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.

Présentation au sujet: "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."— Transcription de la présentation:

1 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 jmdura@igh.cnrs.fr Callahan et al., Nature 1995 Bonkowsky et al., Nature 1999 Yoshikawa et al., Nature 2003

2 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......).

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

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

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

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

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

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11 The DRL protein

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13 30% cross in PC

14 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 +

15 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)

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

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21 drl instructs crossing axons to choose AC: - attractive to AC? - repulsive to PC?

22 ap-GAL4/UAS-tau-lacZ ap-GAL4/UAS-tau-lacZ; 2xUAS-drl drl recognizes a repulsive cue in the PC

23 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

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25 The DRL protein

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

27 Looking for the ligand

28 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

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31 Wnt5 transcripts are localized in the PC

32 WNT5 protein is localized in the PC and in the AC

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

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35 Embryos stained with BP102 (all axons)

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

37 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 -

38 Fc embryos stained with DRL-Fc DRL-Fc

39 embryos stained with DRL-Fc

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

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