fig.cox.miami.edu

Background

• Cell suicide

• Construction, maintenance, repair

• All nucleated cells

ghr.nlm.nih.gov

• Vogt 1842

• J.F. Kerr et al (1972)

• Genetically controlled

• Molecular activators

• Evolutionarily conserved

C. Elegans

expasy.org

• 4 functional group genes:

• Ced-3 → caspases

• Ced-4 → Apaf-1

• Ced-9 → Bcl-2

• Egl-1 → BH3

• Human viral, degenerative diseases

• Therapy - cancer

2006 Wikipedia CD Selection

Embryonic development

• Physiologically, genetically controlled

• Malformations

• Different stages, organs

• Sculpts organs – morphogenetic

• Removal of cells - histogenetic

www.ccs.k12.in.us

Appendages• Mesoderm:

• Amount of cells for skeleton

• Between digits

• Chondrogenetic skeletal condensations

• Ectoderm:

• Apical ectodermal ridge (AER)

• Mesenchymal cells

www.tmd.ac.jp

Fish• Many organs

• Sensory organs, brain – morphogenetic

• Fins – epidermal cells, cartilage

• Median fin fold – unpaired dorsal, anal, caudal

• 20 hrs post fertilization (hpf) median fin fold appears

• 22 hpf – distal parts of fin fold, proximally

• 24 hpf – distal tip• Present until 72 hpf• Not morphogenetic

Apoptosis in median fin fold

Cole and Ross, 2001

Hpf Median fin fold

12

14

16

-

-

-

20

22

24

0

11.0 +/- 5.57

7.00 +/- 6.08

30

36

48

13.67 +/- 13.50

20.00 +/- 6.24

12.00 +/- 6.00

60

72

20.33 +/- 8.08

5.00 +/- 2.83

Amphibians

• Not with free digits

• Some anurans, urodeles – absent

• Some salamanders similar to amniotes

• X. laevis – different proliferation rates in digital, interdigital

• Hindlimb – comparable

Reptiles

• Patterns correlate with adult limb morphology

• Turtles – distal interdigital areas

• Lizards – interdigital

• Apoptosis + digit formation 1st in amniotes (Fallon and Cameron, 1977)

• Ectoderm of AER

• Cells undifferentiated, proliferating

• Snakes – massive apoptosis

• Chameleon

• Autopodial cleft

• Specialized interdigital cell death

• Begins early, wide along distal margin

Avians

• Skeletal primordia of limbs

• 2 areas undifferentiated mesenchyme

• Anterior, posterior margins – proximal segment of limb

• Anterior, posterior necrotic zones (ANZ, PNZ)

• Reduction in digit number

www.dls.ym.edu.tw

• Opaque patch (OP) – central mesenchyme

• 2 pieces of zeugopod

• Digit formation - mesenchyme between rays

• Interdigital necrotic zone (INZ)

www.dls.ym.edu.tw

• Constant w/in species

• Different for different species

• Correlate w/ limb morphology

• Free digits – throughout interdigital space

• Webbed feet – distal interdigital

• Free digits + membranous fold – central interdigital

• Inhibited – syndactyly

• AER – spatial, temporal extension of limb

Kingfisher

www.turtletrack.org

Mammals

• Ectodermal – morphogenetic

• AER – postaxial, preaxial margins

• Regression of extreme ends

• Inhibition - polydactyly

www.nature.com

• Postaxial ridge – digit V

• Interdigital – digit IV

• Later, entire length

• Decreases except digit I

www.gsc.riken.go.jp

• Footplate – “foyer preaxial primaire” (fpp)

• Similar to ANZ

• Reduces quantity of preaxial mesodermal cells

• Talpa (mole) – fpp absent, falciform digit

fpp

www.palaeos.com

www.nature.com

• Subridge mesoderm – foyer marginal I (fmI) – preaxial margin

• Digits 1-3 in forelimb

• 1-1/2 digit 2 in hindlimb

• Foyer marginal V (fmV) – postaxial margin

• Digit 5 to border of 4

• Growth of digital buds

• Decrease in influence of ectodermal layer

fmI

fmV

• Interdigital mesoderm – separation of digits

• 2 waves – superficial layer of subectodermal cells

• Between precartilaginous rudiments of phalanges

Mechanisms

• Bone morphogenic proteins (Bmps)

• Transforming growth factor β superfamily

• Bmp-2, 4, 7, 5 – undifferentiated limb mesoderm, interdigital mesoderm, AER

• Coincide with cell death

• High redundancy

• Regulated by Bmp antagonists

• Noggin, gremlin, DAN, Drm

• Gremlin – ducks, down regulated in chicks prior to INZ

• Bmps – limb patterning, regulate chondrogenic differentiation

• Signal – serine/threonine receptor kinase with type I and II receptors

• Binding – association of 2 receptors

• Phosphorylation of type I by II

• Propagation of intracellular signal

www.medscape.com

• Chondrogenic effects – type Ib receptor

• Type Ia – control of apoptosis

• Interdigital induction of Ib – ectopic digit

arthritis-research.com

• Bmps signal through Smads

• Bmp binds to receptor

• Smad cascade – BMP-responding smads 1, 5, 8

• Co-smad 4

• Inhibitory smads 6, 7

• Translocated to nucleus

• Activate transcription

• Also signal through MapK pathway

• Erk, Jnk, p38 kinase mediate Bmp signaling

MapK pathways

e-kisstoth.staff.shef.ac.uk

• Limb – caspase-3, 9, 2

• Death Inducer-Obliterator-1 (DIO-1)

