood. This also appears to parallel the findings of Bu et al. (2012), where ten week old Aqp9
-/- mice were protected against microgravity-induced bone resorption. In aging bone marrow
stromal cells, ROS accumulation prevents their ability to maintain HSCs (Khatri et al., 2016;
Anthony & Link, 2014). Simultaneously, this invites adipocyte accumulation in the bone marrow
that further impairs hematopoiesis and bone regeneration (Ambrosi et al., 2017). In Aqp9 -/-
mice, a lack of functional Aqp9 may prevent endogenous cytoplasm H2O2 accumulation or
intercellular H2O2 transport in the bone marrow. Hence, the Aqp9 -/- bone marrow
microenvironment may favor HSC maintenance instead of adipogenesis, resulting in the reduced
bone marrow adipose observed at 18 months. This mechanism may suggest that reduced bone
marrow adipose in 18 month Aqp9 -/- mice is not age-oriented, but rather a functional
consequence of Aqp9 absence. Nevertheless, the irregular epiphyseal plate narrowing suggests
that Aqp9 deletion induces a diminished long bone growth rate in adulthood.
5.2.4 E16.5 Aqp9 -/- mice have irregular epiphyseal plate resting zones
To determine if Aqp9 affects endochondral ossification during development, E16.5 WT and
Aqp9 -/- littermate proximal tibia epiphyseal plates were examined with Safranin-O. Proximal
tibias were selected due to their availability from previous experiments in the Kannu lab. At
E16.5, the developing embryo is at the halfway point between when endochondral ossification
commences—at approximately E10.5—and when birth occurs—at approximately E21.0. Hence,
E16.5 is a prime timepoint to examine endochondral ossification in development. Here, the
average Aqp9 -/- resting zone height is significantly shorter than that of WT littermates. No
significant differences were observed in the proliferating and hypertrophic zones. Consequently,
the fraction of the whole epiphyseal plate that contains proliferating and hypertrophic
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chondrocytes is significantly greater in the Aqp9 -/- mice than in WT mice. Although the
proliferating and hypertrophic zones are a popular focus in mutant mouse models, the height of
the resting zone has been scrutinized in Smad4 mutants. SMAD4 is a member of the SMAD
family and signals under BMP and TGF-β as early as in mesenchymal differentiation (Wu, Chen,
& Li, 2016). In Col2a1-Cre; Smad4Co/Co mice, the conditional deletion of Smad4 results in
newborn dwarfism characterized by a drastically expanded resting zone (Zhang et al., 2005). In
E16.5 Aqp9 -/- mice, a diminished resting zone may suggest the opposite. Skeletal staining at
E16.5 would be required to verify long bone lengths in comparison to WT littermates.
The resting zone is host to chondrocytes that are stem-like; they can divide and differentiate into
proliferating chondrocytes but with a limit reflecting a senescence factor (Abad et al., 2002). A
depletion of resting chondrocytes is indicative of proliferative capacity expenditure, which is
suggested to underlie the natural decline in long bone growth rate with age (Schrier et al., 2006).
The irregular central ‘caving’ of the proliferating zone into the resting zone in the Aqp9 -/-
embryos suggests that a greater proportion of resting chondrocytes are differentiating during
development. Hence, the resting zone is diminished in height while the proliferating and
hypertrophic zones together are expanded in height. This observation reflects the hypothesis that
Aqp9 deletion results in an early accelerated phenotype at the epiphyseal plate, which may occur
in development before P5. In epiphyseal plate senescence, Lui et al. (2011) discuss that during
accelerated growth, resting chondrocytes sacrifice proliferative capacity to produce more
proliferating chondrocyte columns in the short term. This comes at a cost of the resting zone cell
density and lifespan, and may require spatial decline as the proliferating zone consumes a greater
area. Here, an irregularly diminished resting zone in E16.5 Aqp9 -/- mice reflects the theory.
Although the P5 epiphyseal plate resting zones were not measured in this study, the observation
coincides with the P5 Aqp9 -/- proliferating zone expansion. Therefore, the tibial overgrowth
observed in Aqp9 -/- mutants may commence in development as early as E16.5.
Overall, shortened proximal tibia resting zones in developing Aqp9 -/- mice suggest that their
epiphyseal plates host chondrocytes that may differentiate faster. This phenotype may persist
postnatally into the P5 age where Aqp9 -/- mice show expanded tibial proliferating zones and
longer tibias than their WT littermates. However, accelerated differentiation likely leads to early
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epiphyseal plate senescence and slower growth rate, as observed in the shortened P21 Aqp9 -/-
femurs. By adulthood, the epiphyseal plate shows more prominent narrowing than WT
littermates, suggesting that the growth rate has rescinded more rapidly into maturity due to the
precocious overgrowth observed.
5.3 Aqp9 mutant chondrocytes show a differential phenotype
5.3.1 P5 Aqp9 +/- and Aqp9 -/- epiphyseal plate chondrocytes proliferate irregularly
To support the role of Aqp9 as a regulator of chondrocyte differentiation and long bone growth,
the investigation was continued at the cellular level. The P5 timepoint was chosen because:
1) Early long bone phenotypes in mutant mouse models are infrequent, and
2) Epiphyseal plate chondrocytes at the P5 timepoint are excised without complication.
A cell proliferation experiment was performed by seeding WT, Aqp9 +/-, and Aqp9 -/-
epiphyseal plate chondrocytes for cell counts over 96 hours. Over the experiment duration, WT
chondrocytes did not differ significantly in cell number from Aqp9 +/- or Aqp9 -/- chondrocytes
during any timepoints except at the 48 hour timepoint. By the final timepoint, all samples
approached the expected confluency of 1.2 x 106 cells.
From 0-96 hours, the WT plot appears relatively linear and deviates from an expected
exponential rate of growth in culture. While exponential growth is observed from 0-48 hours, the
slope plateaus greatly by the 72 hour timepoint before approaching confluency. This may be
attributed to cellular density or natural declines in chondrocyte proliferation that may be
triggered by culture conditions (Lui & Baron, 2011). The Aqp9 -/- plot shows a striking
difference at the 48 hour timepoint in comparison to the WT plot; rather than experiencing
exponential growth, the cell number was only marginally higher than that of the 24 hour
timepoint. At the 48 hour timepoint, the Aqp9 -/- number is ~4 x 105 lower than that of the WT.
However, at the 72 hour timepoint, the Aqp9 -/- number jumped to ~1 x 106. The slope declines
greatly by the 96 hour timepoint, likely due to cellular density as plate confluency is approached.
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The Aqp9 +/- plot parallels the Aqp9 -/- plot, although the jump from 48-72 hours is not as
drastic. Nevertheless, the Aqp9 +/- chondrocytes matched the WT chondrocyte cell number at the
72 hour timepoint despite a lower count by comparison at the 48 hour timepoint. As the most
striking change occurs between the 48-96 hours, the rates there can be calculated as follows:
Table 5.1. Primary epiphyseal plate chondrocyte proliferation rates (48-96 hours)
Sample genotype (Cell number at 96 hours – cell number at
48 hours) / (96 hours – 48 hours) Rate (cells/hour)
WT (~1x106 – ~5.5x105) / 48 ~9375
Aqp9 +/- (~9.5x105 – ~2x105) / 48 ~15625
Aqp9 -/- (~1.5x106 – ~2.5x105) / 48 ~26042
Here, both Aqp9 +/- and -/- chondrocytes appear to have accelerated proliferation following an
apparent pause in growth, in comparison to WT chondrocytes. However, their rates from 0-96
hours do not differ as they start and end at approximately the same cell number counts. In the
growing rat epiphyseal plate, this phenomenon of ‘catch-up growth’ is observed in proliferating
chondrocytes immediately after cessation of dexamethasone, a growth inhibitor (Chagin,
Karimian, Sundström, Eriksson, & Sävendahl, 2010). Deletion of Aqp9 may promote cell
proliferation after suppression through an unexplored mechanism. The pause in growth at the 48
hour timepoint may also be caused by an unexplored mechanism. Interestingly, the error bars for
all chondrocyte counts at this timepoint appear narrower than those of the other timepoints. This
may be due to a small sample size resulting in a statistical anomaly. Nevertheless, the error bars
for the Aqp9 -/- chondrocyte count at 72 hours are remarkably wide and suggest that further
repetition of the experiment would be supportive. Increasing the sample size would be beneficial
to reduce the variance.
In normal epiphyseal plate chondrocytes, H2O2 is involved in a variety of signal transduction
pathways. Intercellular H2O2 transport is only reported among aquaporin homologues (Bienert,
Schjoerring, & Jahn, 2006). However, the cellular production of H2O2 is mainly derived from the
mitochondrial matrix and intermembrane space (Boveris & Cadenas, 2000). Hence, the transport
of H2O2 from the mitochondria to the cytoplasm likely requires Aqp9 function, or other
aquaporin isoforms that are capable such as Aqp3 and Aqp8. AQP9 protein localization in
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mitochondria has been described, although aquaporin transport functionality in mitochondria has
been argued against due to a lack of physiological consequence in several aquaporin mutants
(Lindskog, Asplund, Catrina, Nielsen, & Rützler; 2016; Yang, Zhao, & Verkman, 2006). It is
also possible for H2O2 to alternatively exit the mitochondria through initial enzymatic conversion
to superoxide, and then passage through the electron transport chain proteins and voltage-
dependent anion channels (Han, Canali, Rettori, & Kaplowitz, 2003). Nevertheless, Aqp9 is a
channelome member and is likely expressed on the chondrocyte mitochondrial membrane,
allowing passage of H2O2 into the cytoplasm. Further study such as Aqp9 immunostaining of the
murine chondrocyte mitochondria would confirm its membrane expression. Outside the
mitochondria, members of the HIF family are stimulated through mTOR signalling (Land & Tee,
2007; Mohlin et al., 2015). mTOR signalling is repressed by AMPK signalling, which in turn is
repressed by H2O2 (Lennicke, Rahn, Lichtenfels, Wessjohann, & Seliger, 2015). Hence, H2O2 is a
stimulator of HIF activity. In particular, HIF2α is a HIF homologue stabilized by H2O2 oxidative
stress and induces chondrocyte hypertrophy by binding to the COL10A1, MMP13, and VEGFA
promoters in the nucleus (Diebold et al., 2010; Saito et al., 2010). From mitochondria to nuclear
transcription, the transport of H2O2 to the cytoplasm likely relays stimulus for chondrocyte
hypertrophy. When extra-mitochondrial transport of H2O2 is diminished, hypertrophy may be
delayed. In Aqp9 -/- chondrocytes, it is possible that a proliferative phenotype is attained this
way. This would support the irregular proliferation rate observed in this experiment, the
expanded proliferating zone, and early long bone phenotype observed at P5.
It is also possible that deletion of Aqp9 induces a ‘mitotic wave’. Rabbit articular chondrocytes
treated with TGF-β1 show increased DNA replication rates, a sequestering of chondrocytes in
the G2/M checkpoint, and a subsequent wave of mitosis (Vivien et al., 1993). This would require
a temporary increase in proliferation rate as shown in the Aqp9 -/- chondrocytes. The catch-up
growth between 48-96 hours of culture may be reflective of this pattern. However, the
relationship between TGF-β and Aqp9 in the epiphyseal plate has not been explored. Overall,
Aqp9 may be important in chondrocyte proliferation in the early pup stage. From the
observations, Aqp9 may be a negative regulator as its deletion induces a different proliferation
trend compared to WT chondrocytes. Deletion of Aqp9 may induce a proliferating-leaning
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chondrocytic phenotype by barring physiological levels of H2O2 from stimulating hypertrophy at
a regular rate.
5.3.2 P5 Aqp9 +/- and Aqp9 -/- epiphyseal plate chondrocytes have differential gene
expression profiles
To clarify the proliferation rate difference observed in P5 Aqp9 +/- and -/- chondrocytes—
however limited to specific experimental timepoints—their gene expression profiles were
investigated. P5 WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes were subjected to
qPCR gene expression analysis. The fold change levels of several hypertrophic markers in the
Aqp9 +/- and -/- chondrocytes were not significantly different relative to the WT control. In Aqp9
-/- chondrocytes, the fold change of the one marker of proliferation, Sox9, was significantly
increased. Aqp9 expression was significantly decreased. The Aqp9 +/- chondrocytes did not
follow these changes as consistently. There was a significant reduction of Mmp13 expression in
the Aqp9 +/- chondrocytes compared to the WT chondrocytes.
In Aqp9 -/- chondrocytes, the increase of Sox9 is indicative that their gene profiles lean towards a
proliferative phenotype. In Aqp9 +/- chondrocytes, the significant decrease of Mmp13 is
indicative that they are resisting the hypertrophic phenotype, where Mmp13 would be increased.
While the fold changes of the remaining hypertrophic markers—Col10a1 and Runx2—were not
statistically significant in both Aqp9 -/- and +/- chondrocytes, their direction of change is
noteworthy. Since Aqp9 expression changes in the same direction as the hypertrophic marker
genes and also opposes the fold change direction of the proliferative marker Sox9, Aqp9 may be
a contributor to chondrocyte hypertrophy upstream of Col10a1, Mmp13, and Runx2. It is also
possible that the mutant chondrocytes invoke surveillance mechanisms to ensure appropriate
mRNA quality, influencing the gene profile readout observed. Mammalian cells are capable of
using the Nonsense-mediated decay pathway, a RNA surveillance mechanism that targets
mRNAs with premature stop codons for deletion to prevent production of truncated proteins
(Hug, Longman, & Cáceres, 2016). If this pathway is activated in the mutant chondrocytes, the
significant fold change decrease of Aqp9 may not be reflective of reduced hypertrophy but of
Nonsense-mediated decay checking the Aqp9 mutation instead. Furthermore, the non-significant
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change of Col10a1 and Runx2 in both Aqp9 -/- and +/- chondrocytes relative to the WT
chondrocytes may be analytical error due to a low number of biological replicates. Repetition
with a greater number of replicates may produce a more consistent gene profile.
Other mutant mouse models with deletion of genes critical to chondrogenic differentiation have
similar patterns of chondrocyte marker expression. MAP3K7 is a gene encoding TAK1, an
enzyme critical to the TGF-β and BMP pathways (Le Goff et al., 2016). In chondrocyte-specific
deletion of Tak1 in P7 mice, there is an accompanying significant decrease in expression of Sox9
and Col2a1 while Mmp13 expression increases (Gao et al., 2013). In Aqp9 -/-, the same pattern
of proliferative and hypertrophic marker expression is observed in the opposite fold change
direction. Hence, deletion of Aqp9 appears to be deletion of a functional hypertrophy inducer.
It is possible that certain signalling molecules transported among epiphyseal plate chondrocytes
in culture by Aqp9 influence the expression of hypertrophic genes. If functional Aqp9 is ablated
as in Aqp9 -/- chondrocytes, then a reduction of those transported molecules may trigger non-
physiological pathways leading to reduced transcription factor-promotion of genes like Col10a1,
Mmp13, and Runx2. Epiphyseal plate chondrocytes can exchange micro RNAs among one
another through extracellular vesicles to elicit chondrogenic differentiation (Lin et al., 2018).
However, the function of Aqp9 in chondrocyte matrix vesicle communication has not yet been
explored. The hypothesized blockage of endogenous H2O2 from exiting the chondrocyte
mitochondria may also be responsible for the decreased expression of hypertrophic genes.
