Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 abneuron 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 ing neurons UAS-babo-deltaI = BABO dominant negative form UAS-babo = activated form of BABO What insures Hr39 repression in the gneurons? ftz-f1 itself HR39 was overexpressed (1.78x) in ftz-f1-/- clones when compared with wild-type clones. Nuclear Receptor pathway TGF-bpathway 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!