1. Tubular Organs

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

3. Embryonic Tracheal Development

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

5. From Uv et al, 2003 4-5 µM1 µM0.5 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 1 µM or 4-5 µM

6. 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

7. 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)

8. Invagination of Tracheal Placode EGFR = epidermal growth factor receptor Spitz=EGFR ligand Rho = Rhomboid (EGF pathway activator) From Affolter and Shilo, 2000

9. 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

10. 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

11. 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

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

13. 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

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

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

16. Steps in Tracheal Branching Morphogenesis Ghabrial A, Krasnow M

17. Model of the FGF signaling Pathway in the Control of Cell Migration in Drosophila. Bnl: Branchless Btl: Breathless Dof: Downstream of FGF, Stumps Csw: Corkscrew(phosphatase) HSPG: heparan sulfate proteoglycan Petit V, Affolter M

18. 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

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

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

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

22. AJ Remodeling during Intercalation Process From Ribeiro C et al 2003

23. 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

24. From Uv et al, 2003 Formation of Specialized Tracheal Cells

25. Fusion Process E-Cadherin Membrane CytoskeletonLumen MigrationContactAdhesionInvaginationLumen Formation

26. Dysfusion is Expressed in Tracheal Fusion Cells DT DB LT btl-lacZDys

27. Dysfusion is Required for Fusion Process btl-gal4, UAS-actin-GFPbtl-gal4, UAS-actin-GFP ; dys Wild-type dys mutant

28. dys Misexpression Causes Inhibition of Migration DysMab 2A12 Wild type btl-gal4; UAS-dys

29. Dysfusion Regulates Downstream Targets with Various Functions Shotgun and CG13196 (cell adhesion)↑ members only (protein trafficking) ↑ CG15252 (unknown function) ↑ Trachealess (tracheal migration) ↓

30. 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

31. From Uv et al, 2003 I II III IV

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

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

34. Model for Patterning of Terminal Branching by Bnl Jarecki J, Johnson E and Krasnow MA 1999

35. 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