Without Aqp9 to transport H2O2 and stabilize Hif2α, binding to Col10a1 and Mmp13 promoter
regions is likely reduced, resulting in diminished hypertrophy. It is also important to consider
that cellular hypertrophy is maintained by the cytoskeleton. This cellular network of
microtubules, actin, and other diverse filaments provides the physical structure for chondrocyte
shape and henceforth governs—at least partially—chondrocyte phenotype (Benjamin, Archer, &
Ralphs, 1994). Interestingly, actin-containing cytoplasmic projections known as filopodia were
upregulated in murine fibroblasts when transfected with a human aquaporin-9 (AQP9)
overexpression vector (Loitto et al., 2007). It was hypothesized that a specific water/solute
transporter increased hydrostatic pressure within the membrane-cytoskeleton space to permit
outward filament elongation and form extracellular protrusions. Karlsson et al. (2013) confirmed
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this with a model for AQP9-induced membrane projection in embryonic kidney cells,
demonstrating that local membranous accumulation of AQP9 initiates protrusion and subsequent
extension of the actin cytoskeleton. In Aqp9 -/- chondrocytes, lack of functional Aqp9 may
inhibit the cytoskeleton plasticity necessary to initiate and maintain the hypertrophic shape.
In summary, P5 Aqp9 -/- epiphyseal plate chondrocytes express gene profiles that appear to
oppose hypertrophy and favor proliferation, as noted by the significant expression increase of
Sox9—a promoter of chondrocyte proliferation and inhibitor of hypertrophy. This may explain
the differential proliferation rate of Aqp9 -/- chondrocytes, leading to expansion of the
proliferating zone and accelerated long bone growth at the pup stage. A chondrocyte
proliferation increase in the fetal metatarsal epiphyseal plate via insulin and IGF-1 treatment
results in increased proliferating zone length and total bone length (Zhang et al., 2014). Deletion
of chondrocyte Aqp9 may mimic this mitogenic effect.
5.3.3 Aqp9 knockdown in P5 WT epiphyseal plate chondrocytes induces a gene profile
similar to Aqp9 -/- chondrocytes
In the Aqp9 +/- and Aqp9 -/- chondrocytes, it is possible that their gene expression profiles were
not induced within the chondrocyte but were rather a consequence of signalling from other
organs, such as the bone. Hence, P5 WT chondrocytes were excised and transfected with an
Aqp9-siRNA to determine if a similar gene profile could be adopted. If so, any Aqp9 -/- cellular
phenotypes can be accredited to processes occurring specifically in the chondrocytes in a cell
autonomous manner.
Overall, there were no significant differences in gene expression after Aqp9 silencing. However,
the direction of change of each gene probe is noteworthy. The direction of change of the
hypertrophic markers—Col10a1, Mmp13, and Runx2—was downward, and the proliferative
marker—Sox9—was upward. The direction of change of Aqp9 expression was also downward.
Importantly, the overall gene profile of WT-siRNA reflected the gene profile of the Aqp9 -/-
chondrocytes. This supports that Aqp9 knockdown alone is able to influence the expression of
chondrocyte proliferative and hypertrophic markers, and that the similar gene profile observed in
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Aqp9 -/- chondrocytes was not attributed to external factors such as the generation of the Aqp9 -/-
mutant mouse itself. The lack of observed statistical significance is likely due to a small sample
size. The incubation time post-transfection may also be on the shorter end of the protocol
recommendation. The Aqp9 siRNA oligomer concentration may also be insufficient. The error
bars for all gene probes are wide, hinting at deviant fold change values among the biological
replicates.
In this experiment, knockdown of Aqp9 in WT chondrocytes was able to simulate the Aqp9 -/-
chondrocyte gene profile. A natural inhibitor of Aqp9 named phloretin is able to decrease
expression of MMP13 and prevent the degradation of type II collagen, albeit in human articular
chondrocytes (Zheng, Chen, Zhang, Cai, & Chen, 2018). The use of phloretin to target Aqp9 in
epiphyseal plate chondrocytes is unexplored. Nevertheless, the concomitant downregulation of
hypertrophy-related genes following Aqp9 inhibition is well-reported.
5.4 A model for Aqp9 function in murine endochondral ossification
In this study, analysis of the literature and findings support a speculative model for the function
of Aqp9 in murine long bone growth. In Figure 5.1, the impact of Aqp9 deletion is described
from the chondrocytic level to the long bone at both P5 and P21. The steps synthesize the
aforementioned literature and results, and are described as follows:
1) The pathway starts in the developing epiphyseal plate. The ATP-synthesizing process of oxidative phosphorylation in the
epiphyseal plate chondrocyte mitochondrial matrix generates endogenous superoxide as a waste product, which is then
converted into H2O2.
2) H2O2 attempts to leave through H2O2 transporters along the mitochondrial membranes, but Aqp9 is not available for
transport due to the Aqp9 -/- mutation. Here, H2O2 may be sequestered in the mitochondria.
3) H2O2 can exit the mitochondria through alternative means, such as enzymatic conversion back to superoxide and then
transport through voltage-dependent anion channels. The rate of H2O2 departure is therefore diminished in Aqp9 -/-
chondrocytes.
4) Superoxide that is transported into the cytoplasm can be reconverted into H2O2. H2O2 can function to repress AMPK,
which is a repressor of mTOR. H2O2 is then an activator of mTOR. However, loss of Aqp9 suggests that less-than-
physiological H2O2 will be present in the cytoplasm, so mTOR activation may be diminished.
123
5) H2O2 can repress PTP, which is a repressor of Akt (Lennicke et al., 2015). Akt is a factor that ultimately activates mTOR.
Once again, H2O2 is an activator of mTOR—but without physiological concentrations of H2O2 in the cytoplasm due to
Aqp9 deletion, mTOR activation may be repressed.
6) mTOR is an activator of Hif2α, a hypertrophy inducer in chondrocytes. Oxidative stress conferred by cytoplasmic H2O2
normally stabilizes Hif2α. However, Aqp9 deletion suggests less cytoplasmic H2O2 and therefore less Hif2α.
7) Normally, Hif2α binds to the promoters of hypertrophy genes in the nucleus to promote the hypertrophic phenotype.
Without physiological amounts of Hif2α, fewer hypertrophy genes are transcribed and the proliferative phenotype is more
likely to be retained.
8) In the P5 Aqp9 -/- mouse, more proliferation allows endochondral ossification to occur at a faster rate than usual.
Circulating estrogen finds that there is no Aqp9 target to bind and instead binds available growth hormone receptors,
further accelerating growth at this stage. The growth phenotype becomes localized in the hindlimb, which Tbx4 governs
in development. Furthermore, the Shox transcription factor finds there is no Aqp9 promoter to bind, possibly inducing a
rhizomelic effect in the tibia along with Runx2 and Ihh. Ultimately, a long bone phenotype is observed in the tibia bones.
9) As the Aqp9 -/- mouse ages, it continues to undergo endochondral ossification as the epiphyseal plate narrows. Due to the
precocious overgrowth, the epiphyseal plate has become more senescent than usual. The resting chondrocytes have lost
more replicative capacity than usual. The rate of long bone growth has become gradually retarded relative to WT mice.
10) By P21, the Aqp9 -/- mouse experiences the consequences of accelerated growth. Circulating estrogen now hastens
epiphyseal plate narrowing, further resulting in slower growth rate. The shortening phenotype is still localized in the
hindlimb, likely through Tbx4. However, the Shox transcription factor now induces a mesomelic effect in the femur,
possibly through dysregulation of Fgfr1. Finally, the femur bones are shorter.
11) In WT mice, epiphyseal plate chondrocytes maintained physiological levels of cytoplasmic H2O2 due to functioning Aqp9
transporters. Chondrocyte hypertrophy occurred naturally without a proliferative-leaning phenotype. As a result, P5 WT
mice have shorter tibia bones compared to their Aqp9 -/- littermates. By P21, a regular rate of growth has allowed for the
WT mice to have longer femur bones instead. Throughout growth in the WT mice, Aqp9 is able to interact normally with
its transcription factors.
ECM / Extracellular space
Chondrocyte cell membrane
Chondrocyte cytoplasm
Mitochondrial matrix
Intermembrane space
Mitochondria
H2O2
Oxidative
phosphorylation
H2O2
Nucleus (not to scale)
AMPK
mTOR
Hif2α
Etc…
Col10a1
Mmp13
Vegfa
PTP
Akt
Alternative
exit
1
2
3
4
5
6
7
Aqp9 -/-
H2O2
H2O2
H2O2
Figure 5.1. A model for Aqp9 function in murine endochondral ossification 124
P5 P21
Tbx4
Aqp9 -/-
Shox, Runx2, Ihh
Estrogen (+growth)
Tbx4
Aqp9 -/-
Shox, Fgfr1
Estrogen (-growth)
WT growth scenario
Tibia Femur
P5 P21
Aqp9 Aqp9
Tibia Femur
8
9
10
11
Regular TF-promoter interaction Regular TF-promoter interaction
Figure 5.1. cont. 125
126
6 Conclusions
To address whether Aqp9 is important in endochondral ossification, Aqp9 -/- mice were
examined at the long bone, epiphyseal plate, and chondrocyte levels from the pup stage to old
age. At P5, Aqp9 -/- mice have significantly longer tibia bones than their WT littermates. At P21,
Aqp9 -/- mice have significantly shorter femur bones than their WT littermates. These defects
suggested that Aqp9 deletion may induce accelerated neonatal growth that stunts skeletal length
by the juvenile age, albeit in specific hindlimb bones. Hindlimb epiphyseal plates were then
examined to see if dysregulation in the epiphyseal plate zones would underscore the bone
phenotypes. Analysis of the P5 Aqp9 -/- femur epiphyseal plate showed expansion of the
proliferating zone, supporting a long bone phenotype throughout the hindlimb. However,
analysis of Col10a1 staining in the P21 Aqp9 -/- tibia epiphyseal plate did not show any
remarkable dysregulation. Examination of 18 month Aqp9 -/- femur heads showed irregular
narrowing of the epiphyseal plate and reduced adipose tissue in the bone marrow, suggesting that
old age Aqp9 -/- mice experience irregular growth earlier in their lifespan and resist bone marrow
adipose accumulation. E16.5 Aqp9 -/- embryo epiphyseal plates were also examined to determine
if Aqp9 is important in development; they showed significantly reduced resting zone heights,
indicating an early acceleration of chondrocyte differentiation. Finally, P5 Aqp9 +/- and Aqp9 -/-
epiphyseal plate chondrocytes were examined to see if their behaviour and gene expression could
explain the P5 bone phenotype and epiphyseal plate dysregulation. Aqp9 -/- chondrocytes
displayed an irregular rate of proliferation characterized by a lag in growth and then subsequent
recovery. Gene expression analysis of Aqp9 -/- chondrocytes showed a significant increase of
proliferative marker expression. Silencing of Aqp9 in P5 WT chondrocytes did not yield
significant expression differences, but was able to simulate the same directions of change
observed in the Aqp9 -/- chondrocyte gene probes. This suggests that Aqp9 -/- chondrocytes may
favour a proliferative phenotype in differentiation, supporting the expanded proliferating zone in
the P5 epiphyseal plate.
Aqp9 may be a negative regulator of neonatal bone growth. Deletion of Aqp9 appears to induce
chondrocyte proliferation and accelerate the process of endochondral ossification, leading to
bone-specific elongation in the hindlimb at P5. However, the accelerated growth may trigger
earlier senescence of the epiphyseal plate chondrocytes and ultimately delay growth, leading to
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the bone-specific shortening in the hindlimb at P21. The accelerated differentiation may start as
early as in development at E16.5, but the latent phenotypes are also observable at 18 months of
age. Aqp9 appears to function similarly to hypertrophic genes and its deletion promotes a
proliferative-leaning gene profile. Aqp9 is also a target of estrogen and SHOX, suggesting
another avenue by which Aqp9 deletion may dysregulate long bone growth. Aqp9 may normally
function with Tbx4 and unexplored factors related to mesomelic and rhizomelic disorders in
order to regulate growth in the hindlimb bones. At the chondrocyte level, Aqp9 may normally
shuttle endogenous H2O2 to the cytoplasm to activate hypertrophic genes and regulate the rate of
differentiation. Deletion of Aqp9 would then delay the hypertrophic phenotype.
The limitations of this study include methodological sample size, data measurement, and prior
available research of Aqp9. Regarding sample size, the immunohistochemistry and Safranin-O
staining experiments in this study did not meet a minimum of three biological replicates. Using a
larger sample size for each experiment would improve the statistical power of all analyses and
how representative they are of the Aqp9 mutation. Regarding data measurement, long bone
lengths and epiphyseal plate parameters were collected manually. Despite using digitally-assisted
calipers and software in a blinded setting, fully computerized scanning of long bones and
epiphyseal plates would improve the accuracy of all measurements. Regarding prior research,
investigations in Aqp9 are limited (<500 PubMed search results as of August 2018). It is
possible that a greater number of prior studies would help form a more comprehensive literature
review and discussion. Nevertheless, this limitation may describe a need for further biomedical
research of Aqp9.
Overall, this study demonstrated that Aqp9 plays a role in endochondral ossification. Deletion of
Aqp9 showed significant differences at the long bone, epiphyseal plate, and chondrocyte levels
as hypothesized. As a transporter of a known hypertrophy inducer, subject of regulation by
estrogen and Shox, and a chondrocyte channelome member, Aqp9 does not appear completely
dispensable in function. Aqp9 stands out as a unique aquaporin isoform and sheds light on how
the channelome can critically regulate chondrocyte differentiation in the epiphyseal plate.
Further investigation of Aqp9 chondrocyte function may unveil a novel therapeutic target for
combatting LLDs before resorting to invasive surgical intervention.
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7 Future Directions
7.1 In situ hybridization of Aqp9 during mesenchymal condensation
In this study, in situ hybridization of Aqp9 to the P14 murine WT epiphyseal plate revealed
robust expression in the proliferating and pre-hypertrophic zones. This suggests that Aqp9
functions postnatally to modulate long bone growth. However, endochondral ossification
commences during development at approximately E10.5. Here, mesenchymal condensation
occurs as the initiating step. To determine if Aqp9 is present at the embryonic level, in situ
hybridization could be performed to visualize Aqp9 RNA in the developing murine limb buds. A
multi-probe fluorescent in situ protocol targeting other genes important in condensation could
also be used to determine if Aqp9 is co-localized with them. During mesenchymal condensation,
the well-known Bmps and Fgfs are present as essential factors toward chondrogenesis (Hata et
al., 2017). However, limb outgrowth is also dependent on the Hox and Tbx genes, which
coordinate the spatial and temporal development of the limb (Guy & Clarke, 2008). Hence,
genes can be differentially expressed in the forelimb and hindlimb. The Hox genes contain
promoter regions that will only activate if a specific combination of transcription factors bind. In
general, Hox genes create positional memory in MSCs and influence their ability to differentiate
(Seifert, Werheid, Knapp, & Tobiasch, 2015). A variety of Hoxb isoforms and Hoxc4 have been
identified in murine MSCs, making them distinct markers of condensation (Phinney, Gray, Hill,
& Pandey, 2005). The Tbx genes determine limb identity, where Tbx5 is localized to the forelimb
only and Tbx4 is localized to the hindlimb only (Logan, 2003). If Aqp9 functions in the
developing limb, it may regulate these genes. A preliminary whole-mount in situ hybridization of
Aqp9 to WT E10.5 embryos can be performed to identify its expression, following the protocol
mentioned in this study with minor modifications for embryos (Koyama et al., 1996). If Aqp9
can be visualized in the developing forelimbs and hindlimbs, then a multi-plex assay can be
performed to check if Hox and Tbx are expressed in the same patterns. A multi-plex fluorescent
in situ assay can detect molecules for up to four RNA targets simultaneously, where Aqp9, Tbx4,
Tbx5, and Hoxc4 can be selected and designed as riboprobes. If Aqp9 fluorescence is co-
localized with that of the Hox or Tbx genes, it suggests that Aqp9 may help coordinate limb
outgrowth. Its deletion in Aqp9 -/- mutants would then dysregulate how endochondral
129
ossification is initiated, possibly relaying into differential long bone growth. An EMSA
(electrophoretic mobility shift assay) could then be performed to confirm if Hox or Tbx proteins
bind to the Aqp9 promoter. If they do not appear to regulate Aqp9, then promoter bashing could
be performed to specifically identify which region of the Aqp9 gene promoter controls
transcription the most. The new region can be sequenced, matched to other proteins that regulate
limb patterning through databases such as BioGRID, and an EMSA can be repeated.
