Drosophila

1. variation Drosophila Egg chamber Eggshell (chorion) ? ? Y* The experimental system Cellular responses

2. EGFR/ErbB Signaling Network Invertebrates Vertebrates ErbB3 EGFR ErbB2 EGFR signal diversification • inhibitors ? signal diversification: • heterodimerization

3. The EGFR network and dorsal patterning Amplification Spitz Vein Inhibition Argos Site of Appendage Formation chorion Profile of EGFR activity: WT Initiation Gurken

4. Molecular Genetic Phylogenetic identify targets Create differences = mutant screens Identify differences = other species How can we identify components of the EGFR network?

5. ligand Kek1 Kek1 and feedback regulation of EGFR EGFR kek1 expression ? No overt phenotype ?

6. Kek1 overexpression inhibits receptor signaling ligand Kek1 EGFR

7. WT kek1- WT egfr- egfr-; kek1- BR-C rho aos Evidence that endogenous kek1 inhibits EGFR signaling

8. Profile of EGFR activity: WT Profile of EGFR activity: kek1- What role does Kek1 have in dorsal patterning? Initiation Gurken Amplification Spitz Vein Inhibition Argos Site of Appendage Formation

9. Kek1 inhibits EGFR signaling 1. Mechanism 2. Sequence requirements ligand EGFR Kek1 EGFR

10. Kek1 Kek1 Y* No signaling - ventralized Signaling - dorsalized Kek1 inhibits EGFR signaling 1. Mechanism Ligand sink vs heterodimer EGFR

11. Kek1 EGFR Kek1 inhibits EGFR signaling 2. Sequence requirements ? Domain swaps

12. Kek1 inhibits EGFR signaling 2. Sequence requirements ? The Kek1 LRRs are sufficient for binding EGFR… …but insufficient for full inhibition in vivo! L LIT

13. [GMR-GAL4*] [UAS-kek1*gfp] [GMR-GAL4] [UAS-kek1gfp] ____________________ + ____________________ CyO w; iso2; iso3 EMS EMS Identifying residues important for Kek1/EGFR interaction P suppressor F1

14. Suppressors of Kek1 = egfr alleles Identifying residues important for Kek1/EGFR interaction Knockouts of Kek1

15. Kek1 = a novel inhibitory component of the EGFR network Binding & Inhibition Kek1 EGFR Trafficking & Stability

16. X Mutations in vital gene = lethality Identification of EGFR components Genetic approach Directed mosaics Drosophila Egg chamber dCBP bwk WT

17. wild type EGFR dCBP pnt dCBP mediates feedback inhibition of EGFR signaling

18. dCBP - dCBP mediates feedback inhibition of EGFR signaling wt Initiation Gurken Amplification Spitz Vein Inhibition Argos Site of Appendage Formation chorion

19. amplification inhibition attenuation Rhomboid Kek1 dCBP/Pointed Bwk Argos Egfr mediated epithelial patterning Gurken EGFR Egfr transcriptional targets

20. Identification of EGFR components 1 4 2 2 3 2 4 L.Throckmorton, 1950 Phylogenetic approach 4 latifasciaformis

21. EGFR signaling and appendage patterning What components of the pathway underlie variation in dorsal appendage patterning? Intraspecific analysis Interspecific analysis map natural variation Characterize patterning at molecular level dose-dependent screen ? genes

22. Interspecific analysis: Kek1 A F G B C H D I J E D. melanogaster D. virilis

23. grkHK36 egfrCO + + Intraspecific analysis • Does quantitative variation in the EGFR network exist? • If so, what molecular components underlie this variation? - template for interspecific variation? Appendage variation WT/V0 V1a V1b V2 V3 V4

24. Intraspecific variation in EGFR signaling

25. 3 EGFR signaling and appendage patterning Identifying novel dose-dependent EGFR components grkHK36 egfrCO + +

26. amplification inhibition attenuation Rhomboid Kek1 dCBP/Pointed Bwk Argos Egfr mediated epithelial patterning Gurken EGFR Egfr transcriptional targets

27. Acknowledgements Andrea Cowden Omar Hasnie Matt Rubin Kyle Payne Diego Alvarado Alex Doetsch Tim Evans Christina MacLaren Amy Rice Anne Scherer Fred Derheimer Libbie Eakin Paul Miller Christopher Skipwith Brandon Weasner