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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..... jmdura@igh.cnrs.fr
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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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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)
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from Martin Heisenberg
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Srausfeld et al., MRT 2003 10 m
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From Waddell and Quinn, 2001 Un centre de la mémoire : les corps pédonculés
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rut - ; UAS-rut + ; brain-GAL4 memory rescue? rut - memory defect
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Temporal Control ?
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Temporal And Regional Expression Targeting GAL80: GAL4 inhibitor Binding of GAL80 to the carboxy-terminal 30 aa of GAL4 prevents GAL4-mediated transcriptional activation
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GAL4 mediated misexpression of wild-type genes
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UAS yellow + gène X Gal4 white + Visualise MB UAS mCD8-GFP Induces a MB phenotype
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Srausfeld et al., MRT 2003 10 m
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7B Src Non receptor tyrosine kinase Control
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wild-type p p Src P1 p p
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Clonal analysis -/- +/- -/- +/+
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in yeast FLP recombinase (FLPase) FRT: FLPase recombination targets transgenesis in Drosophila clonal expression
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Repressible marker = GFP under GAL4 control Repressor = GAL80
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-/- +/- -/- viable lethal Dscam -/-
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Down Syndrome cell adhesion molecule
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Srausfeld et al., MRT 2003 10 m
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1:1 3:1 2:0 2:1 + stop short2:2
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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
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Lee et al., Neuron 2000
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Thummel Dev. Cell 2001
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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
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-/- +/- cell-autonomous MARCM
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Nb G N N FLP A multi-cellular Nb clone Single-cell / two cell clones FLP Lee et al., Development 1999
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The nuclear receptors FTZ-F1 and HR39 LIGAND ADN 1 376 441 588 701 DN 1 510 575 803 1027 1043 22% 63 % HR39 FTZ-F1 1 510 575 803 LIGAND ADN
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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
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Boulanger et al., Nature Neuroscience 2011
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Nuclear Receptor pathway TGF- pathway ftz-f1 TGF- / babo EcR-B1 Hr39 HR39 + USP and ecdysone Neuronal remodeling [] Boulanger et al., Nature Neuroscience 2011
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-/- +/- -/- mutant phenotype ?mutant phenotype -/- cell-autonomous ? linotte RTK
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The RYK (DRL) protein
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Srausfeld et al., MRT 2003 10 m
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+/+
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drl + clone in a drl + brain drl - clone in a drl + brain +/+ -/- +/-
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drl - clone in a drl - brain : phenotype is non-cell autonomous! -/-
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Srausfeld et al., MRT 2003 10 m
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+/+ drl - /drl - ? or
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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
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So what is DRL doing? DRL expression surrounds the MBs at 48 h APF. This is consistent with a role for axons guidance.
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DRL MB-GFP
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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
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wnt5 400 /Y; C739 mGFP/+; UAS-wnt5/+ Conclusion : wnt5 est nécessaire à la pousse axonale des CP de façon autonome cellulaire.
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+/+ wnt5 - IMARIS
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sauvageInterprétation à partir de l ’ ensemble des neurones wnt5 -. Pousse axonal? Réalité vue en clones d ’ un seul neurone wnt5 - Guidage axonal
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Model for the MB branch guidance Reynaud et al., appeal submission to Science
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MERCI DE VOTRE ATTENTION
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