7.2 Histomorphometry of WT and Aqp9 -/- long bones
In this study, the long bone length comparisons of WT and Aqp9 -/- mice showed a differential
phenotype. The long bones were measured and superficially analyzed to suggest that
endochondral ossification occurs differently in the Aqp9 -/- mutants. However, the role of Aqp9
may go beyond bone length and also affect bone mineral density and structure. Liu et al. (2009)
show that one year old female Aqp9 -/- mouse bones do not significantly differ from that of WT
mice. Therefore, the contribution of Aqp9 to long bone characteristics at younger timepoints has
not been explored. To determine if Aqp9 affects bone in vivo, long bones can be isolated from
P5, P21, and one year old WT and Aqp9 -/- mice for histomorphometric analysis. Male mice can
be used exclusively to compare to the findings of Liu et al. (2009) and determine if Aqp9
functions in a sex-specific manner. Humerus, femur, and tibia bones can be dissected for micro-
CT analysis. Both trabecular and cortical bone characteristics can be analyzed for bone mineral
density and percent bone volume. The P5 and P21 skeletons stained in this study may be isolated
for this experiment, as the soft tissue has been removed and the fixation process is similar. In P5
Aqp9 -/- mice, their long bones may exhibit decreased bone mineral density as in Fgfr3 null mice
(Su et al., 2010). At P21, Aqp9 -/- long bones may also present with differential bone density,
although even Bmp2fl/fl; OSX-Cre mice—which would be expected to have defected
endochondral characteristics due to the importance of Bmp2—have comparable bone density to
WT control mice (McBride-Gagyi, McKenzie, Buettmann, Gardner, & Silva, 2015). Hence, any
differences observed may be minimal. In this study, the 18 month old mice Aqp9 -/- mice
exhibited an unusual narrowing of the epiphyseal plate. It is possible that in one year old mice,
the narrowing phenotype is also present and may be indicative of shorter bone length in
comparison to WT mice. Micro-CT or 3D skeletal reconstruction would identify the pertinent
130
parameters of bones at this age which could be charted for comparison to the P5 and P21
characteristics. Overall, histomorphometric analysis of the long bones would provide deeper
insight into the endochondral characteristics of Aqp9 -/- mice beyond length. By analyzing long
bones from pups to old mice, the role that Aqp9 plays in murine skeletal development may be
better understood.
7.3 Flow cytometry cell cycle analysis
The cell proliferation experiment performed in this study revealed an irregular proliferation
phenotype among Aqp9 +/- and Aqp9 -/- chondrocytes. Chondrocytes were cultured and counted
at specific timepoints to measure their growth rate. However, the variance, weak statistical
power, and time consumption of the experiment design suggests that other methods can be
employed to accurately measure proliferation rates. To determine if Aqp9 affects the cell cycle
profile of epiphyseal plate chondrocytes, WT, Aqp9 +/-, and Apq9 -/- chondrocytes can be
isolated and subjected to flow cytometry cell cycle analysis. With flow cytometry, propidium
iodide can be used to bind DNA inside the chondrocyte samples and correlate with the amount
they contain. Therefore, the fluorescent intensity measures the number of cells at any one time
that are in each cell cycle phase. A high percentage of cells in the G2/M phase would suggest an
accelerated proliferation phenotype, as cells have just doubled their DNA content in the
preceding S phase. Initially, WT and Aqp9 -/- epiphyseal plate chondrocytes from P5 and P21
mice can be compared first. If they present with distinct cell cycle profiles, then Aqp9 +/-
chondrocytes can be checked as well to determine if heterozygous Aqp9 deletion has an
intermediary profile. Then, chondrocytes from embryonic and old age timepoints can be
harvested to compare profiles from development till adulthood. Chondrocytes can be sorted
according to their phenotype to ensure that only specific chondrocytes are analyzed. Belluoccio
et al. (2010) describe that the cluster of differentiation antigen CD200 is a cell surface marker
restricted to chondrocytes from the pre-hypertrophic and hypertrophic zones. If the analysis is to
be performed only on proliferating chondrocytes, the CD200-positive chondrocytes can be sorted
out. Furthermore, the cell proliferation experiment in this study showed accelerated proliferation
in Aqp9 mutant chondrocytes only between the 48-72 hour timepoints. If chondrocytes are
prepared and permeabilized for flow cytometry immediately after pronase/collagenase treatment,
131
a distinct profile may not be observed. Samples can be cultured for 48 hours and then subjected
to the analysis. The analysis would determine whether the G2M:G1 ratios reflect the
proliferation phenotype observed among Aqp9 -/-chondrocytes.
7.4 RNA-sequencing of WT, Aqp9 +/-, and Aqp9 -/- primary
epiphyseal plate chondrocytes
In this study, the P5 Aqp9 -/- mice exhibited tibial overgrowth and the P21 Aqp9 -/- mice
exhibited femoral shortening. Furthermore, qPCR of P5 Aqp9 -/- epiphyseal plate chondrocytes
revealed a proliferation-leaning gene profile. To determine if Aqp9 functions in any pathways
affecting endochondral ossification, epiphyseal plate chondrocytes isolated in this study could be
RNA-sequenced to identify their gene expression both differentially and temporally. Additional
chondrocytes from different timepoints would also be required to assess expression throughout
endochondral ossification. WT, Aqp9 +/-, and Aqp9 -/- chondrocytes were investigated in this
study at the P5 timepoint through a cell proliferation assay, gene expression analysis, and Aqp9
silencing. While Mmp13 was significantly reduced and Sox9 was significantly increased in the
qPCR experiment among the mutant chondrocytes, siRNA knockdown of Aqp9 only showed a
gene expression trend without significance. Furthermore, the experiments were only performed
at one timepoint whereas Aqp9 deletion appears to affect skeletal stature differently depending
on age. By isolating WT, Aqp9 +/-, and Aqp9 -/- epiphyseal plate chondrocytes at E16.5 and P21,
their RNA could be sequenced alongside the P5 samples to show how the Aqp9 mutation may
affect pathways involving proliferation, hypertrophy, and ossification over time. At E16.5,
embryos have commenced with endochondral ossification for 5-6 days and would capture any
function of Aqp9 in early long bone development. At P21, mice are juvenile and would represent
any function of Aqp9 during early pubertal bone growth. The use of 5 biological replicates per
genotype and timepoint would improve the statistical power of the analysis. However, excision
of epiphyseal plate cartilage from embryos may be manually challenging due to size. To isolate
chondrocytes, whole embryo limbs may be roughly dissected and then digested prior to flow
cytometry sorting. The use of the aforementioned chondrocyte-associated antibodies would
improve the isolation of cells from only the developing limb buds. Altogether, RNA-seq of WT,
Aqp9 +/-, and Aqp9 -/- chondrocytes at the developmental, pup, and juvenile stages can establish
132
how Aqp9 affects differential and temporal gene expression during endochondral ossification.
The RNA-seq may reveal a temporal method by which Aqp9 deletion influences chondrocyte
proliferation and hypertrophy. Disrupted pathways that link Aqp9 to bone defects may also be
identified. Validation of the RNA-seq output could be performed at the protein expression level
through Western blotting with antibodies for AQP9 and other markers differentially expressed.
The frozen P5 Aqp9 -/- epiphyseal plate chondrocyte protein samples saved in this study could
also be used for Western blotting, then compared to the biopsy blot results through band signal
quantification on ImageJ. In standard RNA-seq, all RNA submitted is ultimately reverse
transcribed into double stranded cDNA. However, this means that one of the cDNA strands
represents sense mRNA while the other cDNA strand represents antisense mRNA. Antisense
transcripts may be non-coding but play critical roles in transcriptome regulation (He, Vogelstein,
Velculescu, Papadopoulos, & Kinzler, 2008). With standard sequencing, the PCR amplification
step cannot differentiate between sense and antisense strands and devalues the output. This can
lead to an inaccurate representation of the transcriptome (Mills, Kawahara, & Janitz, 2013). As
Aqp9 deletion appears to relay subtle differences in endochondral ossification, a cleaner
transcriptome analysis may be required. Hence, creating a strand-specific cDNA library through
strand marking or using strand-specific RNA-seq should be considered.
7.5 Therapeutic strategies
The current treatment for LLDs are primarily limited to invasive surgical interventions. Aside
from resecting bone from the longer limb or applying a lengthening fixator to the shorter limb,
bone growth can be temporarily or permanently arrested through stapling the epiphyseal plate.
Epiphysiodesis is the process of guiding bone growth through inhibition of the epiphyseal plate
until a deformity has been corrected (Gottliebsen et al., 2013). Traditionally, stapling the plate
longitudinally prevents that area from ossifying properly and can be used to correct angular
deformities and LLDs (Blount & Clarke, 1949). Stapling is well-established and has been used
for many decades as a safe intervention for inhibiting growth (Raab, Wild, Seller, & Krauspe,
2001). This process has also been developed into tension band plating, where the staple is
replaced with a non-rigid plate-and-screw apparatus that better shifts epiphyseal load and
improves correction time (Stevens, 2007). Permanent epiphysiodesis can also be achieved
133
through the archaic Phemister technique, where a portion containing epiphyseal plate and
surrounding bone are resected and reinserted into the joint with the ends reversed (Gottliebsen,
Shiguetomi-Medina, Rahbek, & Møller-Madsen, 2016). Nevertheless, the discovery of non-
invasive therapies has not been well-explored. Epiphysiodesis was achieved in rabbits through
radiofrequency delivery to their epiphyseal plates with minimal pain and postoperative
complication (Ghanem et al., 2009). Recombinant human BMP2 has also been used in
orthopedic settings to induce bone formation through biomaterial delivery (Wang et al., 1990;
Khan & Lane, 2004; Agrawal & Sinha, 2017). Consuming recombinant human growth hormone
can also rescue the effects of idiopathic short stature in children (Sotos & Tokar, 2014). As Aqp9
may be a negative regulator of long bone growth at the early stages of life, children diagnosed
with dysplasias may benefit from its targeted knockdown. Phloretin is a dihydrochalcone derived
from apples, known AQP9 inhibitor, and protector against osteoarthritis (Zheng, Chen, Zhang,
Cai, & Chen, 2018; Geng et al., 2017). Previously, the Kannu lab has shown that mice that
underwent a 60-day phloretin oral gavage treatment exhibited significantly reduced osteoarthritic
severity (Xie et al., 2017). However, the use of phloretin as an epiphyseal plate deliverable has
not been investigated. To test if Aqp9 can be ameliorated and affect endochondral ossification, a
fracture healing experiment can be performed in young mice using phloretin in the treatment
group. Radiography can be used to capture healing rates over a 60-day timespan. qPCR can be
performed on resected epiphyseal plates to measure chondrogenic, angiogenic, and osteogenic
gene expression. In this study, skeletal staining of Aqp9 -/- mice at P5 and P21 suggests that
phloretin would modulate the long bone growth rate.
134
References
Abad, V., Meyers, J. L., Weise, M., Gafni, R. I., Barnes, K. M., Nilsson, O., & Baron, J. (2002). The role of the
resting zone in growth plate chondrogenesis. Endocrinology, 143(5), 1851-7.
Agrawal, V., & Sinha, M. (2017). A review on carrier systems for bone morphogenetic protein-2. J Biomed Mater
Res Part B Appl Biomater, 105(4), 904-925.
Agre, P. (2006). The aquaporin water channels. Proc Am Thorac Soc, 3(1), 5–13.
Agre, P., Preston, G. M., Smith, B. L., Jung, J. S., Raina, S., Moon, C., . . . Nielsen, S. (1993). Aquaporin CHIP: the
archetypal molecular water channel. American Journal of Physiology, 265, F463–476.
Agre, P., Saboori, A. M., Asimos, A., & Smith, B. L. (1987). Purification and partial characterization of the Mr
30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen. Journal of Biological
Chemistry, 262, 17497–17503.
Aharon, R., & Bar-Shavit, Z. (2006). Involvement of aquaporin 9 in osteoclast differentiation. J Biol Chem, 281(28),
19305-9.
Akiyama, H., Chaboissier, M. C., Martin, J. F., Schedl, A., & de Crombrugghe, B. (2002). The transcription factor
Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for
expression of Sox5 and Sox6. Genes Dev, 16(21), 2813-28.
Akiyama, H., Lyons, J. P., Mori-Akiyama, Y., Yang, X., Zhang, R., Zhang, Z., . . . de Crombrugghe, B. (2004).
Interactions between Sox9 and β-catenin control chondrocyte differentiation. Genes Dev, 18, 1072–1087
Almasalmeh, A., Krenc, D., Wu, B., & Beitz, E. (2014). Structural determinants of the hydrogen peroxide
permeability of aquaporins. FEBS J, 281(3), 647-56.
Ambrosi, T. H., Scialdone, A., Graja, A., Gohlke, S., Jank, A., Bocian, C., . . . Schulz, T. J. (2017). Adipocyte
Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and
Bone Regeneration. Cell Stem Cell, 20(6), 771-784.e6.
Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., . . . Ottersen, O. P.
(2005). Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9
isoform in the inner mitochondrial membrane. FASEB J, 19(11), 1459-67.
Anthony, B. A., & Link, D. C. (2014). Regulation of hematopoietic stem cells by bone marrow stromal cells. Trends
Immunol, 35(1), 32-7.
Arnold, M. A., Kim, Y., Czubryt, M. P., Phan, D., McAnally, J., Qi, X., . . . Olson, E. N. (2007). MEF2C
transcription factor controls chondrocyte hypertrophy and bone development. Dev Cell, 12, 377–389.
Avtanski, D., Novaira, H. J., Wu, S., Romero, C. J., Kineman, R., Luque, R. M., . . . Radovick, S. (2014). Both
estrogen receptor α and β stimulate pituitary GH gene expression. Mol Endocrinol, 28(1), 40-52.
135
Aybar Odstrcil, A. C., Carino, S. N., Ricci, J. C., & Mandalunis, P. M. (2010). Effect of arsenic in endochondral
ossification of experimental animals. Exp Toxicol Path, 62(3), 243-9.
Badaut, J., & Regli, L. (2004). Distribution and possible roles of aquaporin 9 in the brain. Neuroscience, 129(4),
971-81.
Balwierz, P. J., Pachkov, M., Arnold, P., Gruber, A. J., Zavolan, M., & van Nimwegen, E. (2014). ISMARA:
automated modeling of genomic signals as a democracy of regulatory motifs. Genome Res, 24(5), 869–884.
Bandyopadhyay, A., Tsuji, K., Cox, K., Harfe, B. D., Rosen, V., & Tabin, C. J. (2006). Genetic analysis of the roles
of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet, 2(12), e216.
Barcroft, L. C., Offenberg, H., Thomsen, P., & Watson, A. J. (2003). Aquaporin proteins in murine trophectoderm
mediate transepithelial water movements during cavitation. Dev Biol, 256(2), 342-54.
Barham, G., & Clarke, N. M. (2008). Genetic regulation of embryological limb development with relation to
congenital limb deformity in humans. J Child Orthop, 2(1), 1-9.
Barna, M., & Niswander, L. (2007). Visualization of cartilage formation: insight into cellular properties of skeletal
progenitors and chondrodysplasia syndromes. Dev Cell, 12(6), 931-41.
Barrett-Jolley, R., Lewis, R., Fallman, R., & Mobasheri, A. (2010). The emerging chondrocyte channelome. Front
Physiol, 1, 135.
