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Biology 624 - Developmental Genetics. Lecture #8 - Tube Formation I. Tubular Organs. Tube Formation is critical to forming: Lung* Kidney* Mammary gland Blood vessels* Fly trachea C. elegans excretory system (1 cell!). From Nelson, 2003. There are three types of tubes: Multicellular - PowerPoint PPT Presentation
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Biology 624 - Developmental Genetics
Lecture #8 - Tube Formation I
Tubular Organs
Tube Formation is critical to forming:
1.Lung*2.Kidney*3.Mammary gland4.Blood vessels*5.Fly trachea6.C. elegans excretory system (1 cell!)
From Nelson, 2003
There are three types of tubes:
1.Multicellular2.Unicellular, with autocellular
junction3.Unicellular, seamless
From Lubarsky and Krasnow, 2003
Processes of Tubulogenesis
From Lubarsky and Krasnow, 2003
Processes of Tubulogenesis
From Lubarsky and Krasnow, 2003
Genetic Programs involved in Tube Formation
From Nelson, 2003
MDCK Cells in Collagen Gels
- HGF +HGF
MDCK in collagen starts with a polarized cyst
Gp135 = apical, redb-catenin = basolateral, green
From Pollack et al, 98; Zegers et al, 03
Upon addition of HGF, some cellsform an extension outward on their
basolateral side
Gp135, apicalE-cadherin, basolateral
From Pollack et al, 98
The extended cell divides, and cells in the extension express E-cadherin
but not gp135
E-cadherin, basolateral
Gp135, apical
From Pollack et al, 98
From Pollack et al, 98From Pollack et al, 98
As the lumen begins to reform in theextension, gp135 is re-expressed
on the new apical surface
E-cadherin, basolateral
Gp135, apical
From Pollack et al, 98
As lumen formation continues, cells re-establish basolateral expression of
E-cadherin
E-cadherin, basolateral
Gp135, apical
Model derived from this study:
1.Stimulation of migration is the first step in tubulogenesis
2.Apical/basal polarity is transiently lost and then restored
3.Discontinuous lumens form in tubules
4.Cell-cell contacts are retained throughout the process
Apical membrane biogenesis is importantFor lumen formation in MDCK cells
From Lubarsky and Krasnow, 03
Tube formation requires and stabilizes apical-basolateral polarity
From Bryant and Mostov, 08
From Bryant and Mostov, 08
Tube formation requires and stabilizes apical-basolateral polarity
From Uv et al, 2003
Drosophila Tracheal System
•Simple structure
•Powerful genetics
•Easy observation
Embryonic Tracheal Development
From Uv et al, 2003
I
II
III
IV
Drosophila Trachea Has Four Types of Tubes
Drosophila Trachea Has Four Types of Tubes
From Uv et al, 2003
4-5 µM 1 µM 0.5 µM
2-5 cells compose the lumen circumference
single tube-shaped cellswith AJ encircle the lumen
doughnut shaped cells with no AJ
protrusions from single cells without AJ
1 µM or 4-5 µM
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to
FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching
Specification of Tracheal Cells
From Affolter and Shilo, 2000
Trh = trachealess (bHLH PAS transcription factor)Tgo=Tango (bHLH Pas transcription factor, binding partner for Trh)Vvl/Dfr = Ventral veinless-Drifter (POU domain transcription regulator)
Invagination of Tracheal Placode
EGFR = epidermal growth factor receptorSpitz=EGFR ligandRho = Rhomboid (EGF pathway activator)
From Affolter and Shilo, 2000
Model of Tracheal Cell Invagination
Trh, Vvl
Rho (EGF pathway)
Apical Actin Enrichment
Localized Apical Cell Constriction
Ordered Cell invagination
Brodu V and Casanova J 2006
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to
FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching
DB = dorsal branch
DTa/p = dorsal trunk (anterior/posterior)
VB = visceral branch
SB = spiracular branch
LTa/p = lateral trunk (anterior/posterior)LTp is also called GB or ganglionic branch
Branches of the Drosophila Trachea
From Cabernard et al, 2004
From Affolter M 2002
Determination of branch identity
EGF=Epidermal growth factor : GB and DTWg= wigless: DTDpp = Decapentaplegic (Transforming growth factor β-like): DB, LT
EGF αPS1 integrin
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to
FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching
FGF Signaling Directs Primary Branch Outgrowth
From Cabernard et al, 2004
bnl/FGF = blueBtl/FGFR in Trachea cells = brown
Primary Branching Requires Bnl/Btl Signaling
From Cabernard et al, 2004green = actin-GFP (WT)Red = cells lacking Btl/FGFr
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to
FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching
Dpp Signaling is Required for Dorsal Branch Migration in Addition to FGF Signaling
Affolter M 2002
Dpp (ligand)Pnt (receptor)
From Englund et al, 2002
Branch Migration Requires Slit/Robo Signaling
blue= tracheal lumenBrown =DSRF (GB marker)
From Cabernard et al, 2004
Branch Elongation via Cell Rearrangements
Type I Type II Type IIType I
AJ Remodeling during Intercalation Process
From Ribeiro C et al 2003
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to
FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching
From Uv et al, 2003
Formation of Specialized Tracheal Cells
Fusion Process
E-Cadherin Membrane
Cytoskeleton Lumen
Migration Contact Adhesion Invagination Lumen Formation
Dysfusion is Expressed in Tracheal Fusion Cells
DT
DB
LT
btl-lacZ Dys
dys Misexpression Causes Inhibition of Migration
DysMab 2A12
Wild type
btl-gal4; UAS-dys
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• Branch identity determination• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals in addtition to
FGF signaling DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching
From Uv et al, 2003
I
II
III
IV
VB that has ramified to form dozens of fine terminal branches on the gut
Larval Tracheal System
Ghabrial and Krasnow MA 2003
Terminal branch expansion in response to Hypoxia induced branchless
Jarecki J, Johnson E and Krasnow MA 1999 Type IV tube
Model for Patterning of Terminal Branching by Bnl
Jarecki J, Johnson E and Krasnow MA 1999
Steps of Tracheal Tube Formation
• Tracheal sac formation Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode
• General branch outgrowth via FGF signaling• Branch-specific outgrowth is controlled by regional signals and branch
identity genes Branch identity dentermination DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation
• Determination and differentiation of distinct cell types (fusion cell, terminal cell) Fusion process Terminal branching