• Growth Arrest Specific1, 2 (Gas1, 2)

• Apaf-1

arthritis-research.com

• Bax – Bcl-2 family – proapoptotic

• Antiapoptotic – Bcl-2, Bcl-x, A1 digital rays not interdigits

• Before apoptosis Bag-1 expresses antiapoptotic protein

• Binds to Bcl-2 in interdigits

• Defender Against apoptotic cell Death (Dad-1)

• Syndactyly

• Fgf signaling – outgrowth of limbs

• Cooperate with Bmps

• Blocked – Bmps do not trigger apoptosis

• Webbed feet of ducks – decrease in Fgf

• Fgfs activate ERK

www.nature.com

• Retinoic acid signaling – limb patterning

• Acts with Bmps – interdigital regions

• Promotes apoptotic effects of Bmps

• Inhibits chondrogenic effects

• Bmps – induce apoptosis, promote cartilage growth

a-b dying cells

c-d macrophage distribution

e-f macrophage specific antibodies

g-h S phase nucei

Dupe et al, 1999

• Internal skeleton – support, locomotion

• Joints classified by structure, degree of movement

• 1. synarthrosis – joined by cartilage

• 2. schizarthrosis – interzone contains single (small #) of cavities

• 3. hemiarthrosis – single joint cavity, elements united around perifery

• 4. eudiarthrosis – separate articulating elements, cavity limited by synovial tissue

• Degree of movement a joint allows:

• 1. Synarthrosis – no movement

• 2. Amphiarthrosis – limited movement

• 3. Diarthrosis – freely movable

• Diarthroidal joints – aquatic to terrestrial life

• Agnatha to Gnathostomata – hinged mandible

• Greater range of prey

• Agnathans – branchial arches

• Mandibular arch + chondrocranium = jaws

• Upper palatoquadrate + lower mandibular cartilage

• Mammals - malleus + incus - diartroidial

• Chondrichthyes

• Charchariniformes, Squaliformes – hemiarthrosis

• Holocephali – more diarthroidial

• Synovial membrane on one side

• More analysis

• Rajidae – diarthroidial

• Arose in cartilaginous fish

• May be lost in elasmobranches

• Osteichthyes – some may have lost diarthroses

• Quadrate/mandibular - microscopic structure

• Polypterus, Protopterus Haines, 1937

• Lepidosteus (longnose gar) –

• Layer of calcified cartilage• Hypertrophic chondrocytes

integrated into bone• Hyaline cartilage• Articular fibrocartilage – birds, mammals• No fibrous capsule – loose

connective tissue• Synovial membrane

bilayered

CCHC

AF

Haines, 1942

• Early dipnoans – bony, supported by overlying cartilage

• Living – secondary modification

• Fins - no diarthrosis

• Synarthrosis – distal, smaller joints

• Schizoarthrosis, hemiarthrosis – proximal, larger joints

• Amia calva (bowfin) – proximal radial/girdle diarthroidal?

• Joint cavity, minimal connective tissue, 2-layered synovium

• Modern bony fish – more diarthroses

• Maneuverability – swimming, feeding

• Larger size – large joints at base of fins

• Tetrapod limbs – diarthroidial

• Urodeles, anura – distal joints synarthroses

• May be secondary modification

• Primitive structure – no joint capsule supporting 2-layered synovial membrane

• Amphibia, reptilia

• Fibrous/fibrocartilaginous articulating surface overlying hyaline cartilage of epiphysis - birds

Crocodile knee

Haines, 1942

• Crocodilus, Sphenodon, lizards – primitive

• Cruciate ligaments, menisci, single joint cavity femur/tibia/fibula

• Chelonians – firm articulation between median condyle of femur/tibia

• Reduction in medial meniscus

• Urodeles – reduced/lost cavity, menisci, ligaments

• Marsupials,monotremes – femora-fibular articulation

• Joint cavity subdivided by connective tissue

• Eutherians – articulation lost

• Femur closely bound to tibia

• Histological features of joint epiphyses

• Bony fish – cartilaginous epiphyses at end of diaphyses

• Articular surfaces fibrous

• Perichondrium?