Beederman, M., Lamplot, J. D., Nan, G., Wang, J, Liu, X., Yin, L., Li, R., . . . He, T. (2013). BMP signaling in
mesenchymal stem cell differentiation and bone formation. J Biomed Sci Eng, 6(8A), 32–52.
Bell, E. L., Klimova, T. A., Eisenbart, J., Schumacker, P. T., & Chandel, N. S. (2007). Mitochondrial reactive
oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during
hypoxia. Mol Cell Biol, 27(16), 5737-45.
Belluoccio, D., Bernardo, B. C., Rowley, L., & Bateman, J. F. (2008). A microarray approach for comparative
expression profiling of the discrete maturation zones of mouse growth plate cartilage. Biochim Biophys Acta,
1779(5), 330-40.
Belluoccio, D., Etich, J., Rosenbaum, S., Frie, C., Grskovic, I., Stermann, J., . . . Brachvogel, B. (2010). Sorting of
growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status.
J Bone Miner Res, 25(6), 1267-81.
Benjamin, M., Archer, C. W., & Ralphs, J. R. (1994). Cytoskeleton of cartilage cells. Microsc Res Tech, 28(5), 372-
7.
Berendsen, A. D., & Olsen, B. R. (2015). Bone development. Bone, 80, 14-18.
Bi, W., Deng, J. M., Zhang, Z., Behringer, R. R., & de Crombrugghe, B. (1999). Sox9 is required for cartilage
formation. Nat Genet, 22(1), 85-9.
136
Bi, W., Huang, W., Whitworth, D. J., Deng, J. M., Zhang, Z., Behringer, R. R., & de Crombrugghe, B. (2001).
Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization.
Proc Natl Acad Sci U S A, 98(12), 6698-703.
Bienert, G. P., Møller, A. L., Kristiansen, K. A., Schulz, A., Møller, I. M., Schjoerring, J. K., & Jahn, T. P. (2007).
Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J Biol Chem, 282(2),
1183-92.
Bienert, G. P., Schjoerring, J. K., & Jahn, T. P. (2006). Membrane transport of hydrogen peroxide. Biochim Biophys
Acta, 1758(8), 994-1003.
Blount, W. P., & Clarke, G. R. (1949). Control of bone growth by epiphyseal stapling; a preliminary report. J Bone
Joint Surg Am, 31A(3), 464-78.
Bobick, B. E., Cobb, J. (2012). Shox2 regulates progression through chondrogenesis in the mouse proximal limb. J
Cell Sci, 125(Pt 24), 6071-83.
Bond, S. R., Lau, A., Penuela, S., Sampaio, A. V., Underhill, T. M., Laird, D. W., & Naus, C. C. (2011). Pannexin 3
is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes. J Bone Miner
Res, 26(12), 2911-22.
Bouleftour, W., Boudiffa, M., Wade-Gueye, N. M., Bouët, G., Cardelli, M., Laroche, N., . . . Malaval, L. (2014).
Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and
progressive establishment of high trabecular bone mass. PLoS ONE, 9(5), e95144.
Bourayou, R., Giabicani, E., Pouillot, Brailly-Tabard, S., & Brauner, R. (2015). Premature Pubarche before One
Year of Age: Distinguishing between Mini-Puberty Variants and Precocious Puberty. Med Sci Monit, 21,
955–963.
Boveris, A., & Cadenas, E. (2000). Mitochondrial production of hydrogen peroxide regulation by nitric oxide and
the role of ubisemiquinone. IUBMB Life, 50(4-5), 245-50.
Brady, R. J., Dean, J. B., Skinner, T. M., & Gross, M. T. (2003). Limb length inequality: clinical implications for
assessment and intervention. J Orthop Sports Phys Ther, 33(5), 221-34.
Brighton, C. T., Sugioka, Y., & Hunt, R. M.( 1973). Cytoplasmic structures of epiphyseal plate chondrocytes.
Quantitative evaluation using electron micrographs of rat costochondral junctions with special reference to
the fate of hypertrophic cells. J Bone Joint Surg Am, 55(4), 771-84.
Brougham, D. I., & Nicol, R. O. (1987). Valgus deformity after proximal tibial fractures in children. J Bone Joint
Surg Br, 69(3), 482.
Bu, G., Shuang, F., Wu, Y., Ren, D., & Hou, S. (2012). AQP9: a novel target for bone loss induced by microgravity.
Biochem Biophys Res Commun, 419(4), 774-8.
Buckwalter, J. A., Mower, D., Ungar, R., Schaeffer, J., & Ginsberg, B. (1986). Morphometric analysis of
chondrocyte hypertrophy. J Bone Joint Surg Am, 68(2), 243-55.
137
Bundschu, K., Knobeloch, K. P., Ullrich, M., Schinke, T., Amling, M., Engelhardt, C. M., . . . Schuh, K. (2005).
Gene disruption of Spred-2 causes dwarfism. J Biol Chem, 280(31), 28572-80.
Burghardt, B., Elkaer, M. L., Kwon, T. H., Rácz, G. Z., Varga, G., Steward, M. C., & Nielsen, S. (2003).
Distribution of aquaporin water channels AQP1 and AQP5 in the ductal system of the human pancreas. Gut,
52(7), 1008-16.
Burova, E. B., Grudinkin, P. S., Bardin, A. A., & Gamaleĭ, N. N. (2001). H2O2-induced activation of transcription
factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2. Tsitologiia, 43(12), 1153-61.
Caetano-Lopes, J., Canhão, H., & Fonseca, J. E. (2007). Osteoblasts and bone formation. Acta Reumatol Port, 32(2),
103-10.
Cai, L., Lei, C., Li, R., Chen, W., Hu, C., Chen, X., & Li, C. (2017). Overexpression of aquaporin 4 in articular
chondrocytes exacerbates the severity of adjuvant-induced arthritis in rats: an in vivo and in vitro study. J
Inflamm (Lond), 14, 6.
Calamita, G., Gena, P., Ferri, D., Rosito, A., Rojek, A., Nielsen, S., . . . Svelto, M. (2012). Biophysical assessment
of aquaporin-9 as principal facilitative pathway in mouse liver import of glucogenetic glycerol. Biol Cell,
104(6), 342-51.
Carel, J. C., Lahlou, N., Roger, M., & Chaussain, J. L. (2004). Precocious puberty and statural growth. Hum Reprod
Update, 10(2), 135-47.
Caron, M. M., Emans, P. J., Cremers, A., Surtel, D. A., Coolsen, M. M., van Rhijn, L. W., & Welting, T. J. (2013).
Hypertrophic differentiation during chondrogenic differentiation of progenitor cells is stimulated by BMP-2
but suppressed by BMP-7. Osteoarthritis Cartilage, 21(4), 604-13.
Chagin, A. S., Chrysis, D., Takigawa, M., Ritzen, E. M., & Sävendahl, L. (2006). Locally produced estrogen
promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation
and decreased apoptosis. J Endocrinol, 188(2), 193-203.
Chagin, A. S., Karimian, E., Sundström, K., Eriksson, E., & Sävendahl, L. (2010). Catch-up growth after
dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones. J Endocrinol. 2010, (1), 21-9.
Chandel, N. S., Mcclintock, D. S., Feliciano, C. E., Wood, T. M., Melendez, J. A., Rodriguez, A. M., &
Schumacker, P. T. (2000). Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-
inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem, 275(33), 25130-8.
Chanprasertyothin, S., Saetung, S., Rajatanavin, R., & Ongphiphadhanakul, B. (2010). Genetic variant in the
aquaporin 9 gene is associated with bone mineral density in postmenopausal women. Endocrine, 38(1), 83-6.
Chau, M., Forcinito, P., Andrade, A. C., Hegde, A., Ahn, S., Lui, J. C., . . . Nilsson, O. (2011). Organization of the
Indian hedgehog--parathyroid hormone-related protein system in the postnatal growth plate. J Mol
Endocrinol, 47(1), 99-107.
Chen, S., Fu, P., Cong, R., Wu, H., & Pei, M. (2015). Strategies to minimize hypertrophy in cartilage engineering
and regeneration. Genes Dis, 2(1), 76-95.
138
Cheng, A., & Genever, P. G. (2011). SOX9 determines RUNX2 transactivity by directing intracellular degradation.
J Bone Miner Res, 26(2), 439.
Claramunt, D., Gil-Peña, H., Fuente, R., García-López, E., Hernández, F. O., Ordoñez, F. A., . . . Santos, F. (2017).
Effects of growth hormone treatment on growth plate, bone, and mineral metabolism of young rats with
uremia induced by adenine. Pediatr Res, 82(1), 148-154.
Cobb, J., Dierich, A., Huss-Garcia, Y., & Duboule, D. (2006). A mouse model for human short-stature syndromes
identifies Shox2 as an upstream regulator of Runx2 during long-bone development. Proc Natl Acad Sci U S
A, 103(12), 4511–4515.
Cohen, D., Bogeat-Triboulot, M. B., Vialet-Chabrand, S., Merret, R., Courty, P. E., Moretti, S., . . . Hummel, I.
(2013). Developmental and environmental regulation of Aquaporin gene expression across Populus species:
divergence or redundancy? PLoS ONE, 8(2), e55506.
Cooper, K. L., Oh, S., Sung, Y., Dasari, R. R., Kirschner, M. W., & Tabin C. J. (2013). Multiple phases of
chondrocyte enlargement underlie differences in skeletal proportions. Nature, 495(7441), 375-8.
Courties, A., Sellam, J., & Berenbaum, F. (2017). Metabolic syndrome-associated osteoarthritis. Curr Opin
Rheumatol, 29(2), 214-222.
Courtland, H. W., Sun, H., Beth-On, M., Wu, Y., Elis, S., Rosen, C. J., & Yakar, S. (2011). Growth hormone
mediates pubertal skeletal development independent of hepatic IGF-1 production. J Bone Miner Res, 26(4),
761-8.
Cutler, G. B. (1997). The role of estrogen in bone growth and maturation during childhood and adolescence. J
Steroid Biochem Mol Biol, 61(3-6), 141-4.
da Silva, I. V., Rodrigues, J. S., Rebelo, I., Miranda, J. P. G., & Soveral, G. (2018) Revisiting the metabolic
syndrome: the emerging role of aquaglyceroporins. Cell Mol Life Sci, 75(11), 1973-1988.
Damiano, A., Zotta, E., Goldstein, J., Reisin, I., & Ibarra, C. (2001). Water channel proteins AQP3 and AQP9 are
present in syncytiotrophoblast of human term placenta. Placenta, 22(8-9), 776-81.
Day, T. F., & Yang, Y. (2008). Wnt and hedgehog signaling pathways in bone development. J Bone Joint Surg Am,
90 Suppl 1, 19-24.
Delise, A. M., Fischer, L., & Tuan, R. S. (2000). Cellular interactions and signaling in cartilage development.
Osteoarthritis Cartilage, 8(5), 309-34.
Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A., & Leder, P. (1996). Fibroblast growth factor receptor 3 is a
negative regulator of bone growth. Cell, 84(6), 911-21.
Deng, Y., Wu, A., Li, P., Li, G., Qin, L., Song, H., & Mak, K. K. (2016). Yap1 Regulates Multiple Steps of
Chondrocyte Differentiation during Skeletal Development and Bone Repair. Cell Rep, 14(9), 2224-2237.
Dibas, A., Yang, M. H., Bobich, J., & Yorio, T. (2007). Stress-induced changes in neuronal Aquaporin-9 (AQP9) in
a retinal ganglion cell-line. Pharmacol Res, 55(5), 378-84.
139
Diebold, I., Flügel, D., Becht, S., Belaiba, R. S., Bonello, S., Hess, J., . . . Görlach, A. (2010). The hypoxia-inducible
factor-2alpha is stabilized by oxidative stress involving NOX4. Antioxid Redox Signal, 13(4), 425-36.
Duque, G. (2007). As a matter of fat: New perspectives on the understanding of age-related bone loss. BoneKEy-
Osteovision, 4, 129–140.
Elkjaer, M., Vajda, Z., Nejsum, L. N., Kwon, T., Jensen, U. B., Amiry-Moghaddam, M., . . . Nielsen, S. (2000).
Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. Biochem Biophys Res Commun,
276(3), 1118-28.
Emons, J., Chagin, A. S., Sävendahl, L., Karperien, M., & Wit, J. M. (2011). Mechanisms of growth plate
maturation and epiphyseal fusion. Horm Res Paediatr, 75(6), 383-91.
Eriksen, E. F. (2010). Cellular mechanisms of bone remodeling. Rev Endocr Metab Disord, 11(4), 219-27.
Eswarakumar, V. P., & Schlessinger, J. (2007). Skeletal overgrowth is mediated by deficiency in a specific isoform
of fibroblast growth factor receptor 3. Proc Natl Acad Sci U S A, 104(10), 3937–3942.
Eswarakumar, V. P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G. M., & Lonai, P. (2002).
The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development, (16), 3783-93.
Fandrey, J., Frede, S., & Jelkmann, W. (1994). Role of hydrogen peroxide in hypoxia-induced erythropoietin
production. Biochem J, 303(Pt 2), 507-10.
Fox, A. J. S., Bedi, A., & Rodeo, S. A. (2009). The basic science of articular cartilage: structure, composition, and
function. Sports Health, 1(6), 461-8.
Gajghate, S., Hiyama, A., Shah, M., Sakai, D., Anderson, D. G., Shapiro, I. M., & Risbud, M. V. (2009). Osmolarity
and intracellular calcium regulate Aquaporin2 expression through TonEBP in nucleus pulposus cells of the
intervertebral disc. J Bone Miner Res, 24(6), 992–1001.
Gao, H., Ren, G., Xu, Y., Jin, C., Jiang, Y., Lin, L., . . . Gui, L. (2011). Correlation between expression of
aquaporins 1 and chondrocyte apoptosis in articular chondrocyte of osteoarthritis. Zhongguo Xiu Fu Chong
Jian Wai Ke Za Zhi, 25(3), 279-84.
Gao, L., Sheu, T. J., Dong, Y., Hoak, D. M., Zuscik, M. J., Schwarz, E. M., . . . Jonason, J. H. (2013). TAK1
regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate
and articular cartilages. J Cell Sci, 126(Pt 24), 5704-13.
Garfinkel, B. P., Arad, S., Le, P. T., Bustin, M., Rosen, C. J., Gabet, Y., & Orly, J. (2015). Proportionate Dwarfism
in Mice Lacking Heterochromatin Protein 1 Binding Protein 3 (HP1BP3) Is Associated With Alterations in
the Endocrine IGF-1 Pathway. Endocrinology, 156(12), 4558-70.
Ge, X., Tsang, K., He, L., Garcia, R. A., Ermann, J., Mizoguchi, F., . . . Aliprantis, A. O. (2016). NFAT restricts
osteochondroma formation from entheseal progenitors. JCI Insight, 1(4), e86254.
Geng, X., Mcdermott, J., Lundgren, J., Liu, L., Tsai, K. J., Shen, J., & Liu, Z. (2017). Role of AQP9 in transport of
monomethyselenic acid and selenite. Biometals, 30(5), 747-755.
140
Ghanem, I., El Hage, S., Diab, M., Saliba, E., Khazzaka, A., Aftimos, G., . . . Kharrat, K. (2009). Radiofrequency
application to the growth plate in the rabbit: a new potential approach to epiphysiodesis. J Pediatr Orthop,
29(6), 629-35.
Glasson, S. S., Chambers, M. G., Van den berg, W. B., & Little C. B. (2010). The OARSI histopathology initiative -
recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthr Cartil, 18 Suppl 3,
S17-23.
Glatt, V., Canalis, E., Stadmeyer, L., & Bouxsein, M. L. (2007). Age-related changes in trabecular architecture
differ in female and male C57BL/6J mice. J Bone Miner Res, 22(8), 1197-207.
