1. 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

2. 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......).

3. in yeast GAL4 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)

6. from Martin Heisenberg

7. Srausfeld et al., MRT 2003 10  m

8. From Waddell and Quinn, 2001 Un centre de la mémoire : les corps pédonculés

9. rut - ; UAS-rut + ; brain-GAL4 memory rescue? rut - memory defect

12. Temporal Control ?

13. Temporal And Regional Expression Targeting GAL80: GAL4 inhibitor Binding of GAL80 to the carboxy-terminal 30 aa of GAL4 prevents GAL4-mediated transcriptional activation

16. GAL4 mediated misexpression of wild-type genes

18. UAS yellow + gène X Gal4 white + Visualise MB UAS mCD8-GFP Induces a MB phenotype

20. Srausfeld et al., MRT 2003 10  m

21. 7B Src Non receptor tyrosine kinase Control

22. wild-type p    p  Src P1 p    p

23. Clonal analysis -/- +/- -/- +/+

27. in yeast FLP recombinase (FLPase) FRT: FLPase recombination targets transgenesis in Drosophila clonal expression

30. Repressible marker = GFP under GAL4 control Repressor = GAL80

36. -/- +/- -/- viable lethal Dscam -/-

37. Down Syndrome cell adhesion molecule

38. Srausfeld et al., MRT 2003 10  m

39. 1:1 3:1 2:0 2:1 + stop short2:2

41. Vanessa Lee et al., Development 1999 Developmental axon pruning is a general mechanism required to transform an immature neural circuit into a mature one. During Drosophila metamorphosis, larval-specific dendrites and axons of early  neurons of the mushroom bodies are pruned and replaced by adult-specific processes

42. Lee et al., Neuron 2000

45. Thummel Dev. Cell 2001

46. 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

47. -/- +/- cell-autonomous MARCM

48. Nb G N N FLP A multi-cellular Nb clone Single-cell / two cell clones FLP Lee et al., Development 1999

50. The nuclear receptors FTZ-F1 and HR39 LIGAND ADN 1 376 441 588 701 DN 1 510 575 803 1027 1043 22% 63 % HR39 FTZ-F1 1 510 575 803 LIGAND ADN

51. 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

52. Boulanger et al., Nature Neuroscience 2011

53. Nuclear Receptor pathway TGF-  pathway ftz-f1 TGF-  / babo EcR-B1 Hr39 HR39 + USP and ecdysone Neuronal remodeling [] Boulanger et al., Nature Neuroscience 2011

54. -/- +/- -/- mutant phenotype ?mutant phenotype -/- cell-autonomous ? linotte RTK

55. The RYK (DRL) protein

56. Srausfeld et al., MRT 2003 10  m

57. +/+

58. drl + clone in a drl + brain drl - clone in a drl + brain +/+ -/- +/-

59. drl - clone in a drl - brain : phenotype is non-cell autonomous! -/-

60. Srausfeld et al., MRT 2003 10  m

61.    +/+ drl - /drl -  ? or

62.  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  and  branches do not stop at the midline  branch  branch

63. So what is DRL doing? DRL expression surrounds the MBs at 48 h APF. This is consistent with a role for  axons guidance.

64. DRL MB-GFP

65. wnt5 400 /Y; C739 mGFP/+ Conclusion : wnt5 est nécessaire à la pousse axonale? WNT5, protéine sécrétée de la famille Wnts est le ligand de DRL

66. wnt5 400 /Y; C739 mGFP/+; UAS-wnt5/+ Conclusion : wnt5 est nécessaire à la pousse axonale des CP de façon autonome cellulaire.

67.   +/+ wnt5 - IMARIS

68.   sauvageInterprétation à partir de l ’ ensemble des neurones wnt5 -. Pousse axonal? Réalité vue en clones d ’ un seul neurone wnt5 - Guidage axonal

69. Model for the MB  branch guidance Reynaud et al., appeal submission to Science

70. MERCI DE VOTRE ATTENTION