Biology 624 - Developmental Genetics Tubular Organs Lecture #8 - Tube Formation I Tube Formation is critical to forming: 1. Lung* 2. Kidney* 3. Mammary gland 4. Blood vessels* 5. Fly trachea 6. C. elegans excretory system (1 cell!) There are three types of tubes: 1. Multicellular 2. Unicellular, with autocellular junction 3. Unicellular, seamless From Nelson, 2003 From Lubarsky and Krasnow, 2003 Processes of Tubulogenesis Processes of Tubulogenesis From Lubarsky and Krasnow, 2003 From Lubarsky and Krasnow, 2003 1
Genetic Programs involved in Tube Formation MDCK Cells in Collagen Gels - HGF +HGF From Nelson, 2003 MDCK in collagen starts with a polarized cyst Upon addition of HGF, some cells form an extension outward on their basolateral side Gp135 = apical, red b-catenin = basolateral, green From Pollack et al, 98; Zegers et al, 03 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 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 E-cadherin, basolateral Gp135, apical From Pollack et al, 98 2
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: 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 important For lumen formation in MDCK cells Tube formation requires and stabilizes apicalbasolateral polarity From Lubarsky and Krasnow, 03 From Bryant and Mostov, 08 Tube formation requires and stabilizes apicalbasolateral polarity Drosophila Tracheal System Simple structure Powerful genetics Easy observation From Bryant and Mostov, 08 3
Embryonic Tracheal Development Drosophila Trachea Has Four Types of Tubes II I IV III 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 Specification of Tracheal Cells Invagination of Tracheal Placode 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) EGFR = epidermal growth factor receptor Spitz=EGFR ligand Rho = Rhomboid (EGF pathway activator) From Affolter and Shilo, 2000 From Affolter and Shilo, 2000 4
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 Determination of branch identity 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 EGF αps1 integrin From Cabernard et al, 2004 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 5
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 Branch Migration Requires Slit/Robo Signaling Dpp (ligand) Pnt (receptor) Affolter M 2002 blue= tracheal lumen Brown =DSRF (GB marker) From Englund et al, 2002 Branch Elongation via Cell Rearrangements AJ Remodeling during Intercalation Process Type I Type I Type II Type II From Cabernard et al, 2004 From Ribeiro C et al 2003 6
Formation of Specialized Tracheal Cells Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation Fusion process Terminal branching Fusion Process Migration Contact Adhesion Dysfusion is Expressed in Tracheal Fusion Cells Invagination Lumen Formation DB DT E-Cadherin Membrane Cytoskeleton Lumen LT btl-lacz dys Misexpression Causes Inhibition of Migration Dys Specification of tracheal cells (tracheal placodes) Invagination of the tracheal placode Wild type DB migration requires Dpp signaling GB migration requires slit-robo signaling Tube elongation through cell intercalation btl-gal4; UAS-dys Fusion process Terminal branching Mab 2A12 Dys 7
Larval Tracheal System II I IV III 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 Model for Patterning of Terminal Branching by Bnl Type IV tube Jarecki J, Johnson E and Krasnow MA 1999 Jarecki J, Johnson E and Krasnow MA 1999 Branch-specific outgrowth is controlled by regional signals and branch identity genes Branch identity dentermination 8