Transcript Aucun titre de diapositive - Master 1 Biologie Sant&#233

The mushroom bodies (MBs) in the
adult Drosophila brain
Rein et al., 2002 Current Biology
brain = central brain + optic lobes = 200.000 neurons
central brain = central complex + MB
central complex = 20.000 neurons
2 X MB = 2 X 2.000 neurons = 4000 neurons
1 neuroblast
2 x 4 neuroblats
3 types of neurons
2 x 2000 neurons
- a/b
- a’/b’
-g
2 x 1 MB
A wild-type ab neuron
a lobe
10mm
a vertical branch
b lobe
Srausfeld et al., MRT 2003
b horizontal branch
10mm
10mm
midline
Marking mutant clones in a mosaic organism by the MARCM technique
Parental cell
Mitotic recombination
After DNA replication
Two distinct
mosaic progeny
x
Repressor
protein
Repressible
marker
x
x
FRT
x
Mutant
x
x
x
x
x
Centro
mere
Repressor
NB: one can make clones in wild-type background also!:
visualization clones
x
Two mutually exclusive types of marked clones. Either all postmitotic neurons
generated subsequently in the same lineage will be labeled (upper), or only two
neurons derived from the GMC will be labeled in the whole lineage (lower).
N
G
Nb
N
FLP
A multi-cellular Nb clone
FLP
Two cell clones
In addition, mitotic recombination in a dividing GMC can generate a single
cell clone
FLP
Single-cell clone
UAS-gfp-Mir and UAS-Cd2-Mir that silence the reporters
UAS-Cd8::gfp and UAS-Cd2::rfp respectively.
Twin spot MARCM to reveal the developmental origin and identity of neurons
Yu et al., Nature Neursc. 2009
Review by Kao and Lee, COiN 2010
Yu et al., Nature Neursc. 2009
MARCM
-/+/-
cell-autonomous
Ecdysone pics during development
NHL
L3
0h APF
Summary of the mushroom body development
Axon reorganization of g neurons during metamorphosis. Clones were generated
in NHL and examined 12, 18 and 24 hours APF
Lee et al., Neuron (2000) 28:807-818
Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for
mushroom body neuronal remodeling in Drosophila
Zheng et al., Cell (2003) 112:303-315
TGF-b signaling activates steroid hormone receptor expression during neuronal
remodeling in the Drosophila brain
Boulanger et al., Nature Neuroscience (2011) 14(1):37-44
ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom
body neuron remodeling
and neuroblast clones also
Summary of the genetic crosses for the MARCM-based genetic screen
Identification of the l(X)48 mutant defective in the pruning of larval-specific
axons and dentrites
l(X)48 = usp5 , usp4 and usp3 two previously identified usp alleles.
All three alleles result in changes of invariant arginines that contact
phosphates in target DNA
Nuclear hormone receptors are ligand-dependent transcription factors
Nuclear receptors share common structure/function domains
A: The ECR-USP heterodimer binds ecdysone (E) and an EcRE in the DNA,
activating a downstream promoter (arrow)
B, C and D: Several models for negative regulation of ecdysone signaling
pathways.
BR-C, E74 and E75 primary response genes for ecdysone are not individually
essential for MB neuronal remodeling. Therefore, the USP/EcR-B heterodimer
probably mediates the ecdysone-dependent MB neuronal remodeling through
other target genes.
l(X)MB388 (dSmad21) carries a missense mutation in the dSmad2 gene and
expression of wild-type dSmad2 cDNA rescues neuronal remodeling defects in
l(X)MB388 mutant MB neurons.
EcR-A: no rescue, EcR-B1: partial rescue, EcR-B2: no rescue
THE ECDYSONE REGULATED GENE CASCADE
King-Jones et al., Cell 2005
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.
bFTZ-F1 is required for g neuron pruning. The expression of GAL4-201Y-driven
GFP (green) and FASII (red) is shown in adult g neurons.
Clones were induced at NHL
The nuclear receptors FTZ-F1 and HR39
1
ADN 441
376
HR39
588
701
LIGAND
ADN
DN
1
510
FTZ-F1
575
803
22%
63 %
1
510
575
1027 1043
803
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?
HR39 ectopic expression blocks g neuron remodeling
g: Molecular map of the Hr39 locus with the intron/exon structure of the two
main categories of mRNAs
h: Expression of HR39 detected by western blot of adult heads. Controls (+/+)
and mutant (Hr39C105 /Def(2L))
Hr39 is required for normal abneuron development
but not for g neuron pruning
+
ftz-f1
Hr39
-
g neuron pruning
clones:
control
Hr39-/Hr39-/- ;UAS-ftz-f1+
Phenotype
predicted obtained
P
P
P
P
P
1
ECR-B1
-
+
ftz-f1
Hr39
g neuron pruning
clones:
Phenotype
predicted obtained
usp-/UP
usp-/- ;UAS-ftz-f1+
P
UP
usp-/- ; Hr39-/P
UP
usp-/- ; Hr39-/- ;UAS-ftz-f1+ P
UP
HYPOTHESIS
1
ECR-B1
ftz-f1
FTZ-F1
-
+
Hr39
g neuron pruning
2
HR39 surexp
-
+
EcR-B1
g neuron pruning
-FTZ-F1 is binding the polytene chromosome band 42 A (EcR) (Lavorgna et al PNAS 1993)
- at 10h APF reduced expression of EcR in hs-ftz-f1 RNAi (Lam and Thummel Cu.Bio. 2000)
Expression of ECR-B1 depends on normal FTZ-F1 activity in g neurons
(a, b): wild-type, (c, d): ftz-f1-/-, e): Quantification of ECR-B1 signal in arbitrary
units (A.U.)
Expression of ECR-B1 depends on lack of HR39 activity in g neurons
(f, g): wild-type, (h, i): + UAS-Hr39, (j): Quantification of ECR-B1 signal in
arbitrary units (A.U.)
2
FTZ-F1
HR39 surexp
-
+
clones:
EcR-B1
Phenotype
predicted obtained
ftz-f1-/ftz-f1-/-; UAS-EcRB1+
UAS-Hr39+
UAS-Hr39+;UAS-EcRB1+
UP
P
P
UP
P
P
g neuron pruning
UAS-Hr39+;UAS-ftz-f1+
P + UP
in vivo competition
in vivo competition between HR39 and bFTZ-F1 for g neuron remodeling
hypothesis:
TGF-b/babo signaling
-
+
ftz-f1
Hr39
+
EcR-B1
Zheng et al., Cell 2003
g neuron pruning
clones:
Phenotype
predicted obtained
babo-/babo-/-; UAS-ftzf1+
Hr39-/- babo-/Hr39-/- babo-/-; UAS-ftzf1+
P
P
P
UP
UP
UP
UP
Is ftz-f1/Hr39 pathway independent of TGF-b
signaling?
Over-expression of HR39 and FTZ-F1 does not depend on babo activity ing neurons
UAS-babo-deltaI = BABO dominant negative form
UAS-babo = activated form of BABO
What insures Hr39 repression in the gneurons?
ftz-f1 itself
HR39 was overexpressed (1.78x) in ftz-f1-/- clones when compared with wild-type
clones.
Nuclear Receptor pathway
TGF-bpathway
ftz-f1
TGF-b / babo
Hr39
[ HR39
]
EcR-B1
+ USP and ecdysone
Neuronal remodeling
ChiP seq technique
In vivo binding of FTZ-F1 upstream of EcR-B1 transcription start site
Awasaki and Lee after Boulanger at al., Nature Neuroscience 2011
Thank you for your attention!