Guigage axonal dans le système nerveux ventral chezDrosophila: rôles du récepteur DRL et de son ligand WNT5

Jean-Maurice Dura Institut de Génétique Humainejmdura@igh.cnrs.fr

Callahan et al., Nature 1995Bonkowsky et al., Nature 1999Yoshikawa et al., Nature 2003

Strong similarities between Drosophila and human

• More than 50% of the 15.000 Drosophila genes have a strong similarity with a human gene.

• Amongst 289 human genes involved in a severe pathology, 177 have a clear homologue in Drosophila. • Numerous molecular pathway are very well conserved (signal transduction; neurotransmitters; etc......).

Crible “enhancer trap” avec P[etau-lacZ] qui permetde bien visualiser les trajets axonaux - crible de 2400nouvelles insertions au hasard.

+/+ anti-HRP drlP[etau-lacZ]/+ anti-gal

200 interneurons / hemisegment 20 interneurons / hemisegment

drlP : insertion - allèle perte de fonction - lacZ exprimé comme drl+ drlPexc : excision précise - allèle sauvagedrlR : délétion - allèle null

drlP/+drlP/+

drlP/drlP drlP/drlR

[+]

[-]

[+]

[-]

10% cross in PCdefasciculation

drlP/drlPexc

[+]

drlR/drlR

[-]

anti-Fas2

anti-DRL

@ 10h

anti-DRL

@ 11.5h

The DRL protein

30% cross in PC

drlP : insertion - allèle perte de fonction - lacZ exprimé comme drl+

drlPexc : excision précise - allèle sauvagedrlR : délétion - allèle nulldrlRed2 : allèle null - lacZ exprimé comme drl+

in yeastGAL4 881 aa: regulator of transcription of genes induced by galactose (GAL10 and GAL1) by directly binding to 4 related 17 bp sites defining an Upstream Activating Sequences (UAS)

in Drosophila (transgenesis via P transposable element)

(no deleterious effect)

eagle-GAL4 + UAS-tau-lacZ +UAS-drl +UAS-drlE:cross in AC + PC cross only in AC cross in AC + PC

drl instructs crossing axons to choose AC: - attractive to AC?

- repulsive to PC?

ap-GAL4/UAS-tau-lacZ ap-GAL4/UAS-tau-lacZ; 2xUAS-drl

drl recognizes a repulsive cue in the PC

ap-GAL4/UAS-tau-lacZ

+ UAS-drl : no crossing - no effect

+ UAS-comm : crossing in AC +PC+ UAS-comm + UAS-drl : crossing only in AC+ UAS-comm + UAS-drlE : crossing in AC +PC

The DRL protein

DRL-myc soluble extracellular domain probe:should correspond to putative DRL ligand

red: DRL-mycgreen: endogenousdrl expression

Looking for the ligand

genetic screen for DRL ligand:suppression of drl gain-of-function80% genome deficiencies screeneg-GAL4 UAS-drl; def/+250 defs one by oneOne particular def is selected, then overlaping defs,and finally producing mutant of interesting gene

Wnt5 transcripts are localized in the PC

WNT5 protein is localized in the PC and in the AC

DRL-myc soluble extracellular domain probe:should correspond to putative DRL ligand

red: DRL-mycgreen: endogenousdrl expression

myc

Embryos stained with BP102 (all axons)

embryos stained for subsets of AC axons:+/Y; drlP/+ +/Y; drlP/drlR Wnt5D7/Y; drlP/+

embryos stained forsubsets of PC axons:Wnt5 mutants are normal

embryos stained for all axons: anti-HRPmidline glia GAL4/UAS-Wnt5;

drl+/drl+ drl-/drl-

Fc

embryos stained with DRL-Fc

DRL-Fc

embryos stained with DRL-Fc

drl- embryos stained with DRL-Fc DRL-Fc immunoprecipitates WNT5