Golightly, Y. M., Allen, K. D., Helmick, C. G., Renner, J. B., & Jordan, J. M. (2009). Symptoms of the knee and hip
in individuals with and without limb length inequality. Osteoarthr Cartil, 17(5), 596-600.
Gottliebsen, M., Rahbek, O., Hvid, I., Davidsen, M., Hellfritzsch, M. B., & Møller-Madsen, B. (2013).
Hemiepiphysiodesis: similar treatment time for tension-band plating and for stapling: a randomized clinical
trial on guided growth for idiopathic genu valgum. Acta Orthop, 84(2), 202-6.
Gottliebsen, M., Shiguetomi-Medina, J. M., Rahbek, O., & Møller-Madsen, B. (2016). Guided growth: mechanism
and reversibility of modulation. J Child Orthop, 10(6), 471-477.
Graziano, A. C. E., Avola, R., Pannuzzo, G., & Cardile, V. (2018). Aquaporin1 and 3 modification as a result of
chondrogenic differentiation of human mesenchymal stem cell. J Cell Physiol, 233(3), 2279-2291.
Gurney, B. (2002). Leg length discrepancy. Gait Posture, 15(2), 195-206.
Hall, K. C., & Blobel, C. P. (2012). Interleukin-1 Stimulates ADAM17 through a Mechanism Independent of its
Cytoplasmic Domain or Phosphorylation at Threonine 735. PLoS One, 7(2), e31600.
Hall, K. C., Hill, D., Otero, M., Plumb, D. A., Froemel, D., Dragomir, C. L., . . . Blobel, C. P. (2013). ADAM17
controls endochondral ossification by regulating terminal differentiation of chondrocytes. Mol Cell Biol,
33(16), 3077-90.
Han, D., Canali, R., Rettori, D., & Kaplowitz, N. (2003). Effect of glutathione depletion on sites and topology of
superoxide and hydrogen peroxide production in mitochondria. Mol Pharmacol, 64(5), 1136-44.
Han, Y., & Lefebvre, V. (2008). L-Sox5 and Sox6 drive expression of the aggrecan gene in cartilage by securing
binding of Sox9 to a far-upstream enhancer. Mol Cell Biol, 28(16), 4999-5013.
Haneda, M., Hayashi, S., Matsumoto, T., Hashimoto, S., Takayama, K., Chinzei, N., . . . Kuroda, R. (2018).
Depletion of aquaporin 1 decreased ADAMTS-4 expression in human chondrocytes. Mol Med Rep, 17(4),
4874–4882.
Hara-Chikuma, M., Satooka, H., Watanabe, S., Honda, T., Miyachi, Y., Watanabe, T., & Verkman, A. S. (2015).
Aquaporin-3-mediated hydrogen peroxide transport is required for NF-κB signalling in keratinocytes and
development of psoriasis. Nat Commun, 6, 7454.
141
Hartmann, C., & Tabin, C. J. (2000). Dual roles of Wnt signaling during chondrogenesis in the chicken limb.
Development, 127(14), 3141-59.
Hasler, C. C. (2000). Leg length inequality. Indications for treatment and importance of shortening procedures.
Orthopade, 29(9), 766-74.
Hata, K., Takahata, Y., Murakami, T., & Nishimura, R. (2017). Transcriptional network controlling endochondral
ossification. J Bone Metab, 24(2), 75–82.
Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., . . . von der Mark, K. (2010). SOX9 is a
major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.
Development, 137(6), 901-11.
He, Y., Vogelstein, B., Velculescu, V. E., Papadopoulos, N., & Kinzler, K. W. (2008). The antisense transcriptomes
of human cells. Science, 322(5909), 1855-7.
Hellemans, J., Coucke, P. J., Giedion, A., De Paepe, A., Kramer, P., Beemer, F., & Mortier, G. R. (2003).
Homozygous mutations in IHH cause acrocapitofemoral dysplasia, an autosomal recessive disorder with
cone-shaped epiphyses in hands and hips. Am J Hum Genet, 72(4), 1040-6.
Hill, T. P., Spater, D., Taketo, M. M., Birchmeier, W., & Hartmann, C. (2005). Canonical Wnt/β-catenin signaling
prevents osteoblasts from differentiating into chondrocytes. Dev Cell, 8, 727–738
Ho, M. S., Tsang, K. Y., Lo, R. L., Susic, M., Mäkitie, O., Chan, T. W., . . . Chan, D. (2007). COL10A1 nonsense
and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse
metaphyseal chondrodysplasia type Schmid. Hum Mol Genet, 16(10), 1201-15.
Honsawek, S., Tanavalee, A., Yuktanandana, P., Ngarmukos, S., Saetan, N., & Tantavisut, S. (2010). Dickkopf-1
(Dkk-1) in plasma and synovial fluid is inversely correlated with radiographic severity of knee osteoarthritis
patients. BMC Musculoskelet Disord, 11, 257.
Horner, A., Bishop, N. J., Bord, S., Beeton, C., Kelsall, A. W., Coleman, N., & Compston, J. E. (1999).
Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage.
J Anat, 194, 519–524.
Hossain, M. S., Bergstrom, D. J., & Chen, X. B. (2015). Modelling and simulation of the chondrocyte cell growth,
glucose consumption and lactate production within a porous tissue scaffold inside a perfusion bioreactor.
Biotechnol Rep (Amst), 5, 55–62.
Huang, W., Chung, U. I., Kronenberg, H. M., & de Crombrugghe, B. (2001). The chondrogenic transcription factor
Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral
bones. Proc Natl Acad Sci U S A, 98, 160–165.
Hug, N., Longman, D., & Cáceres, J. F. (2016). Mechanism and regulation of the nonsense-mediated decay
pathway. Nucleic Acids Res, 44(4), 1483-95.
Hughes, A. J., Miyazaki, H., Coyle, M. C., Zhang, J., Laurie, M. T., Chu, D., . . . Gartner, Z. J. (2018). Engineered
tissue folding by mechanical compaction of the mesenchyme. Dev Cell, 44(2), 165-178.e6.
142
Hussain, S. A., Palmer, D. H., Syn, W. K., Sacco, J. J., Greensmith, R. M. D., Elmetwali, T., . . . James, N. D.
(2017). Gene expression profiling in bladder cancer identifies potential therapeutic targets. Int J Oncol, 50(4),
1147-1159.
Hyde, G., Boot-Handford, R. P., & Wallis, G. A. (2008). Col2a1 lineage tracing reveals that the meniscus of the
knee joint has a complex cellular origin. J Anat, 213(5), 531-8.
Iena, F. M., & Lebeck, J. (2018). Implications of Aquaglyceroporin 7 in Energy Metabolism. Int J Mol Sci, 19(1),
154.
Inada, M., Wang, Y., Byrne, M. H., Rahman, M. U., Miyaura, C., López-Otín, C, & Krane, S. M. (2004). Critical
roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification.
Proc Natl Acad Sci U S A, 101(49), 17192-7.
Inada, M., Yasui, T., Nomura, S., Miyake, S., Deguchi, K., Himeno, M., . . . Komori, T. (1999). Maturational
disturbance of chondrocytes in Cbfa1-deficient mice. Dev Dyn, 214(4), 279-90.
Ishibashi, K., Kuwahara, M., Gu, Y., Tanaka, Y., Marumo, F., & Sasaki, S. (1998). Cloning and functional
expression of a new aquaporin (AQP9) abundantly expressed in the peripheral leukocytes permeable to water
and urea, but not to glycerol. Biochem Biophys Res Commun, 244(1), 268-74.
Ishibashi, K., Tanaka, Y., & Morishita, Y. (2014). The role of mammalian superaquaporins inside the cell. Biochim
Biophys Acta, 1840(5), 1507-12.
Isidor, B., Pichon, O., Redon, R., Day-Salvatore, D., Hamel, A., Siwicka, K. A., . . . Le Caignec, C. (2010).
Mesomelia-Synostoses Syndrome Results from Deletion of SULF1 and SLCO5A1 Genes at 8q13. Am J Hum
Genet, 87(1), 95–100.
Iwasaki, M., Le, A. X., & Helms, J. A. (1997). Expression of indian hedgehog, bone morphogenetic protein 6 and
gli during skeletal morphogenesis. Mech Dev, 69(1-2), 197-202.
Jackson, A. R., Huang, C. Y., & Gu, W. Y. (2011). Effect of endplate calcification and mechanical deformation on
the distribution of glucose in intervertebral disc: a 3D finite element study. Comput Methods Biomech
Biomed Engin, 14(2), 195-204.
Jackson, S. J., Andrews, N., Ball, D., Bellantuono, I., Gray, J, Hachoumi, L., … Chapman, K. (2017). Does age
matter? The impact of rodent age on study outcomes. Lab Anim, 51(2), 160-169.
Jacob, A. L., Smith, C., Partanen, J., & Ornitz, D. M. (2006). Fibroblast growth factor receptor 1 signaling in the
osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev Biol, 296(2), 315-28.
James, C. G., Woods, A., Underhill, T. M., & Beier, F. (2006). The transcription factor ATF3 is upregulated during
chondrocyte differentiation and represses cyclin D1 and A gene transcription. BMC Mol Biol, 7, 30.
Jiang, Y., & Tuan, R. S. (2015). Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev
Rheumatol, 11(4), 206-12.
143
Jilka, R. L. (2013). The relevance of mouse models for investigating age-related bone loss in humans. J Gerontol A
Biol Sci Med Sci, 68(10), 1209-17.
Jones, D. C., Schweitzer, M. N., Wein, M., Sigrist, K., Takagi, T., Ishii, S., & Glimcher, L. H. (2010). Uncoupling
of growth plate maturation and bone formation in mice lacking both Schnurri-2 and Schnurri-3. Proc Natl
Acad Sci U S A, 107(18), 8254-8.
Joseph, A., Shur, B. D., & Hess, R. A. (2011). Estrogen, efferent ductules, and the epididymis. Biol Reprod, 84(2),
207-17.
Kannu, P., Weng, A., Poon, R., Ali, S. A., & Ma, H. (2015). Knee deep in water: aquaporin 9 and cartilage
damage. Poster session presented at the Matrix Dynamics Group Seminar, Toronto, ON.
Karlsson, T., Bolshakova, A., Magalhães, M. A., Loitto, V. M., & Magnusson, K. E. (2013). Fluxes of water
through aquaporin 9 weaken membrane-cytoskeleton anchorage and promote formation of membrane
protrusions. PLoS ONE, 8(4), e59901.
Kaufman, K. R., Miller, L. S., & Sutherland, D. H. (1996). Gait asymmetry in patients with limb-length inequality. J
Pediatr Orthop, 16(2), 144-50.
Khan, S. N., & Lane, J. M. (2004). The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in
orthopaedic applications. Expert Opin Biol Ther, 4(5), 741-8.
Khatri, R., Krishnan, S., Roy, S., Chattopadhyay, S., Kumar, V., & Mukhopadhyay, A. (2016). Reactive Oxygen
Species Limit the Ability of Bone Marrow Stromal Cells to Support Hematopoietic Reconstitution in Aging
Mice. Stem Cells Dev, 25(12), 948-58.
Kilborn, S. H., Trudel, G., & Uhthoff, H. (2002). Review of growth plate closure compared with age at sexual
maturity and lifespan in laboratory animals. Contemp Top Lab Anim Sci, 41(5), 21-6.
Klatt, A. R., Nitsche, D. P., Kobbe, B., Mörgelin, M., Paulsson, M., & Wagener, R. (2000). Molecular structure and
tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal
development. J Biol Chem, 275(6), 3999-4006.
Klein, G. L. (2014). Insulin & bone: recent developments. World J Diabetes, 5(1), 14–16.
Knutson, G, A. (2005). Anatomic and functional leg-length inequality: a review and recommendation for clinical
decision-making. Part I, anatomic leg-length inequality: prevalence, magnitude, effects and clinical
significance. Chiropr Osteopat, 13, 11.
Kobayashi, T., Lyons, K. M., McMahon, A. P., & Kronenberg, H. M. (2005). BMP signaling stimulates cellular
differentiation at multiple steps during cartilage development. Proc Natl Acad Sci U S A, 102(50), 18023-7.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., . . . Kishimoto, T. (1997). Targeted
disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.
Cell, 89(5), 755-64.
144
Koyama, E., Yamaai, T., Iseki, S., Ohuchi, H., Nohno, T., Yoshioka, H., . . . Pacifici, M. (1996). Polarizing activity,
Sonic hedgehog, and tooth development in embryonic and postnatal mouse. Dev Dyn, 206(1), 59-72.
Kozono, D., Yasui, M., King, L. S., & Agre, P. (2002). Aquaporin water channels: atomic structure molecular
dynamics meet clinical medicine. J Clin Invest, 109(11), 1395-9.
Kronenberg, H. M. (2003). Developmental regulation of the growth plate. Nature, 423(6937), 332-6.
Kronenberg, H. M., & Chung, U. (2001). The parathyroid hormone-related protein and Indian hedgehog feedback
loop in the growth plate. Novartis Found Symp, 232, 144-5.
Kruse, E., Uehlein, N., & Kaldenhoff, R. (2006). The aquaporins. Genome Biol, 7(2), 206.
Kuriyama, H., Kawamoto, S., Ishida, N., Ohno, I., Mita, S., Matsuzawa, Y., . . . Okubo, K. (1997). Molecular
cloning and expression of a novel human aquaporin from adipose tissue with glycerol permeability. Biochem
Biophys Res Commun, 241(1), 53-8.
Kuriyama, H., Shimomura, I., Kishida, K., Kondo, H., Furuyama, N., Nishizawa, H., . . . Matsuzawa, Y. (2002).
Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin
9. Diabetes, 51(10), 2915-21.
Laforenza, U., Bottino, C., & Gastaldi, G. (2016). Mammalian aquaglyceroporin function in metabolism. Biochim
Biophys Acta, 1858(1), 1-11.
Lai, Z., Yin, H., Cabrera-Pérez, J., Guimaro, M. C., Afione, S., Michael, D. G., . . . Chiorini, J. A. (2016).
Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice. Proc Natl Acad Sci U S A, 113(20),
5694-9.
Lambert, C., Dubuc, J. E., Montell, E., Vergés, J., Munaut, C., Noël, A., & Henrotin, Y. (2014). Gene expression
pattern of cells from inflamed and normal areas of osteoarthritis synovial membrane. Arthritis Rheumatol,
66(4), 960-8.
Land, S. C., & Tee, A. R. (2007). Hypoxia-inducible factor 1alpha is regulated by the mammalian target of
rapamycin (mTOR) via an mTOR signaling motif. J Biol Chem, 282(28), 20534-43.
Le Goff, C., Rogers, C., Le Goff, W., Pinto, G., Bonnet, D., Chrabieh, M., . . . Cormier-Daire, V. (2016).
Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause
Cardiospondylocarpofacial Syndrome. Am J Hum Genet, 99(2), 407-13.
Lebeck, J., Gena, P., O'neill, H., Skowronski, M. T., Lund, S., Calamita, G., & Praetorius, J. (2012). Estrogen
prevents increased hepatic aquaporin-9 expression and glycerol uptake during starvation. Am J Physiol
Gastrointest Liver Physiol, 302(3), G365-74.
Lee, H., & Behringer, R. R. (2007). Conditional Expression of Wnt4 during Chondrogenesis Leads to Dwarfism in
Mice. PLoS One, 2(5), e450.
145
Lee, K., Lanske, B., Karaplis, A. C., Deeds, J. D., Kohno, H., Nissenson, R. A., . . . Segre, G. V. (1996). Parathyroid
hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone
development. Endocrinology, 137(11), 5109-18.
