drl

advertisement
Jean-Maurice Dura
jmdura@igh.cnrs.fr
Institut de Génétique Humaine
Guigage axonal dans le système nerveux ventral chez
Drosophila: rôles du récepteur DRL et de son ligand
WNT5
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 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 permet
de bien visualiser les trajets axonaux - crible de 2400
nouvelles insertions au hasard.
+/+
anti-HRP
200 interneurons / hemisegment
drlP[etau-lacZ]/+
anti-gal
20 interneurons / hemisegment
drlP : insertion - allèle perte de fonction - lacZ exprimé comme drl+
drlPexc : excision précise - allèle sauvage
drlR : délétion - allèle null
drlP/+
drlP/+
[+]
defasciculation
[+]
drlP/drlP
drlP/drlR
[-]
[-]
10% cross in PC
drlP/drlPexc
[+]
drlR/drlR
[-]
anti-Fas2
anti-DRL
anti-DRL
@ 10h
@ 11.5h
The DRL protein
30% cross in PC
drlP : insertion - allèle perte de fonction - lacZ exprimé comme drl+
drlPexc : excision précise - allèle sauvage
drlR : délétion - allèle null
drlRed2 : 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
cross in AC + PC
+UAS-drl
cross only in AC
+UAS-drlE:
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
myc
red: DRL-myc
green: endogenous
drl expression
Embryos stained with BP102 (all axons)
embryos stained for subsets of AC axons:
+/Y; drlP/+
+/Y; drlP/drlR
Wnt5D7/Y; drlP/+
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-
DRL-Fc
Fc
embryos stained with DRL-Fc
embryos stained with DRL-Fc
drl- embryos stained with DRL-Fc
DRL-Fc immunoprecipitates WNT5
Download