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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.....
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 yeastGAL4 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)
Temporal And Regional Expression Targeting
GAL80: GAL4 inhibitorBinding of GAL80 to the carboxy-terminal 30 aa of GAL4 prevents GAL4-mediated transcriptional activation
in yeast
FLP recombinase (FLPase)FRT: FLPase recombination targets
transgenesis in Drosophilaclonal expression
Vanessa Vanessa
Lee et al., Development 1999
Developmental axon pruning is a general mechanism required to transform animmature 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-specificprocesses
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:» a 1043 aa in early embryogenesis.» b 816 aa from late embryo to pre-pupae.
The nuclear receptor FTZ-F1
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
ADN1 376 441 588 701
DN
1 510 575 803 1027 1043
22%63 %
HR39
FTZ-F1
1 510 575 803
LIGANDADN
• 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
Nuclear Receptor pathway TGF- b pathway
ftz-f1 TGF-b / babo
EcR-B1
Hr39
HR39
+ USP and ecdysone
Neuronal remodeling
[ ]
Boulanger et al., Nature Neuroscience 2011
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
andbranches 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 a axons guidance.
wnt5400/Y; C739 mGFP/+
Conclusion : wnt5 est nécessaire à la pousse axonale?
WNT5, protéine sécrétée de la famille Wnts est le ligand de DRL
wnt5400/Y; C739 mGFP/+; UAS-wnt5/+
Conclusion : wnt5 est nécessaire à la pousse axonale desCP de façon autonome cellulaire.
sauvage Interprétation à partir de l’ensemble des neuroneswnt5-. Pousse axonal?
Réalité vue en clones d’un seul neurone wnt5-
Guidage axonal