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

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)

from Martin Heisenberg

Srausfeld et al., MRT 2003

10mm

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 inhibitorBinding of GAL80 to the carboxy-terminal 30 aa of GAL4 prevents GAL4-mediated transcriptional activation

GAL4 mediated misexpression of wild-type genes

UASyellow+ gène X

Gal4 white+

Visualise MBUAS mCD8-GFP

Induces a MB phenotype

Srausfeld et al., MRT 2003

10mm

7B

SrcNon receptor tyrosine kinase

Control

wild-type

p

a

b

a

pb

SrcP1

pa a

bb

p

Clonal analysis

-/-

+/--/-

-/-

+/+

+/+

in yeast

FLP recombinase (FLPase)FRT: FLPase recombination targets

transgenesis in Drosophilaclonal expression

Repressible marker = GFP under GAL4 controlRepressor = GAL80

-/-

+/- -/-

viable lethal

Dscam-/-

Down Syndrome cell adhesion molecule

Srausfeld et al., MRT 2003

10mm

1:1 3:1 2:0

2:1 + stop short 2:2

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

Lee et al., Neuron 2000

Lee et al., Neuron 2000

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:» a 1043 aa in early embryogenesis.» b 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

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

Boulanger et al., Nature Neuroscience 2011

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

-/-

+/--/-

mutant phenotype ?mutant phenotype

-/-

cell-autonomous ?linotte RTK

The RYK (DRL) protein

Srausfeld et al., MRT 2003

10mm

+/+ +/+

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 2003

10mm

a

b

b

+/+

drl-/drl-

a

?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

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.

DRLMB-GFP

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.

a

b

+/+

wnt5-

IMARIS

sauvage Interprétation à partir de l’ensemble des neuroneswnt5-. Pousse axonal?

Réalité vue en clones d’un seul neurone wnt5-

Guidage axonal

Model for the MB a branch guidance

Reynaud et al., appeal submission to Science

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