Presentation on theme: "Rein et al., 2002 Current Biology The mushroom bodies (MBs) in the adult Drosophila brain."— Presentation transcript:
Rein et al., 2002 Current Biology The mushroom bodies (MBs) in the adult Drosophila brain
1 neuroblast 3 types of neurons - - ’ ’ - 2 x 4 neuroblats 2 x 2000 neurons2 x 1 MB brain = central brain + optic lobes = neurons central brain = central complex + MB central complex = neurons 2 X MB = 2 X neurons = 4000 neurons
midline horizontal branch Srausfeld et al., MRT m A wild-type neuron vertical branch 10 m lobe lobe
Parental cell Mitotic recombination After DNA replication Two distinct mosaic progeny x x x FRT Mutant Centro mere Repressor x protein x Repressible marker x x x x x Marking mutant clones in a mosaic organism by the MARCM technique NB: one can make clones in wild-type background also!: visualization clones
Nb G N N FLP A multi-cellular Nb clone Two cell clones FLP 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).
In addition, mitotic recombination in a dividing GMC can generate a single cell clone Single-cell clone FLP
Twin spot MARCM to reveal the developmental origin and identity of neurons Yu et al., Nature Neursc Review by Kao and Lee, COiN 2010 UAS-gfp-Mir and UAS-Cd2-Mir that silence the reporters UAS-Cd8::gfp and UAS-Cd2::rfp respectively.
Yu et al., Nature Neursc. 2009
-/- +/- cell-autonomous MARCM
NHL L3 0h APF Ecdysone pics during development
Summary of the mushroom body development
Axon reorganization of neurons during metamorphosis. Clones were generated in NHL and examined 12, 18 and 24 hours APF
Lee et al., Neuron (2000) 28: Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila Zheng et al., Cell (2003) 112: TGF- 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 = usp 5, usp 4 and usp 3 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 (dSmad2 1 ) 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 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
FTZ-F1 is required for neuron pruning. The expression of GAL4-201Y-driven GFP (green) and FASII (red) is shown in adult neurons. Clones were induced at NHL
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
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 (Hr39 C105 /Def(2L))
Hr39 is required for normal neuron development but not for neuron pruning
neuron pruning + ftz-f1 - Hr39 control P Hr39 -/- P P Hr39 -/- ;UAS-ftz-f1 + P P clones: Phenotype predicted obtained
ECR-B1 neuron pruning + ftz-f1 - 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) HYPOTHESIS 1 2 EcR-B1 neuron pruning + FTZ-F1 - Hr39 - HR39 surexp
Expression of ECR-B1 depends on normal FTZ-F1 activity in 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 neurons (f, g): wild-type, (h, i): + UAS-Hr39, (j): Quantification of ECR-B1 signal in arbitrary units (A.U.)
clones: Phenotype predicted obtained ftz-f1-/- UP ftz-f1-/-; UAS-EcRB1 + P P UAS-Hr39 + UP UAS-Hr39 + ;UAS-EcRB1 + P P UAS-Hr39 + ;UAS-ftz-f1 + P + UP in vivo competition 2 EcR-B1 neuron pruning + FTZ-F1 - HR39 surexp
in vivo competition between HR39 and FTZ-F1 for neuron remodeling