Jean-Maurice Dura, Institut de Génétique Humaine,
Montpellier
jmdura@igh.cnrs.fr
Expression ciblée de gènes chez la Drosophile
1) Charles Darwin
2) in vivo veritas
rendre à César.....
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......).
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, most
of the time)
from Martin Heisenberg
10mm
Srausfeld et al., MRT 2003
Un centre de la
mémoire : les corps
pédonculés
From Waddell and Quinn, 2001
rut- memory defect
rut- ; UAS-rut+ ; brain-GAL4 memory rescue?
Temporal Control ?
GAL80: GAL4 inhibitor
Binding of GAL80 to the carboxy-terminal 30 aa of GAL4 prevents
GAL4-mediated transcriptional activation
Temporal And Regional Expression Targeting
GAL4 mediated misexpression of wild-type genes
Gal4
UAS
Visualise MB
mCD8-GFP
+
yellow
+
white
UAS
gèneX
Induces a MB phenotype
10mm
Srausfeld et al., MRT 2003
7B
Control
Src
Non receptor tyrosine kinase
wild-type
a
p
a
b
b
SrcP1
a
p
p
a
p
bb
Clonal analysis
-/-/-/-
+/-
+/+
+/+
in yeast
FLP recombinase (FLPase)
FRT: FLPase recombination targets
transgenesis in Drosophila
clonal expression
Repressible marker = GFP under GAL4 control
Repressor = GAL80
-/+/-
-/-
viable
lethal
Dscam-/-
Down Syndrome cell adhesion molecule
10mm
Srausfeld et al., MRT 2003
1:1
2:1 + stop short
3:1
2:2
2:0
Vanessa
Developmental axon pruning is a general mechanism required to transform an
immature neural circuit into a mature one.
During Drosophila metamorphosis, larval-specific dendrites and axons of early g
neurons of the mushroom bodies are pruned and replaced by adult-specific
processes
Lee et al., Development 1999
Lee et al., Neuron 2000
Lee et al., Neuron 2000
Lee et al., Neuron 2000
Thummel Dev. Cell 2001
Caractéristiques
de FTZ-F1
FTZ-F1
The nuclear receptor
•
Nuclear steroid hormone receptor.
•
Isolated in a biochemical screen for embryonic proteins binding regulatory
sequences of ftz (Ueda et al Genes and Dev 90) and Alcohol dehydrogenase
(Ayer et al Nucl Ac Res, 93).
•
2 mARN, 2 proteins that differ in N-terminal:
 a 1043 aa in early embryogenesis.
 b 816 aa from late embryo to pre-pupae.
MARCM
-/+/-
cell-autonomous
N
G
N
FLP
Nb
A multi-cellular Nb clone
FLP
Single-cell / two cell clones
Lee et al., Development 1999
The nuclear receptors FTZ-F1 and HR39
1
376
HR39
ADN
441
588
701
LIGAND
ADN
DN
1
510
FTZ-F1
575
803
63 %
1
510
575
803
1027 1043
22%
The nuclear receptors FTZ-F1 and HR39
•
Both proteins have the same target sequences in vitro.
•
Competition between the two receptors for binding to a common DNA
element (Ohno et al., MCB 94).
•
Antagonist role of the two proteins HR39 et FTZ-F1 in vivo?
Boulanger et al., Nature Neuroscience 2011
Nuclear Receptor pathway
TGF-b pathway
ftz-f1
TGF-b / babo
Hr39
[ HR39
]
EcR-B1
+ USP and ecdysone
Neuronal remodeling
Boulanger et al., Nature Neuroscience 2011
-/-
-/-
-/-
+/-
mutant phenotype
mutant phenotype ?
cell-autonomous ?
linotte RTK
The RYK (DRL) protein
10mm
Srausfeld et al., MRT 2003
+/+
+/+
+/+
+/+
drl+ clone in a drl+brain
-/-
drl- clone in a drl+ brain
+/-
-/-
-/-
drl- clone in a drl- brain : phenotype is non-cell autonomous!
10mm
Srausfeld et al., MRT 2003
+/+
a
b
drl-/drla
or
?
b
A drl -/- single cell MARCM clone in a mutant brain
a branch
a branch
dendrites
b branch
midline
cell body
Two different extrinsic (non cell autonomous) phenotypes:
a branch is horizontally misguided
a and b branches do not stop at the midline
So what is DRL doing?
DRL expression surrounds the MBs at 48 h APF.
This is consistent with a role for a axons guidance.
DRL
MB-GFP
WNT5, protéine sécrétée de la famille Wnts est le ligand de DRL
wnt5400/Y; C739 mGFP/+
Conclusion : wnt5 est nécessaire à la pousse axonale?
wnt5400/Y; C739 mGFP/+; UAS-wnt5/+
Conclusion : wnt5 est nécessaire à la pousse axonale des
CP de façon autonome cellulaire.
+/+
wnt5-
a
b
IMARIS
sauvage
Interprétation à partir de
l’ensemble des neurones
wnt5-. Pousse axonal?
a
b
Réalité vue en clones d’un seul neurone wnt5Guidage axonal
Model for the MB a branch guidance
Reynaud et al., appeal submission to Science
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