Guigage axonal dans le système nerveux ventral chez Drosophila: rôles du récepteur DRL et de son ligand WNT5 Jean-Maurice Dura Institut de Génétique Humaine Callahan et al., Nature 1995 Bonkowsky et al., Nature 1999 Yoshikawa et al., Nature 2003

Strong similarities between Drosophila and human More than 50% of the 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 permet de bien visualiser les trajets axonaux - crible de 2400 nouvelles insertions au hasard.

+/+ anti-HRP drl P[etau-lacZ] /+ anti- gal 200 interneurons / hemisegment 20 interneurons / hemisegment

drl P : insertion - allèle perte de fonction - lacZ exprimé comme drl + drl Pexc : excision précise - allèle sauvage drl R : délétion - allèle null

drl P /+ drl P /drl P drl P /drl R [+] [-] [+] [-] 10% cross in PCdefasciculation

drl P /drl Pexc [+] drl R /drl R [-] anti-Fas2 10h 11.5h

The DRL protein

30% cross in PC

drl P : insertion - allèle perte de fonction - lacZ exprimé comme drl + drl Pexc : excision précise - allèle sauvage drl R : délétion - allèle null drl Red2 : allèle null - lacZ exprimé comme drl +

in yeast GAL4 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-drl E: 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-drl E : crossing in AC +PC

The DRL protein

DRL-myc soluble extracellular domain probe: should correspond to putative DRL ligand red: DRL-myc green: endogenous drl expression

Looking for the ligand

genetic screen for DRL ligand: suppression of drl gain-of-function 80% genome deficiencies screen eg-GAL4 UAS-drl; def/+ 250 defs one by one One 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-myc green: endogenous drl expression myc

Embryos stained with BP102 (all axons)

embryos stained for subsets of AC axons: +/Y; drl P /+ +/Y; drl P /drl R Wnt5 D7 /Y; drl P /+

embryos stained for subsets of PC axons: Wnt5 mutants are normal embryos stained for all axons: anti-HRP midline 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-FcDRL-Fc immunoprecipitates WNT5