Lee, Y. C., Song, I. W., Pai, Y. J., Chen, S. D., & Chen, Y. T. (2017). Knock-in human FGFR3 achondroplasia
mutation as a mouse model for human skeletal dysplasia. Sci Rep, 7, 43220.
Lefevbre, V., & Bhattaram, P. (2010). Vertebrate skeletogenesis. Curr Top Dev Biol, 90, 291-317.
Lennicke, C., Rahn, J., Lichtenfels, R., Wessjohann, L. A., Seliger, B. (2015). Hydrogen peroxide - production, fate
and role in redox signaling of tumor cells. Cell Commun Signal, 14, 13:39.
Li, X. F., Wang, S. J., Jiang, L. S., & Dai, L. Y. (2012). Gender- and region-specific variations of estrogen receptor
α and β expression in the growth plate of spine and limb during development and adulthood. Histochem Cell
Biol, 137(1), 79-95.
Lian, J. B., McKee, M. D., Todd, A. M., & Gerstenfeld, L. C. (1993). Induction of bone-related proteins, osteocalcin
and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in
vitro. J Cell Biochem, 52(2), 206-19.
Liang, H. T., Feng, X. C., & Ma, T. H. (2008). Water channel activity of plasma membrane affects chondrocyte
migration and adhesion. Clin Exp Pharmacol Physiol, 35(1), 7-10.
Lim, J., Tu, X., Choi, K., Akiyama, H., Mishina, Y, & Long, F. (2015). BMP-Smad4 signaling is required for
precartilaginous mesenchymal condensation independent of Sox9 in the mouse. Dev Biol, 400(1), 132–138.
Lin, S. S., Tzeng, B., Lee, K., Smith, R. J. H., Campbell, K. P., & Chen, C. (2014). Cav3.2 T-type calcium channel
is required for the NFAT-dependent Sox9 expression in tracheal cartilage. Proc Natl Acad Sci U S A,
111(19), E1990–E1998.
Lin, Z., McClure, M. J., Zhao, J., Ramey, A. N., Asmussen, N., Hyzy, S. L., . . . Boyan, B. D. (2018). MicroRNA
Contents in Matrix Vesicles Produced by Growth Plate Chondrocytes are Cell Maturation Dependent. Sci
Rep, 8(1), 3609.
Lindskog, C., Asplund, A., Catrina, A., Nielsen, S., & Rützler. M. (2016). A Systematic Characterization of
Aquaporin-9 Expression in Human Normal and Pathological Tissues. J Histochem Cytochem, 64(5), 287-
300.
List, E. O., Berryman, D. E., Wright-Piekarski, J., Jara, A., & Kopchick, J. J. (2012). The effects of weight cycling
on lifespan in male C57BL/6J mice. Int J Obes (Lond), 37(8), 1088–1094.
Liu, L., Trimarchi, J. R., Navarro, P., Blasco, M. A., & Keefe, D. L. (2003). Oxidative stress contributes to arsenic-
induced telomere attrition, chromosome instability, and apoptosis. J Biol Chem, 278(34), 31998-2004.
Liu, W., Toyosawa, S., Furuichi, T., Kanatani, N., Yoshida, C., Liu, Y., . . . Komori, T. (2001). Overexpression of
Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol,
155(1), 157–166.
146
Liu, Y., Song, L., Wang, Y., Rojek, A., Nielsen, S., Agre, P., & Carbrey, J. M. (2009). Osteoclast differentiation and
function in aquaglyceroporin AQP9-null mice. Biol Cell, 101(3), 133-40.
Liu, Z., Shen, J., Carbrey, J. M., Mukhopadhyay, R., Agre, P., & Rosen, B. P. (2002). Arsenite transport by
mammalian aquaglyceroporins AQP7 and AQP9. Proc Natl Acad Sci U S A, 99(9), 6053-8.
Logan, M. (2003). Finger or toe: the molecular basis of limb identity. Development, 130(26), 6401-10.
Loitto, V. M., Huang, C., Sigal, Y. J., & Jacobson, K. (2007). Filopodia are induced by aquaporin-9 expression. Exp
Cell Res, 313(7), 1295-306.
Long, F., & Ornitz, D. M. (2013). Development of the endochondral skeleton. Cold Spring Harb Perspect Biol, 5(1),
a008334.
Long, F., Schipani, E., Asahara, H., Kronenberg, H., & Montminy, M. (2001). The CREB family of activators is
required for endochondral bone development. Development, 128, 541–550.
Lui, J. C., & Baron, J. (2011). Mechanisms limiting body growth in mammals. Endocr Rev, 32(3), 422-40.
Lui, J. C., Nilsson, O., & Baron, J. (2011). Growth plate senescence and catch-up growth. Endocr Dev, 21, 23-9.
Ma, H., Vi, L., Whetstone, H., Kannu, P., & Alman, B. (2014). Identifying β-catenin targets in osteoarthritis. Poster
session presented at the 2014 Clinical and Metabolics Research Day, Toronto, ON.
Mackie, E. J., Ahmed, Y. A., Tatarczuch, L., Chen, K. S., & Mirams, M. (2008). Endochondral ossification: how
cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol, 40(1), 46-62.
Mansour, S., Offiah, A. C., McDowall, S., Sim, P., Tolmie, J., & Hall, C. (2002). J Med Genet, 39(8), 597-602.
Maor, G., & Karnieli, E. (1999). The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth
in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor. Endocrinology,
140(4), 1841-51.
Marchini, A., Rappold, G., & Schneider, K. U. (2007). SHOX at a glance: from gene to protein. Arch Physiol
Biochem, 113(3), 116-23.
Massagué, J., Seoane, J., & Wotton, D. (2005). Smad transcription factors. Genes Dev, 19(23), 2783-810.
Matsubara, T., Kida, K., Yamaguchi, A., Hata, K., Ichida, F., Meguro, H., . . . Yoneda, T. (2008). BMP2 regulates
Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem, 283(43), 29119-25.
Matsukawa, N., Grzesik, W. J., Takahashi, N., Pandey, K. N., Pang, S., Yamauchi, M., & Smithies, O. (1999). The
natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide
system. Proc Natl Acad Sci U S A, 96(13), 7403–7408.
147
McBride-Gagyi, S. H., Mckenzie, J. A., Buettmann, E. G., Gardner, M. J., & Silva, M. J. (2015). Bmp2 conditional
knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical
loading in adult mice. Bone, 81, 533-543.
McGee S. R., & Narayan, P. (2013). Precocious puberty and Leydig cell hyperplasia in male mice with a gain of
function mutation in the LH receptor gene. Endocrinology, 154(10), 3900-13.
Mead, T. J., & Yutzey, K. E. (2009). Notch pathway regulation of chondrocyte differentiation and proliferation
during appendicular and axial skeleton development. Proc Natl Acad Sci, 106, 14420–14425
Meng, J. H., Ma, X. C., Li, Z. M., & Wu, D. C. (2007). Aquaporin-1 and aquaporin-3 expressions in the temporo-
mandibular joint condylar cartilage after an experimentally induced osteoarthritis. Chin Med J (Engl),
120(24), 2191-4.
Mikic, B., Ferreira, M. P., Battaglia, T. C., & Hunziker, E. B. (2008). Accelerated hypertrophic chondrocyte kinetics
in GDF-7 deficient murine tibial growth plates. J Orthop Res, 26(7), 986-90.
Mills, J. D., Kawahara, Y., & Janitz, M. (2013). Strand-Specific RNA-Seq Provides Greater Resolution of
Transcriptome Profiling. Curr Genomics, 14(3), 173-81.
Mirtz, T. A., Chandler, J. P., & Eyers, C. M. (2011). The effects of physical activity on the epiphyseal growth plates:
a review of the literature on normal physiology and clinical implications. J Clin Med Res, 3(1), 1-7.
Mitchell, N. C., Gould, G. G., Smolik, C. M., Koek, W., & Daws, L. C. (2013). Antidepressant-like drug effects in
juvenile and adolescent mice in the tail suspension test: Relationship with hippocampal serotonin and
norepinephrine transporter expression and function. Front Pharmacol, 4, 131.
Mobasheri, A., & Marples, D. (2004). Expression of the AQP-1 water channel in normal human tissues: a
semiquantitative study using tissue microarray technology. Am J Physiol Cell Physiol, 286(3), C529-37.
Mobasheri, A., Matta, C., Uzielienè, I., Budd, E., Martín-Vasallo, P., & Bernotiene, E. (2018). The chondrocyte
channelome: A narrative review. Joint Bone Spine, pii: S1297-319X(18), 30015-0.
Mobasheri, A., Trujillo, E., Bell, S., Carter, S. D., Clegg, P. D., Martín-Vasallo, P., & Marples, D. (2004).
Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes. Vet J, 168(2),
143-50.
Mobasheri, A., Wray, S., & Marples, D. (2005). Distribution of AQP2 and AQP3 water channels in human tissue
microarrays. J Mol Histol, 36(1-2), 1-14.
Mohlin, S., Hamidian, A., Von stedingk, K., Bridges, E., Wigerup, C., Bexell, D., & Påhlman, S. (2015). PI3K-
mTORC2 but not PI3K-mTORC1 regulates transcription of HIF2A/EPAS1 and vascularization in
neuroblastoma. Cancer Res, 75(21), 4617-28.
Morales-Roselló, J., & Llorens, N. P. (2012). Outcome of fetuses with diagnosis of isolated short femur in the
second half of pregnancy. ISRN Obstet Gynecol, 2012, 268218.
148
Morita, K., Miyamoto, T., Fujita, N., Kubota, Y., Ito, K., Takubo, K., . . . Suda, T. (2007). Reactive oxygen species
induce chondrocyte hypertrophy in endochondral ossification. J Exp Med, 204(7), 1613-23.
Mow, V. C., Kuei, S. C., Lai, W. M., & Armstrong, C. G. (1980). Biphasic creep and stress relaxation of articular
cartilage in compression? Theory and experiments. J Biomech Eng, 102(1), 73-84.
Murakami, S., Balmes, G., McKinney, S., Zhang, Z., Givol, D., & de Crombrugghe, B. (2004). Constitutive
activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the
Fgfr3-deficient mouse phenotype. Genes Dev, 18, 290–305.
Murakami, S., Kan, M., McKeehan, W. L., & de Crombrugghe, B. (2000). Up-regulation of the chondrogenic Sox9
gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl
Acad Sci U S A, 97(3), 1113-8.
Muramatsu, S., Wakabayashi, M., Ohno, T., Amano, K., Ooishi, R., Sugahara, T., . . . Matsuda, A. (2007).
Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation. J Biol
Chem, 282(44), 32158-67.
Murata, K., Mitsuoka, K., Hirai, T., Walz, T., Agre, P., Heymann, J. B., . . . Fujiyoshi, Y. (2000). Structural
determinants of water permeation through aquaporin-1. Nature, 407(6804), 599-605.
Murray, K. J., & Azari, M. F. (2015). Leg length discrepancy and osteoarthritis in the knee, hip and lumbar spine. J
Can Chiropr Assoc, 59(3), 226-37.
Musumeci, G., Leonardi, R., Carnazza, M. L., Cardile, V., Pichler, K., Weinberg, A. M., & Loreto, C. (2013).
Aquaporin 1 (AQP1) expression in experimentally induced osteoarthritic knee menisci: an in vivo and in
vitro study. Tissue Cell, 45(2), 145-52.
Nagahara, M., Waguri-Nagaya, Y., Yamagami, T., Aoyama, M., Tada, T., Inoue, K., . . . Otsuka, T. (2010). TNF-
alpha-induced aquaporin 9 in synoviocytes from patients with OA and RA. Rheumatology (Oxford).
2010;49(5):898-906.
Nakamura, Y., Nawata, M., & Wakitani, S. (2005). Expression profiles and functional analyses of Wnt-related genes
in human joint disorders. Am J Pathol, 167(1), 97-105.
Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J. M., Behringer, R. R., & de Crombrugghe, B. (2002). The
novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone
formation. Cell, 108, 17–29.
Nalin, A. M., Greenlee, T. K., & Sandell, L. J. (1995). Collagen gene expression during development of avian
synovial joints: transient expression of types II and XI collagen genes in the joint capsule. Dev Dyn, 203(3),
352-62.
Nam, J. S., Turcotte, T. J., & Yoon, J. K. (2007). Dynamic expression of R-spondin family genes in mouse
development. Gene Expr Patterns, 7(3), 306-12.
Nicchia, G. P., Frigeri, A., Nico, B., Ribatti, D., & Svelto, M. (2001). Tissue distribution and membrane localization
of aquaporin-9 water channel: evidence for sex-linked differences in liver. J Histochem Cytochem, 49(12),
1547-56.
149
Nilsson, O., Weise, M., Landman, E. B., Meyers, J. L., Barnes, K. M., & Baron, J. (2014). Evidence that estrogen
hastens epiphyseal fusion and cessation of longitudinal bone growth by irreversibly depleting the number of
resting zone progenitor cells in female rabbits. Endocrinology, 155(8), 2892-9.
Nishimoto, S., & Logan, M. P. (2016). Subdivision of the lateral plate mesoderm and specification of the forelimb
and hindlimb forming domains. Semin Cell Dev Biol, 49, 102-8.
Nissenson, R. A. (1998). Parathyroid hormone (PTH)/PTHrP receptor mutations in human chondrodysplasia.
Endocrinology, 139(12), 4753-5.
Nowlan, N. C., Prendergast, P. J., & Murphy, P. (2008). Identification of mechanosensitive genes during embryonic
bone formation. PLoS Comput Biol, 4(12), e1000250.
Nurminskaya, M., & Linsenmayer, T. F. (1996). Identification and characterization of up-regulated genes during
chondrocyte hypertrophy. Dev Dyn, 206, 260–271.
Offiah, A. C., & Hall, C. M. (2003). Radiological diagnosis of the constitutional disorders of bone. As easy as A, B,
C?. Pediatr Radiol, 33(3), 153-61.
Oftadeh, R., Perez-Viloria, M., Villa-Camacho, J. C., Vaziri, A., & Nazarian A. (2015). Biomechanics and
mechanobiology of trabecular bone: a review. J Biomech Eng, 137(1), 0108021–01080215.
Ogawa, H., Hatano, S., Suguira, N., Nagai, N., Sato, T., Shimizu, K., . . . Watanabe, H. (2012). Chondroitin Sulfate
Synthase-2 Is Necessary for Chain Extension of Chondroitin Sulfate but Not Critical for Skeletal
Development. PLoS One, 7(8), e43806.
Oh, S. K., Shin, J. O., Baek, J. I., Lee, J., Bae, J. W., Aknamerddy, H., . . . Lee, K. Y. (2015). Pannexin 3 is required
for normal progression of skeletal development in vertebrates. FASEB J, 29(11), 4473-84.
Okamoto, M., Udagawa, N., Uehara, S., Maeda, K., Yamashita, T., Nakamichi, Y., . . . Kobayashi, Y. (2014).
Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis. Sci Rep, 4, 4493.
Oliveira, C. A., Carnes, K., França, L. R., Hermo, L., & Hess, R. A. (2005). Aquaporin-1 and -9 are differentially
regulated by oestrogen in the efferent ductule epithelium and initial segment of the epididymis. Biol Cell,
97(6), 385-95.
Ong, D. B., Colley, S. M., Norman, M. R., Kitazawa, S., & Tobias, J. H. (2004). Transcriptional regulation of a
BMP-6 promoter by estrogen receptor alpha. J Bone Miner Res, 19(3), 447-54.
Ornitz, D. M. (2005). FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev, 16(2),
205-13.
Ornitz, D. M., & Marie, P. J. (2002). FGF signaling pathways in endochondral and intramembranous bone
development and human genetic disease. Genes Dev, 16(12), 1446-65.
