Expression ciblée de gènes chez la Drosophile Jean-Maurice Dura, Institut de Génétique Humaine, Montpellier 1) Charles Darwin 2) in vivo veritas rendre à César.....

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......).

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

Srausfeld et al., MRT  m

From Waddell and Quinn, 2001 Un centre de la mémoire : les corps pédonculés

rut - ; UAS-rut + ; brain-GAL4 memory rescue? rut - memory defect

Temporal Control ?

Temporal And Regional Expression Targeting GAL80: GAL4 inhibitor Binding of GAL80 to the carboxy-terminal 30 aa of GAL4 prevents GAL4-mediated transcriptional activation

GAL4 mediated misexpression of wild-type genes

UAS yellow + gène X Gal4 white + Visualise MB UAS mCD8-GFP Induces a MB phenotype

Srausfeld et al., MRT  m

7B Src Non receptor tyrosine kinase Control

wild-type p    p  Src P1 p    p

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

Srausfeld et al., MRT  m

1:1 3:1 2:0 2:1 + stop short2:2

Vanessa Lee et al., Development 1999 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  neurons of the mushroom bodies are pruned and replaced by adult-specific processes

Lee et al., Neuron 2000

Thummel Dev. Cell 2001

Caractéristiques de FTZ-F1 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:  1043 aa in early embryogenesis.  816 aa from late embryo to pre-pupae. The nuclear receptor FTZ-F1

-/- +/- cell-autonomous MARCM

Nb G N N FLP A multi-cellular Nb clone Single-cell / two cell clones FLP Lee et al., Development 1999

The nuclear receptors FTZ-F1 and HR39 LIGAND ADN DN % 63 % HR39 FTZ-F LIGAND ADN

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? The nuclear receptors FTZ-F1 and HR39

Boulanger et al., Nature Neuroscience 2011

Nuclear Receptor pathway TGF-  pathway ftz-f1 TGF-  / babo EcR-B1 Hr39 HR39 + USP and ecdysone Neuronal remodeling [] Boulanger et al., Nature Neuroscience 2011

-/- +/- -/- mutant phenotype ?mutant phenotype -/- cell-autonomous ? linotte RTK

The RYK (DRL) protein

Srausfeld et al., MRT  m

+/+

drl + clone in a drl + brain drl - clone in a drl + brain +/+ -/- +/-

drl - clone in a drl - brain : phenotype is non-cell autonomous! -/-

Srausfeld et al., MRT  m

   +/+ drl - /drl -  ? or

 branch midline dendrites cell body A drl -/- single cell MARCM clone in a mutant brain Two different extrinsic (non cell autonomous) phenotypes:  branch is horizontally misguided  and  branches do not stop at the midline  branch  branch

So what is DRL doing? DRL expression surrounds the MBs at 48 h APF. This is consistent with a role for  axons guidance.

DRL MB-GFP

wnt5 400 /Y; C739 mGFP/+ Conclusion : wnt5 est nécessaire à la pousse axonale? WNT5, protéine sécrétée de la famille Wnts est le ligand de DRL

wnt5 400 /Y; C739 mGFP/+; UAS-wnt5/+ Conclusion : wnt5 est nécessaire à la pousse axonale des CP de façon autonome cellulaire.

  +/+ wnt5 - IMARIS

  sauvageInterprétation à partir de l ’ ensemble des neurones wnt5 -. Pousse axonal? Réalité vue en clones d ’ un seul neurone wnt5 - Guidage axonal

Model for the MB  branch guidance Reynaud et al., appeal submission to Science

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