DrosophilaEgg chamber Eggshell (chorion)

variation

??

The experimental system

Y*Cellular responses

EGFR/ErbB Signaling Network

Vertebrates

EGFR ErbB2 ErbB3

Invertebrates

EGFR

signal diversification• inhibitors ?

signal diversification:• heterodimerization

The EGFR network and dorsal patterningThe EGFR network and dorsal patterning

Profile of EGFR activity: WTInitiation

Gurken

InhibitionArgos

AmplificationSpitzVein

Site ofAppendageFormation

chorion

How can we identify components of the EGFR network?

identify targets

Molecular

Create differences = mutant screens

Genetic Phylogenetic

Identify differences = other species

kek1 expression

Kek1EGFR

ligand

Kek1 and feedback regulation of EGFR

?No overt phenotype ?

Kek1EGFR

ligand

Kek1 overexpression inhibits receptor signaling

Evidence that endogenous kek1 inhibits EGFR signaling

WT kek1-

aos

rho

BR-C

WT egfr- egfr-; kek1-

InitiationGurken

AmplificationSpitzVein

InhibitionArgos

Site ofAppendageFormation

What role does Kek1 have in dorsal patterning?What role does Kek1 have in dorsal patterning?

Profile of EGFR activity: WT

Profile of EGFR activity: kek1-

EGFRKek1EGFR

ligand

1. Mechanism2. Sequence requirements

Kek1 inhibits EGFR signaling

EGFR

Ligand sink vs heterodimer

1. Mechanism

Kek1 inhibits EGFR signaling

Kek1

No signaling - ventralized

Kek1

Y*

Signaling - dorsalized

Kek1 inhibits EGFR signaling

EGFRKek1

2. Sequence requirements ?

LRRs

Ig

N FLANK

C FLANK

Kek2Kek5

Kek6Kek4Kek3

Kek1

EC

IC

Domain swaps

2. Sequence requirements ?

Kek1 inhibits EGFR signaling

EGFRKek1

Kek1 Kek2LLILIT

160

75

180

IP: anti-GFPIB: anti-EGFR

IP and IB: anti-GFP

IB: anti-EGFR

KDa

180

K1 LIT LI L K2K1 K2

The Kek1 LRRs are sufficient for binding EGFR…

L

LIT

…but insufficient for full inhibition in vivo!

____________________CyO

[GMR-GAL4] [UAS-kek1gfp]

suppressor

____________________+

[GMR-GAL4*] [UAS-kek1*gfp]

EMS

w; iso2; iso3

P

F1

Identifying residues important for Kek1/EGFR interaction

EMS

Knockouts of Kek1

Kek1

L136F

G160S

G160D

P329S

IP: anti-GFPIB: anti-EGFR

IP and IB: anti-GFP

IB: anti-EGFR

180 KDa

55

180

160

70

EGFR + + + + +WT

180 KDa

180 KDa

160 KDa

IP GFP

IB -EGFR

Cell lysate IB:-EGFR

IP and IB: GFP

wt SOK3 SOK4 wt SOK3 SOK4 wt SOK3 SOK4EGFR

Kek1-GFP EGFR-GFP KECG

Suppressors of Kek1= egfr alleles

Identifying residues important for Kek1/EGFR interaction

EGFRKek1

Kek1 = a novel inhibitory component of the EGFR network

Trafficking &

Stability

Binding&

Inhibition

Identification of EGFR components Identification of EGFR components Genetic approach

WT bwkdCBP

Directed mosaics

DrosophilaEgg chamberMutations in

vital gene =lethalityX

dCBP mediates feedback inhibition of EGFR signaling dCBP mediates feedback inhibition of EGFR signaling

wild type EGFR dCBP pnt

InitiationGurken

AmplificationSpitzVein

InhibitionArgos

Site ofAppendageFormation

chorion

wt dCBP -

dCBP mediates feedback inhibition of EGFR signaling dCBP mediates feedback inhibition of EGFR signaling

Egfr mediated epithelial Egfr mediated epithelial patterningpatterning

EGFR

Egfr transcriptional targets

Bwk

amplification

Rhomboid Kek1

attenuation

dCBP/Pointed

Argos

inhibition

Gurken

latifasciaformis

Identification of EGFR components Identification of EGFR components

L.Throckmorton, 1950

1144

33

44

2222

22

Phylogenetic approach

44

map natural variation

? genes

Interspecificanalysis

dose-dependent screen

EGFR signaling and appendage patterning

Intraspecificanalysis

Characterize patterningat molecular level

What components of the pathway underlie variation in dorsal appendage patterning?

A

B

C

D

E

F

G

H

I

JD. melanogaster D. virilis

Interspecific analysis: Kek1

grkHK36 egfrCO

+ +

Appendage variation

WT/V0 V1a V1b V2 V3 V4

• Does quantitative variation in the EGFR network exist?• If so, what molecular components underlie this variation?

- template for interspecific variation?

Intraspecific analysis

Intraspecific variation in EGFR signaling

EGFR signaling and appendage patterning

grkHK36 egfrCO

+ +

Identifying novel dose-dependent

EGFR components

0

5

10

15

20

25

30

35

40

45

50

# of Df's

>75% 51-75% 25-50% <25%% WT chorions

Screen for modifiers of EGFR signaling

3

Egfr mediated epithelial Egfr mediated epithelial patterningpatterning

EGFR

Egfr transcriptional targets

Bwk

amplification

Rhomboid Kek1

attenuation

dCBP/Pointed

Argos

inhibition

Gurken

Diego AlvaradoAlex DoetschTim EvansChristina MacLarenAmy RiceAnne Scherer Fred Derheimer

Libbie EakinPaul MillerChristopher SkipwithBrandon Weasner

Andrea CowdenOmar HasnieMatt RubinKyle Payne