Ortega, N., Behonick, D. J., & Werb, Z. 2004. Matrix remodeling during endochondral ossification. Trends Cell
Biol, 14(2), 86-93.
150
Oshio, K., Binder, D. K., Yang, B., Schecter, S., Verkman, A. S., & Manley, G. T. (2004). Expression of aquaporin
water channels in mouse spinal cord. Neuroscience, 127(3), 685-93.
Oswald, E. S., Chao, P. G., Bulinkski, J. C., Ateshian, G. A., & Hung, C. T. (2008). Dependence of zonal
chondrocyte water transport properties on osmotic environment. Cell Mol Bioeng. 1(4), 339–348.
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Glimour, K. C., Rosewell, I. R., . . . Owen, M. J. (1987). Cbfa1,
a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone
development. Cell, 89(5), 765-71.
Panda, A., Gamanagatti, S., Jana, M., & Gupta, A. K. (2014). Skeletal dysplasias: A radiographic approach and
review of common non-lethal skeletal dysplasias. World J Radiol, 6(10), 808-25.
Pastor-Soler, N. M., Fisher, J. S., Sharpe, R., Hill, E., Van Hoek, A., Brown, D., & Breton, S. (2010). Aquaporin 9
expression in the developing rat epididymis is modulated by steroid hormones. Reproduction, 139(3), 613–
621.
Penuela, S., Bhalla, R., Gong, X. Q., Cowan, K. N., Celetti, S. J., Cowan, B. J., . . . Laird, D. W. (2007). Pannexin 1
and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap
junction proteins. J Cell Sci, 120(Pt 21), 3772-83.
Perlman, R. L. (2016). Mouse models of human disease. Evol Med Public Health, 2016(1), 170–176.
Perry, M. J., McDougall, K. E., Hou, S. C., & Tobias, J. H. (2008). Impaired growth plate function in bmp-6 null
mice. Bone, 42(1), 216-25.
Peters, K. G., Werner, S., Chen, G., & Williams, L. T. (1992). Two FGF receptor genes are differentially expressed
in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development,
114(1), 233-43.
Phinney, D. G., Gray, A. J., Hill, K., & Pandey, A. (2005). Murine mesenchymal and embryonic stem cells express a
similar Hox gene profile. Biochem Biophys Res Commun, 338(4), 1759-65.
Phornphutkul, C., Wu, K. Y., & Gruppuso, P. A. (2006). The role of insulin in chondrogenesis. Mol Cell
Endocrinol, 249(1-2), 107-15.
Pillozzi, S., & Becchetti, A. (2012). Ion channels in hematopoietic and mesenchymal stem cells. Stem Cells Int,
2012, 217910.
Plourde, M., Ubeda, J. M., Mandal, G., Monte-Neto, R. L., Mukhopadhyay, R., & Ouellette, M. (2015). Generation
of an aquaglyceroporin AQP1 null mutant in Leishmania major. Mol Biochem Parasitol, 201(2), 108-11.
Pomès, R., & Roux, B. (1996). Structure and dynamics of a proton wire: a theoretical study of H+ translocation
along the single-file water chain in the gramicidin A channel. Biophys J, 71(1), 19-39.
Qutub, A. A., & Popel, A. S. (2008). Reactive oxygen species regulate hypoxia-inducible factor 1alpha differentially
in cancer and ischemia. Mol Cell Biol, 28(16), 5106-19.
151
Raab, P., Wild, A., Seller, K., & Krauspe, R. (2001). Correction of length discrepancies and angular deformities of
the leg by Blount's epiphyseal stapling. Eur J Pediatr, 160(11), 668-74.
Raczkowski, J. W., Daniszewska, B., & Zolynski, K. (2010). Functional scoliosis caused by leg length discrepancy.
Arch Med Sci, 6(3), 393-8.
Rampersad, S. N. (2012). Multiple applications of Alamar Blue as an indicator of metabolic function and cellular
health in cell viability bioassays. Sensors (Basel), 12(9), 12347-60.
Retting, K. N., Song, B., Yoon, B. S., & Lyons, K. M. (2009). BMP canonical Smad signaling through Smad1 and
Smad5 is required for endochondral bone formation. Development, 136(7), 1093-104.
Richardson, S. M., Knowles, R., Marples, D., Hoyland, J. A., & Mobasheri, A. (2008). Aquaporin expression in the
human intervertebral disc. J Mol Histol, 39(3), 303-9.
Rigueur, D., & Lyons, K. M. (2014). Whole-mount skeletal staining. Methods Mol Biol, 1130, 113-121.
Rodríguez, A., Catalán, V., Gómez-Ambrosi, J., & Frühbeck, G. (2006). Role of aquaporin-7 in the
pathophysiological control of fat accumulation in mice. FEBS Lett, 580(20), 4771-6.
Rodríguez, A., Gena, P., Méndez-Giménez, L., Rosito, A., Valenti, V., Rotellar, F., . . . Frühbeck, G. (2014).
Reduced hepatic aquaporin-9 and glycerol permeability are related to insulin resistance in non-alcoholic fatty
liver disease. Int J Obes (Lond, 38(9), 1213-20.
Rodriguez-Esteban, C., Tsukui, T., Yonei, S., Magallon, J., Tamura, K., & Izpisua Belmonte, J. C. (1999). The T-
box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature, 398(6730), 814-8.
Rojek, A. M., Skowronski, M. T., Füchtbauer, E. M., Füchtbauer, A. C., Fenton, R. A., Agre, P., . . . Nielsen, S.
(2007). Defective glycerol metabolism in aquaporin 9 (AQP9) knockout mice. Proc Natl Acad Sci U S A,
104(9), 3609-14.
Rosen, C. J., Ackert-Bicknell, C., Rodriguez, J. P., & Pino, A. M. (2009). Marrow fat and the bone
microenvironment: developmental, functional, and pathological implications. Crit Rev Eukaryot Gene Expr,
19(2), 109-24.
Rothert, M., Rönfeldt, D., & Beitz, E. (2017). Electrostatic attraction of weak monoacid anions increases probability
for protonation and passage through aquaporins. The Journal of Biological Chemistry, 292, 9358-9364.
Rux, D. R., & Wellik, D. M. (2017). Hox genes in the adult skeleton: Novel functions beyond embryonic
development. Dev Dyn, 246(4), 310-317.
Sabirov, R. Z., Kurbannazarova, R. S., Melanova, N. R., & Okada, Y. (2013). Volume-sensitive anion channels
mediate osmosensitive glutathione release from rat thymocytes. PLoS One, 8(1), e55646.
Sahni, M., Ambrosetti, D. C., Mansukhani, A., Gertner, R., Levy, D., & Basilico, C. (1999). FGF signaling inhibits
chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev, 13(11),
1361-6.
152
Sahni, M., Raz, R., Coffin, J. D., Levy, D., & Basilico, C. (2001). STAT1 mediates the increased apoptosis and
reduced chondrocyte proliferation in mice overexpressing FGF2. Development, 128(11), 2119-29.
Saito, T., Ciobotaru, A., Bopassa, J. C., Toro, L., Stefani, E., & Eghbali, M. (2009). Estrogen contributes to gender
differences in mouse ventricular repolarization. Circ Res, 105(4), 343-52.
Saito, T., Fukai, A., Mabuchi, A., Ikeda, T., Yano, F., Ohba, S., . . . Kawaguchi, H. (2010). Transcriptional
regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development.
Nat Med, 16(6), 678-86.
Salazar, V. S., Gamer, L. W., & Rosen, V. (2016). BMP signalling in skeletal development, disease and repair. Nat
Rev Endocrinol, 12(4), 203-21.
Sarabipour, S., & Hristova, K. (2016). Mechanism of FGF receptor dimerization and activation. Nat Commun, 7,
10262.
Schaefer, S. L., Johnson, K. A., & O’Brien, R. T. (1995). Compensatory tibial overgrowth following healing of
closed femoral fractures in young dogs. Veterinary and Comparative Orthopaedics and Traumatology,
08(03), 159-162
Schey, K. L., Petrova, R. S., Gletten, R. B., & Donaldson, P. J. (2017). The role of aquaporins in ocular lens
homeostasis. Int J Mol Sci, 18(12), 2693.
Schipani, E., Ryan, H. E., Didrickson, S., Kobayashi, T., Knight, M., & Johnson, R. S. (2001). Hypoxia in cartilage:
HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev, 15(21), 2865-76.
Schlomer, B. J., Feretti, M., Rodriguez, E., Blaschko, S., Cunha, G., & Baskin, L. (2013). Sexual differentiation in
the male and female mouse from days 0 to 21: a detailed and novel morphometric description. J Urol, 190(4
Suppl), 1610-7.
Schmidl, M., Adam, N., Surmann-Schmitt, C., Hattori, T., Stock, M., Dietz, U., . . . von der Mark, K. (2006).
Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in
chondrocytes in vitro and in vivo. J Biol Chem, 281(42), 31790-800.
Schrier, L., Ferns, S. P., Barnes, K. M., Emons, J. A., Newman, E. I., Nilsson, O., & Baron, J. (2006). Depletion of
resting zone chondrocytes during growth plate senescence. J Endocrinol, 189(1), 27-36.
Seifert, A., Werheid, D. F., Knapp, S. M., & Tobiasch, E. (2015). Role of Hox genes in stem cell differentiation.
World J Stem Cells, 7(3), 583-95.
Selvamurugan, N., Kwok, S., Alliston, T., Reiss, M., & Partridge, N. C. (2004). Transforming growth factor-beta 1
regulation of collagenase-3 expression in osteoblastic cells by crosstalk between the Smad and MAPK
signaling pathways and their components, Smad2 and Runx2. J Biol Chem, 279, 19327–19334.
Seto, J., Busse, B., Gupta, H. S., Schäfer, C., Krauss, S., Dunlop, J. W. C., . . . Jahnen-Dechent, W. (2012).
Accelerated growth plate mineralization and foreshortened proximal limb bones in fetuin-A knockout mice.
PLoS ONE, 7(10), e47338.
153
Shao, Z., Poon, R., Vertel, L., Yang, J., Weng, A., Wei, Q., & Kannu, P. (2016). Closing the floodgates: the role of
aquaporin-9 in osteoarthritis. Poster session presented at the SickKids Summer Research Program Annual
Summer Student Symposium, Toronto, ON.
Sherr, C. J., & Roberts, J. M. (2004). Living with or without cyclins and cyclin-dependent kinases. Genes Dev,
18(22), 2699-711.
Shi, S., Zheng, S., Li, X. F., & Liu, Z. D. (2017). The Effect of Estradiol on the Growth Plate Chondrocytes of Limb
and Spine from Postnatal Mice in vitro: The Role of Estrogen-Receptor and Estradiol Concentration. Int J
Biol Sci, 13(1), 100-109.
Shim, K. S. (2015). Pubertal growth and epiphyseal fusion. Ann Pediatr Endocrinol Metab, 20(1), 8-12.
Shim, Y. (2016). An update on the genetic causes of central precocious puberty. Ann Pediatr Endocrinol Metab,
21(2), 66–69.
Shimasaki, M., Kanazawa, Y., Sato, K., Tsuchiya, H., & Ueda, Y. (2018). Aquaporin-1 and -5 are involved in the
invasion and proliferation of soft tissue sarcomas. Pathology - Research and Practice, 214(1), 80-88.
Shu, B., Zhang, M., Xie, R., Wang, M., Jin, H., Hou, W., . . . Chen, D. (2011). BMP2, but not BMP4, is crucial for
chondrocyte proliferation and maturation during endochondral bone development. J Cell Sci, 124(Pt 20),
3428-40.
Sinha, K. M., Yasuda, H., Coombes, M. M., Dent, S. Y., & de Crombrugghe, B. (2010). Regulation of the
osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase. EMBO J, 29,
68–79.
Slootweg, M. C., Swolin, D., Netelenbos, J. C., Isaksson, O. G., & Ohlsson, C. (1997). Estrogen enhances growth
hormone receptor expression and growth hormone action in rat osteosarcoma cells and human osteoblast-like
cells. J Endocrinol, 155(1), 159-64.
Smits, P., Dy, P., Mitra, S., & Lefebvre, V. (2004). Sox5 and Sox6 are needed to develop and maintain source,
columnar and hypertrophic chondrocytes in the cartilage growth plate. J Cell Biol, 164, 747–758.
Smits, P., Li, P., Mandel, J., Zhang, Z., Deng, J. M., Behringer, R. R., de Crombrugghe, B., & Lefebvre, V. (2001).
The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell, 1(2), 277-90.
Somoza, R. A., Welter, J. F., Correa, D., & Caplan, A. I. (2014). Chondrogenic differentiation of mesenchymal stem
cells: challenges and unfulfilled expectations. Tissue Eng Part B Rev, 20(6), 596–608.
Song, M. G., Hwang, S. Y., Park, J. I., Yoon, S., Bae, H. R., & Kwak, J. Y. (2011). Role of aquaporin 3 in
development, subtypes and activation of dendritic cells. Mol Immunol, 49(1-2), 28-37.
Sotos, J. F., & Tokar, N. J. (2014). Growth hormone significantly increases the adult height of children with
idiopathic short stature: comparison of subgroups and benefit. Int J Pediatr Endocrinol, 2014(1), 15.
Später, D., Hill, T. P., O'Sullivan, R. J., Gruber, M., Conner, D. A., & Hartmann, C. (2006). Wnt9a signaling is
required for joint integrity and regulation of Ihh during chondrogenesis. Development, 133(15), 3039-49.
154
Stevens, P, M. (2007). Guided growth for angular correction: a preliminary series using a tension band plate. J
Pediatr Orthop, 27(3), 253-9.
St-Jacques, B., Hammerschmidt, M., & McMahon, A. P. (1999). Indian hedgehog signaling regulates proliferation
and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 13(16), 2072-86.
Stroud, R. M., Miercke, L. J., O'connell, J., Khademi, S., Lee, J. K., Remis, J., . . . Akhavan, D. (2003). Glycerol
facilitator GlpF and the associated aquaporin family of channels. Curr Opin Struct Biol, 13(4), 424-31.
Su, N., Xu, X., Li, C., He, Q., Zhao, L., Li, C., . . . Chen, L. (2010). Generation of Fgfr3 conditional knockout mice.
Int J Biol Sci, 6(4), 327-32.
Su. N., Jin M., Chen, L. (2014). Role of FGF/FGFR signaling in skeletal development and homeostasis: learning
from mouse models. Bone Res, 2, 14003.
Subotnick, S. I. (1981). Limb length discrepancies of the lower extremity (the short leg syndrome). J Orthop Sports
Phys Ther, 3(1), 11-6.
Sun, X., Mariani, F. V., & Martin, G. R. (2002). Functions of FGF signalling from the apical ectodermal ridge in
limb development. Nature, 418(6897), 501-8.
Takarada, T., Hinoi, E., Nakazato, R., Ochi, H., Xu, C., Tsuchikane, A., . . . Yoneda, Y. (2013). An analysis of
skeletal development in osteoblast-specific and chondrocyte-specific runt-related transcription factor-2
(Runx2) knockout mice. J Bone Miner Res, 28(10), 2064-9.
Takata, K., Matsuzaki, T., & Tajika, Y. (2004). Aquaporins: water channel proteins of the cell membrane. Prog
Histochem Cytochem, 39(1), 1-83.
Takegami, Y., Ohkawara, B., Ito, M., Masuda, A., Nakashima, H., Ishiguro, N., & Ohno, K. (2016). R-spondin 2
facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-
catenin signaling in endochondral ossification. Biochem Biophys Res Commun, 473(1), 255-264.
Tchkonia, T., Morbeck, D. E., von Zglinicki, T., van Deursen, J., Lustgarten, J., Scrable, H., . . . Kirkland, J. L.