• Mass of rounded chondrocytes surrounded by cartilage matrix

• Metaphyses – become flattened, hypertrophied

• Matrix may become calcified

• Reabsorbed by elements of bone marrow – marrow processes

• True endochondral ossification

• Calcified cartilage in center of epiphyses of epibranchial bone

• Forerunner of secondary center of ossification

• Closing plate of endochondral bone

• Bony fish – true epiphysis – endochondral growth mechanism

• Early tetrapods – cartilaginous epiphyses

• Lacked secondary centers of ossification

• Chelonia, Crocodilia retain primitive condition

• Modifications for land dwelling:

• Reduction in zone of round cells

• Flattened zone closer to articular surface

• Firmer epiphysis

• Tuatara – most primitive secondary ossification

• Large masses of calcified cartilage

• Greater part of adult epiphysis

• Thin layer of articular cartilage

• Flattened cell zone partitioned into columns

• Division of founder population at top of column

• Progeny lie beneath mother cell

• Mammals – alignment occurs early

• Reptiles/birds – initially not aligned

• Loose alignment in postembryonic development

• Maintained during hypertrophy, calcification

• Only septa between hypertrophied cells calcifies

• Form templates for endochondral bone

• Noncalcified septa broken down by metalloproteinases

• Hypertrophs undergo apoptosis, transdifferentiation into ostoblasts

• Cavity – greater range of motion

• Schizarthrosis – primitive condition?

• Mechanism – differential hyaluronan (HA) synthesis

• Mechanical stimuli

• Glycosaminoglycan HA, CD44 differentially expressed at joint interzone, articular surfaces

• Diphospho-glucose dehydrogenase (UDPGD) increases prior to cavitation in interzone

• Articular surfaces, synovium during cavitation

• UDPGD – UDP-glucuronate – HA • HA synthesis increases at time of

separation• HA + CD44 – adhesion, separation• Depends on concentration• CD44 - interzone, articular surfaces +

increased HA synthesis - separation

• Mechanical strain influences HA synthesis

• More strain – increases HA, UDPGD, CD44

• HA displaced from receptor – fused joints

• Evolution of higher vertebrates

• Ability to respond to mechanical cues of joint motion

• Increase in HA synthesis

• Accumulation between opposing elements

• Terrestrial evolution – reduction in number of joints

• Secondarily aquatic – hyperphalangy

• Increased number of joints

• Rare in terrestrial amniotes

• Early ichthyosaurs – 2-4-4-4-1

• Later up to 30

• Early cetaceans – little hyperphalangy

• Extant – up to 14

• Better maneuverability, navigation

• Digit length, phalange/joint number – AER, Fgfs

• Chick – Fgf8 in AER first switched off over digit IV, then II, then III

• Correlates with phalange number

• White sided dolphin – maintained over digits II and III

• Fgf8 regulated by Shh in ZPA

• Ihh condensing cartilage of digits

• Signals for joint position – mesenchyme posterior to each digit

Scenarios in joint formation

• Long bone elements:

• Cartilage differentiates across joint locations

• Chondrocytes flatten

• Matrix becomes nonchondrogenic – type I, III collagens, little proteoglycan

• Interzone – signaling center, acts on opposing elements

• Tarsals, carpals:

• Chondrogenesis – center of condensations

• Expand through matrix accumulation

• Periphery cells stretch – form boundary perichondrium

• Abut perichondrium of neighboring element

• Interzone – present, not clearly defined

• Secondary cartilaginous joints:

• Bone formation before cartilage

• Mechanical stimulation – progenitor cells in periosteum become chondrogenic

• Form joint with neighboring cartilage element

• GDF-5 - primary joint formation

• Molecular mechanisms – long bones

• Reversal of chondrogenic phenotype

• Blocked prochondrogenic signaling

• Noggin, GDF-5, Chordin inhibit Bmps in interzone

• Bmp-7 – prochondrogenic

• Perichondria of cartilaginous primordia

• Absent at presumptive joint

• Bmp-2, Bmp-4 similar

• GDF-5 – 2 roles in skeletogenesis

• Promotes condensation of mesenchyme

• Promotes proliferation of chondrocytes in epiphysis

• Maintenance, early development of some joints

• Mutant mice – joint missing

• GDF-6 – carpals, tarsals

• Knockouts – no wrist joints

• GDF-5 marks digit joints

• GDF-5/6 knockouts form joints, lost secondarily

• Maintenance of joints

• Contact - fish homologue of GDF-5

• Between dorsal fin and fin radials

• Evolution of joint morphogenesis

• Wnt9a – interzone

• Upstream of GDF-5, CD44, chordin, autotaxin

• Wnt9 – hagfish, thresher shark

• Cux-1 – inhibits chondrogenesis

• Interzone

• Joint number – maintenance of joint specifying signals

• Increased Fgf signaling – loss of joints

• Hyperphalangy – duplication of Wnt9a

• More distal expression due to prolonged survival of AER

• Extended expression of Fgf-8

Fin dermoskeleton

• Unmineralized actinotrichia + segmented, bony lepidotrichia

• Joined by collagenous ligaments

• Evx-1 related to pair-rule

• Precedes joint formation

• Marks developing joints

• May be involved in joint specification