Oocytes on "cell" phones - EGFR signaling in Drosophila
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The experimental system variation Drosophila Egg chamber Eggshell (chorion) ? ? Y* Cellular responses EGFR/ErbB Signaling Network Invertebrates EGFR signal diversification • inhibitors ? Vertebrates EGFR ErbB2 ErbB3 signal diversification: • heterodimerization The EGFR network and dorsal patterning Initiation Gurken Profile of EGFR activity: WT Amplification Spitz Vein Inhibition Argos Site of Appendage Formation chorion How can we identify components of the EGFR network? Molecular identify targets Genetic Create differences = mutant screens Phylogenetic Identify differences = other species Kek1 and feedback regulation of EGFR ligand Kek1 EGFR kek1 expression ? No overt phenotype ? Kek1 overexpression inhibits receptor signaling ligand EGFR Kek1 Evidence that endogenous kek1 inhibits EGFR signaling kek1- WT WT egfr- egfr-; kek1- BR-C rho aos What role does Kek1 have in dorsal patterning? Initiation Gurken Amplification Spitz Vein Inhibition Argos Site of Appendage Formation Profile of EGFR activity: WT Profile of EGFR activity: kek1- Kek1 inhibits EGFR signaling 1. Mechanism 2. Sequence requirements ligand EGFR Kek1 EGFR Kek1 inhibits EGFR signaling 1. Mechanism Ligand sink vs heterodimer EGFR Kek1 Kek1 Y* No signaling - ventralized Signaling - dorsalized Kek1 inhibits EGFR signaling 2. Sequence requirements ? Kek1 Kek2 Kek3 Kek4 Kek5 Kek6 N FLANK Kek1 EGFR LRRs C FLANK Ig EC IC Domain swaps Kek1 inhibits EGFR signaling 2. Sequence requirements ? The Kek1 LRRs are sufficient for binding EGFR… KDa 180 K1 LIT LI L …but insufficient for full inhibition in vivo! K2K1 K2 IP: anti-GFP IB: anti-EGFR L 160 IP and IB: anti-GFP 75 LIT 180 IB: anti-EGFR Identifying residues important for Kek1/EGFR interaction EMS EMS [GMR-GAL4] [UAS-kek1gfp] ____________________ CyO w; iso2; iso3 P suppressor F1 [GMR-GAL4*] [UAS-kek1*gfp] ____________________ + Identifying residues important for Kek1/EGFR interaction Suppressors of Kek1 = egfr alleles Knockouts of Kek1 Kek1 L136F EGFR WT + + G160D G160S + + P329S + Kek1-GFP 180 KDa EGFR wt SOK3 SOK4 KECG EGFR-GFP wt SOK3 SOK4 wt SOK3 SOK4 180 KDa IP GFP IB -EGFR 160 KDa IP and IB: GFP IP: anti-GFP IB: anti-EGFR 70 55 160 180 IP and IB: anti-GFP IB: anti-EGFR 180 KDa Cell lysate IB: -EGFR Kek1 = a novel inhibitory component of the EGFR network Binding & Inhibition Kek1 Trafficking & Stability EGFR Identification of EGFR components Genetic approach Directed mosaics X Drosophila dCBP Mutations in vital gene = lethality WT Egg chamber bwk dCBP mediates feedback inhibition of EGFR signaling wild type EGFR dCBP pnt dCBP mediates feedback inhibition of EGFR signaling wt Initiation Gurken Amplification Spitz Vein Inhibition Argos Site of Appendage Formation chorion dCBP - Egfr mediated epithelial patterning Gurken EGFR amplification Rhomboid inhibition attenuation Kek1 Bwk dCBP/Pointed Egfr transcriptional targets Argos Identification of EGFR components Phylogenetic approach 1 4 2 2 3 2 4 4 latifasciaformis L.Throckmorton, 1950 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 Interspecific analysis: Kek1 A F B G C H D I E J D. melanogaster D. virilis Intraspecific analysis • Does quantitative variation in the EGFR network exist? • If so, what molecular components underlie this variation? - template for interspecific variation? grkHK36 egfrCO + + Appendage variation WT/V0 V1a V1b V2 V3 V4 Intraspecific variation in EGFR signaling EGFR signaling and appendage patterning Identifying novel dose-dependent EGFR components grkHK36 egfrCO + Screen for modifiers of EGFR signaling 50 45 40 35 30 # of Df's 25 20 15 3 10 5 0 >75% 51-75% 25-50% % WT chorions <25% + Egfr mediated epithelial patterning Gurken EGFR amplification Rhomboid inhibition attenuation Kek1 Bwk dCBP/Pointed Egfr transcriptional targets Argos Diego Alvarado Alex Doetsch Tim Evans Christina MacLaren Amy Rice Anne Scherer Brandon Weasner Andrea Cowden Omar Hasnie Matt Rubin Kyle Payne Fred Derheimer Libbie Eakin Paul Miller Christopher Skipwith
