Biology 624 - Developmental Genetics

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 Unicellular, with autocellular junction Unicellular, seamless 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, red b-catenin = basolateral, green From Pollack et al, 98; Zegers et al, 03

Upon addition of HGF, some cells form an extension outward on their basolateral side E-cadherin, basolateral Gp135, apical 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

As the lumen begins to reform in the extension, gp135 is re-expressed on the new apical surface E-cadherin, basolateral Gp135, apical From Pollack et al, 98 From Pollack et al, 98

As lumen formation continues, cells re-establish basolateral expression of E-cadherin E-cadherin, basolateral Gp135, apical From Pollack et al, 98

Model derived from this study: Stimulation of migration is the first step in tubulogenesis Apical/basal polarity is transiently lost and then restored Discontinuous lumens form in tubules Cell-cell contacts are retained throughout the process

Apical membrane biogenesis is important For lumen formation in MDCK cells From Lubarsky and Krasnow, 03

Tube formation requires and stabilizes apical-basolateral polarity From Bryant and Mostov, 08

Drosophila Tracheal System • Simple structure • Powerful genetics • Easy observation From Uv et al, 2003

Embryonic Tracheal Development

Drosophila Trachea Has Four Types of Tubes II IV I III From Uv et al, 2003

Drosophila Trachea Has Four Types of Tubes 4-5 µM 1 µM 1 µM or 4-5 µM 0.5 µM 2-5 cells compose the lumen circumference single tube-shaped cells with AJ encircle the lumen doughnut shaped cells with no AJ protrusions from single cells without AJ From Uv 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

Specification of Tracheal Cells 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) From Affolter and Shilo, 2000

Invagination of Tracheal Placode EGFR = epidermal growth factor receptor Spitz=EGFR ligand Rho = 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

Branches of the Drosophila Trachea 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 From Cabernard et al, 2004

Determination of branch identity EGF αPS1 integrin EGF=Epidermal growth factor : GB and DT Wg= wigless: DT Dpp = Decapentaplegic (Transforming growth factor β-like): DB, LT From Affolter M 2002

FGF Signaling Directs Primary Branch Outgrowth bnl/FGF = blue Btl/FGFR in Trachea cells = brown From Cabernard et al, 2004

Primary Branching Requires Bnl/Btl Signaling green = actin-GFP (WT) Red = cells lacking Btl/FGFr From Cabernard et al, 2004

Dpp Signaling is Required for Dorsal Branch Migration in Addition to FGF Signaling Dpp (ligand) Pnt (receptor) Affolter M 2002

Branch Migration Requires Slit/Robo Signaling blue= tracheal lumen Brown =DSRF (GB marker) From Englund et al, 2002

Branch Elongation via Cell Rearrangements Type I Type I Type II Type II From Cabernard et al, 2004

AJ Remodeling during Intercalation Process From Ribeiro C et al 2003

Formation of Specialized Tracheal Cells From Uv et al, 2003

Fusion Process Migration Contact Adhesion Invagination Lumen Formation E-Cadherin Membrane CytoskeletonLumen

DB DT LT btl-lacZ Dys Dysfusion is Expressed in Tracheal Fusion Cells

dys Misexpression Causes Inhibition of Migration Wild type btl-gal4; UAS-dys Mab2A12 Dys

II IV I III From Uv et al, 2003

Larval Tracheal System VB that has ramified to form dozens of fine terminal branches on the gut Ghabrial and Krasnow MA 2003

Terminal branch expansion in response to Hypoxia induced branchless Type IV tube Jarecki J, Johnson E and Krasnow MA 1999

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

Biology 624 - Developmental Genetics