(2010). Aging cell, 9(5), 667-84.
ten Berge, D., Brugmann, S. A., Helms, J. A., & Nusse, R. (2008). Wnt and FGF signals interact to coordinate
growth with cell fate specification during limb development. Development, 135, 3247–3257.
Torres, E. S., Andrade, C. V., Fonseca, E. C., Mello, M. A., & Duarte, M. E. (2003). Insulin impairs the maturation
of chondrocytes in vitro. Braz J Med Biol Res, 36(9), 1185-92.
Tsang, K. Y., Chan, D., & Cheah, K. S. (2015). Fate of growth plate hypertrophic chondrocytes: death or lineage
extension? Dev Growth Differ, 57(2), 179-92.
Tsukaguchi, H., Shayakul, C., Berger, U. V., Mackenzie, B., Devidas, S., Guggino, W. B., . . . Hediger, M. A.
(1998). Molecular characterization of a broad selectivity neutral solute channel. J Biol Chem, 273(38),
24737-43.
155
Tsukaguchi, H., Weremowicz, S., Morton, C. C., & Hediger, M. A. (1999). Functional and molecular
characterization of the human neutral solute channel aquaporin-9. Am J Physiol, 277(5 Pt 2), F685-96.
Tsumaki, N., Nakase, T., Miyaji, T., Kakuichi, M., Kimura, T., Ochi, T., & Yoshikawa, H. (2002). Bone
morphogenetic protein signals are required for cartilage formation and differently regulate joint development
during skeletogenesis. J Bone Miner Res, 17, 898–906.
Tuljapurkar, S. R., Mcguire, T. R., Brusnahan, S. K., Jackson, J. D., Garvin, K. L., Kessinger, M. A., . . . Sharp, J.
G. (2011). Changes in human bone marrow fat content associated with changes in hematopoietic stem cell
numbers and cytokine levels with aging. J Anat, 219(5), 574-81.
Turner, N., & Grose, R. (2010). Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer,
10, 116–129
Van der kraan, P. M., & Van den berg, W. B. (2012). Chondrocyte hypertrophy and osteoarthritis: role in initiation
and progression of cartilage degeneration? Osteoarthr Cartil, 20(3), 223-32.
Van der weyden, L., Wei, L., Luo, J., Yang, X., Birk, D. E., Adams, D. J., . . . Chen, Q. (2006). Functional knockout
of the matrilin-3 gene causes premature chondrocyte maturation to hypertrophy and increases bone mineral
density and osteoarthritis. Am J Pathol, 169(2), 515-27.
Veldhuis-Vlug, A. G., & Rosen, C. J. (2018). Clinical implications of bone marrow adiposity. J Intern Med, 283(2),
121-139.
Vergara, R., Parada, F., Rubio, S., & Pérez, F. J. (2012). Hypoxia induces H2O2 production and activates
antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene. J Exp Bot, 63(11),
4123-31.
Verkman, A. S., Smith, A. J., Phuan, P. W., Tradtrantip, L., & Anderson, M. O. (2017). The aquaporin-4 water
channel as a potential drug target in neurological disorders. Expert Opin Ther Targets, 21(12), 1161-1170.
Viadiu, H., Gonen, T., & Walz, T. Projection map of aquaporin-9 at 7 A resolution. (2007). J Mol Biol, 367(1), 80-8.
Vivien, D., Petitfrère, E., Martiny, L., Sartelet, H., Galéra, P., Haye, B., & Pujol, J. P. (1993). IPG
(inositolphosphate glycan) as a cellular signal for TGF-beta 1 modulation of chondrocyte cell cycle. J Cell
Physiol, 155(3), 437-44.
Walker, G. D., Fischer, M., Gannon, J., Thompson, R. C., & Oegema, T. R. (1995). Expression of type-X collagen
in osteoarthritis. J Orthop Res, 13(1), 4-12.
Wang, B., Zhao, J., & Zhang, P. (2017). Gene signatures in osteoarthritic acetabular labrum using microarray
analysis. Int J Rheum Dis, 20(12), 1927-1934.
Wang, E. A., Rosen, V., D’Alessandro, J. S., Bauduy, M., Cordes, P., Harada, T., . . . LaPan, P. (1990).
Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A, 87(6),
2220-4.
156
Wang, G. S., & Cooper, T. A. (2007). Splicing in disease: disruption of the splicing code and the decoding
machinery. Nat Rev Genet, 8(10), 749-61.
Wang, K., Yamamoto, H., Chin, J. R., Werb, Z., & Vu, T. H. (2004). Epidermal growth factor receptor-deficient
mice have delayed primary endochondral ossification because of defective osteoclast recruitment. J Biol
Chem, 279(51), 53848-56.
Wang, R. N., Green, J., Wang, Z., Deng, Y., Qiao, M., Peabody, M., . . . Shi, L. L. (2014). Bone Morphogenetic
Protein (BMP) signaling in development and human diseases. Genes Dis, 1(1), 87-105.
Wang, W., Hart, P. S., Piesco, N. P., Lu, X., Gorry, M. C, & Hart, T. C. (2003). Aquaporin expression in developing
human teeth and selected orofacial tissues. Calcif Tissue Int, 72(3), 222-7.
Wang, Y., Zhu, J., & DeLuca, H. F. (2014). Identification of the vitamin D receptor in osteoblasts and chondrocytes
but not osteoclasts in mouse bone. J Bone Miner Res, 29(3), 685-92.
Watanabe, S., Moniaga, C. S., Nielsen, S., & Hara-Chikuma, M. (2016). Aquaporin-9 facilitates membrane transport
of hydrogen peroxide in mammalian cells. Biochem Biophys Res Commun, 471(1), 191-7.
Weinstein, M., Xu, X., Ohyama, K., & Deng, C. X. (1998). FGFR-3 and FGFR-4 function cooperatively to direct
alveogenesis in the murine lung. Development, 125(18), 3615-23.
Weise, M., De-Levi, S., Barnes, K. M., Gafni, R. I., Abad, V., & Baron, J. (2001). Effects of estrogen on growth
plate senescence and epiphyseal fusion. Proc Natl Acad Sci U S A, 98(12), 6871-6.
White, K. E., Cabral, J. M., Davis, S. I., Fishburn, T., Evans, W. E., Ichikawa, S., . . . Econs, M. J. (2005). Mutations
that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation. Am J Hum Genet,
76(2), 361-7.
Wilkie, A. O. (2005). Bad bones, absent smell, selfish testes: the pleiotropic consequences of human FGF receptor
mutations. Cytokine Growth Factor Rev, 16(2), 187-203.
Williams, G. R. (20140. Role of thyroid hormone receptor-α1 in endochondral ossification. Endocrinology, 155(8),
2747-50.
Wilsman, N. J., Farnum, C. E., Leiferman, E. M., Fry, M., & Barreto, C. (1996). Differential growth by growth
plates as a function of multiple parameters of chondrocytic kinetics. J Orthop Res, 14(6), 927-36.
Wilson, D. G., Phamluong, K., Lin, W. Y., Barck, K., Carano, R. A., Diehl, L., . . . Solloway, M. J. (2012).
Chondroitin sulfate synthase 1 (Chsy1) is required for bone development and digit patterning. Dev Biol,
363(2), 413-25.
Wilson, M. E. (1998). Effects of estradiol and exogenous insulin-like growth factor I (IGF-I) on the IGF-I axis
during growth hormone inhibition and antagonism. J Clin Endocrinol Metab, 83(11), 4013-21.
Wu, M., Chen, G., & Li, Y. P. (2016). TGF-β and BMP signaling in osteoblast, skeletal development, and bone
formation, homeostasis and disease. Bone Res, 4, 16009.
157
Wu, Q., Ma, Q., He, C., Wang, C., Gao, S., Hou, X., & Ma, T. (2007). Reduced Bone Mineral Density and Bone
Metabolism in Aquaporin-1 Knockout Mice. Chemical Research in Chinese Universities, 23(3), 297-299.
Wu, Q., Zhang, Y., & Chen, Q. (2001). Indian hedgehog is an essential component of mechanotransduction complex
to stimulate chondrocyte proliferation. J Biol Chem, 276(38), 35290-6.
Xiao, E., Chen, C., & Zhang, Y. (2016). The mechanosensor of mesenchymal stem cells: mechanosensitive channel
or cytoskeleton? Stem Cell Res Ther, 7, 140.
Xie, W., Tang, P., Ahmed, K., Vertel, L., Vi, L., Lawrence, M., Poon, R., & Kannu, P. (2017). An Apple a Day
Keeps The Osteoarthritis Away: Identifying Regulators of Osteoarthritis. Poster session presented at the
SickKids Summer Research Program Annual Summer Student Symposium, Toronto, ON.
Xie, Y., Luo, F., Xu, W., Wang, Z., Sun, X., Xu, M., . . . Chen, L. (2017). FGFR3 deficient mice have accelerated
fracture repair. Int J Biol Sci, 13(8), 1029-1037.
Xing, W., Cheng, S., Wergedal, J., & Mohan, S. (2014). Epiphyseal chondrocyte secondary ossification centers
require thyroid hormone activation of Indian hedgehog and osterix signaling. J Bone Miner Res, 29(10),
2262–2275.
Xu, W., Chen, Q, Liu, C., Chen, J, Xiong, F., & Wu, B. (2017). A novel, complex RUNX2 gene mutation causes
cleidocranial dysplasia. BMC Med Genet, 18, 13.
Yamashita, S., Andoh, M., Ueno-Kudoh, H., Sato, T., Miyaki, S., & Asahara, H. (2009). Exp Cell Res, 315(13),
2231–2240.
Yan, C., & Luo, J. (2010). An analysis of reentrant loops. Protein J, 29(5), 350-4.
Yang, B., Zhao, D., & Verkman, A. S. (2006). Evidence against functionally significant aquaporin expression in
mitochondria. J Biol Chem, 281(24), 16202-6.
Yang, J., Andre, P., Ye, L., & Yang, Y. Z. (2015). The Hedgehog signalling pathway in bone formation. Int J Oral
Sci, 7(2), 73-9.
Yang, J., Safavi, A., Ma, H., Vi, L., Poon, R., Kannu, P., & Alman, B. (2014). Knee deep in water: aquaporin 9 and
cartilage damage. Poster session presented at the SickKids Summer Research Program Annual Summer
Student Symposium, Toronto, ON.
Yang, X., Trehan, S. K., Guan, Y., Sun, C., Moore, D. C., Jayasuriya, C. T., & Chen, Q. (2014). Matrilin-3 inhibits
chondrocyte hypertrophy as a bone morphogenetic protein-2 antagonist. J Biol Chem, 289(50), 34768–34779.
Yao, Y., & Wang, Y. (2013). ATDC5: an excellent in vitro model cell line for skeletal development. J Cell
Biochem, 114(6), 1223-9.
Yazdi, P. G., Su, H., Ghimbovschi, S., Fan, W., Coskun, P. E., Nalbandian, A., . . . Kimonis, V. E. (2013).
Differential gene expression reveals mitochondrial dysfunction in an imprinting center deletion mouse model
of Prader-Willi syndrome. Clin Transl Sci, 6(5), 347-55.
158
Yi, F., Khan, M., Gao, H., Hao, F., Sun, M., Zhong, L., . . . Ma, T. (2012). Increased differentiation capacity of bone
marrow-derived mesenchymal stem cells in aquaporin-5 deficiency. Stem Cells Dev, 21(13), 2495-507.
Yoon, B. S., Ovchinnikov, D. A., Yoshii, I., Mishina, Y., Behringer, R. R., & Lyons, K. M. (2005). Bmpr1a and
Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Proc Natl Acad Sci U S A,
102(14), 5062-7.
Yoshida, C. A., Yamamoto, H., Fujita, T., Furuichi, T., Ito, K., Inoue, K., . . . Komori, T. (2004). Runx2 and Runx3
are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian
hedgehog. Genes Dev, 18, 952–963.
Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N., . . . Ornitz, D. M. (2003). Conditional inactivation of FGF
receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone
growth. Development, 130(13), 3063-74.
Zanatta, A. P., Brouard, V., Gautier, C., Goncalves, R., Bouraïma-Lelong, H., Silva, F. R. M. B., & Delalande, C.
(2017). Interactions between oestrogen and 1α,25(OH)2-vitamin D3 signalling and their roles in
spermatogenesis and spermatozoa functions. Basic Clin Androl, 27, 10.
Zanello, L. P., & Norman, A. W. (2004). Rapid modulation of osteoblast ion channel responses by 1α,25(OH)2-
vitamin D3 requires the presence of a functional vitamin D nuclear receptor. Proc Natl Acad Sci U S A,
101(6), 1589–1594.
Zhang, F., He, Q., Tsang, W. P., Garvey, W. T., Chan, W. Y., & Wan, C. (2014). Insulin exerts direct, IGF-1
independent actions in growth plate chondrocytes. Bone Res, 2, 14012.
Zhang, J., Tan, X., Li, W., Wang, Y., Wang, J., Cheng, X., & Yang, X. (2005). Smad4 is required for the normal
organization of the cartilage growth plate. Dev Biol, 284(2), 311-22.
Zhao, Y. P., Liu, B., Tian, Q. Y., Wei, J. L., Richbourgh, B., & Liu, C. J. (2015). Progranulin protects against
osteoarthritis through interacting with TNF-α and β-Catenin signalling. Ann Rheum Dis, 74(12), 2244-2253.
Zheng, Q., Zhou, G., Morello, R., Chen, Y., Garcia-Rojas, X., & Lee, B. (2003). Type X collagen gene regulation by
Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo. J Cell Biol, 162, 833–
842.
Zheng, W., Chen, C., Zhang, C., Cai, L., & Chen, H. (2018). The protective effect of phloretin in osteoarthritis: an in
vitro and in vivo study. Food Funct, 9(1), 263-278.
Zuloaga, D. G., Zuloaga, K. L., Hinds, L. R., Carbone, D. L., & Handa, R. J. (2014). Estrogen receptor β expression
in the mouse forebrain: age and sex differences. J Comp Neurol, 522(2), 358-71.
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Appendix
Statement of Contributions
My supervisor—Dr. Peter Kannu—and my Program Advisory Committee members—Dr. Brian
Ciruna and Dr. Marco Magalhaes— contributed to the experimental design, data interpretation,
thesis revision, and approval. Liliana Vertel, Kashif Ahmed, Michael Liang, Raymond Poon,
Mushriq Al-Jazrawe, Qingxia Wei, and Chunying Yu contributed to the experimental design and
data interpretation. Dr. Eric Campos, William Scott, Stephanie Tran, Erin Chown, William Xie,
Lisa Vi, Marc Lawrence, Tarimobo Otobo, Archita Srinath, Carlos King, Anh Chu, and members
of the University of Toronto Collaborative Program in Musculoskeletal Science provided
supporting data interpretation and advice.
The Kannu lab and Alman lab organized all mouse cages and maintenance with The Centre for
Phenogenomics (TCP). Genotyping of weaned mice was performed by TCP and Transnetyx, Inc.
Liliana Vertel performed in situ hybridization. TCP employees performed H&E and Toluidine
blue staining. Marco Magalhaes performed H&E staining. Angela Weng performed
immunohistochemistry and Safranin-O staining. Kashif Ahmed assisted with cell culture
troubleshooting. Joseph Yang, Ziyi Shao, and members of the Alman lab performed qPCR and
RNA silencing. Additional dissection tools, reagents, and miscellaneous equipment were
provided by the Wall and Justice labs.
I contributed to the experimental design, genotyped all pups/neonatal mice, performed
superficial mouse measurements, performed skeletal staining, photographed and processed all
images, harvested whole knees and primary chondrocytes, performed cell culture, performed all
statistical analysis, interpreted data, hypothesized a working model, and wrote the thesis.
This study is funded by the Sickkids Restracomp Scholarship and the CIHR CGS-M Program.
