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variation. Drosophila. Egg chamber. Eggshell (chorion). ?. ?. Y*. The experimental system. Cellular responses. EGFR/ErbB Signaling Network. Invertebrates. Vertebrates. ErbB3. EGFR. ErbB2. EGFR. signal diversification • inhibitors ?. signal diversification: • heterodimerization.

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Drosophila

variation

Drosophila

Egg chamber

Eggshell (chorion)

?

?

Y*

The experimental system

Cellular responses


Drosophila

EGFR/ErbB Signaling Network

Invertebrates

Vertebrates

ErbB3

EGFR

ErbB2

EGFR

signal diversification

• inhibitors ?

signal diversification:

• heterodimerization


Drosophila

The EGFR network and dorsal patterning

Amplification

Spitz

Vein

Inhibition

Argos

Site of

Appendage

Formation

chorion

Profile of

EGFR activity: WT

Initiation

Gurken


Drosophila

Molecular

Genetic

Phylogenetic

identify

targets

Create differences

= mutant screens

Identify differences

= other species

How can we identify components of the EGFR network?


Drosophila

ligand

Kek1

Kek1 and feedback regulation of EGFR

EGFR

kek1 expression

?

No overt phenotype ?


Drosophila

Kek1 overexpression inhibits receptor signaling

ligand

Kek1

EGFR


Drosophila

WT

kek1-

WT

egfr-

egfr-; kek1-

BR-C

rho

aos

Evidence that endogenous kek1 inhibits EGFR signaling


Drosophila

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


Drosophila

Kek1 inhibits EGFR signaling

1. Mechanism

2. Sequence requirements

ligand

EGFR

Kek1

EGFR


Drosophila

Kek1

Kek1

Y*

No signaling - ventralized

Signaling - dorsalized

Kek1 inhibits EGFR signaling

1. Mechanism

Ligand sink vs heterodimer

EGFR


Drosophila

Kek1

EGFR

Kek1 inhibits EGFR signaling

2. Sequence requirements ?

Domain swaps


Drosophila

Kek1 inhibits EGFR signaling

2. Sequence requirements ?

The Kek1 LRRs are sufficient

for binding EGFR…

…but insufficient for full inhibition in vivo!

L

LIT


Drosophila

[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


Drosophila

Suppressors of Kek1

= egfr alleles

Identifying residues important for Kek1/EGFR interaction

Knockouts of Kek1


Drosophila

Kek1 = a novel inhibitory component of the EGFR network

Binding

&

Inhibition

Kek1

EGFR

Trafficking

&

Stability


Drosophila

X

Mutations in

vital gene =

lethality

Identification of EGFR components

Genetic approach

Directed mosaics

Drosophila

Egg chamber

dCBP

bwk

WT


Drosophila

wild type

EGFR

dCBP

pnt

dCBP mediates feedback inhibition of EGFR signaling


Drosophila

dCBP -

dCBP mediates feedback inhibition of EGFR signaling

wt

Initiation

Gurken

Amplification

Spitz

Vein

Inhibition

Argos

Site of

Appendage

Formation

chorion


Drosophila

amplification

inhibition

attenuation

Rhomboid

Kek1

dCBP/Pointed

Bwk

Argos

Egfr mediated epithelial patterning

Gurken

EGFR

Egfr transcriptional

targets


Drosophila

Identification of EGFR components

1

4

2

2

3

2

4

L.Throckmorton, 1950

Phylogenetic approach

4

latifasciaformis


Drosophila

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


Drosophila

Interspecific analysis: Kek1

A

F

G

B

C

H

D

I

J

E

D. melanogaster

D. virilis


Drosophila

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


Drosophila

Intraspecific variation in EGFR signaling


Drosophila

3

EGFR signaling and appendage patterning

Identifying novel

dose-dependent

EGFR components

grkHK36 egfrCO

+ +


Drosophila

amplification

inhibition

attenuation

Rhomboid

Kek1

dCBP/Pointed

Bwk

Argos

Egfr mediated epithelial patterning

Gurken

EGFR

Egfr transcriptional

targets


Drosophila

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


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