L œil de la Drosophile pour l étude du développement et de l apoptose
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1 L œil de la Drosophile pour l étude du développement et de l apoptose Pr Bertrand Mollereau Apoptosis and Neurogenetic LBMC UMR5239 ENS Lyon
2 Drosophila is a powerful genetic model -Gene conservation and little gene redundancy -Short generation time Adult -Easy gene manipulation : Pupa 10 days Egg - transgenesis - gene invalidation - Genetic screens larva
3
4 The Drosophila compound eye Organization of the Drosophila eye A C 3 2 B /8 6 cornea cone cells pigment cells outer PRs = rod cells inner PRs = cone cells optic lobe lamina medulla
5 Outer/inner Photoreceptors
6 The Drosophila life cycle Adult Pupa 10 days Egg larva
7 Photoreceptor differentiation and establishment of planar cell polarity
8 Photoreceptor specification and differentiation Third instar larva Pupation Adult R R1-6 R 8 R1-6 EGFR/Spitz Sev/Boss Notch/Delta Photoreceptor specification rhodopsin expression Rhabdomere morphogenesis
9 A GFP enhancer trap screen for genes expressed in adult photoreceptors Tissue specific genomic enhancer GAL4 GAL4 UAS GFP flies observed for a GFP expression in the retina lines with a GFP expression in adult Drosophila -16 lines with a specific photoreceptor expression Mollereau et al (2000) Mech Dev 93,
10 Rhodopsins are expressed in photoreceptor subtypes
11 spalt insertion - expression in R7 and R8 GF P la cz ant i -Salm pgawb GAL4 spalt rel ated spalt major 68 5b p PZ
12 Spalt is required for R7/R8 differentiation
13 In salm/salr -/- R7 and R8 acquire R1-6 subtype rhabdomere morphology
14 salm/salr are required for rhodopsin expression in R7 and R8 rh3 (R7) rh4 (R7) rh5/rh6 (R8) wt salm/salr -/-
15 R7 and R8 are transformed into R1-R6 in salm/salr -/- retina wt salm/salr -/- Rh1 Mollereau et al (2001) Nature 412,
16 salm is expressed in R3/R4 and R7 in the larval eye imaginal disc Salm Svp (R3,4, 1, 6) Pros (R7,cc)
17 salm is expressed in R3/R4 and R7 in the larval eye imaginal disc
18 Photoreceptor differentiation and establishment of planar cell polarity
19 spalt mutant ommatidia have a defect in planar cell polarity Wild type salm/salr -/-
20 Sequential roles of spalt in R3/R4 and R7 differentiation Domingos et al. Development Dev Mollereau and Domingos Dev. Dyn. 2005
21 Identification of new regulators of development and apoptosis in the Drosophila retina
22 Immersion objective Screen strategy Cornea neutralization and PR-specific expression of fluorescent protein (Pichaud & Desplan, 2001) agarose Glass slide water agarose
23
24 Mitotic recombination and recessive lethal mutant clone genesis in non vital organs +/- screening of lethal recessive mutations - + Mitotic Recombination -/- +/+ +/- Eye -/- +/+
25 FRT/FLP technique and mitotic recombination Mitosis +/+ +/- FLP FRT -/- mutation Phenotypic marker, for example red fluorescent protein
26 The Tomato/GFP FLP/FRT method of PR visualization Mitotic recombinaison, cornea neutralization and confocal microscope Rh1-Gal4,ey-FLP; UAS-GFP; FRT rh1-tdtomato/frt gene* 488nm 543nm merge Mutant PRs -/- Wild type and Heterozygous PRs Gambis, Dourlen et al. Dev. Biol. 2011
27 Wild type and Heterozygous PRs Mutant PRs -/-
28 Spalt mutants analyzed with Tomato/GFP FLP/FRT method
29 141 mutations with loss of photoreceptors Fatty acid transport protein Fatp : missing PRs +/+ -/-
30 Focusing on mutations leading to caspase dependent cell death developmental defects Gene mutation Missing PRs Cell death/survival mechanisms - Caspase dependent - Caspase independent
31 Focusing on mutations leading to caspase dependent cell death developmental defects Gene mutation Missing PRs Cell death/survival mechanisms Inhibitor of caspase (p35) - Caspase dependent - Caspase independent Rescue?
32 Genes which mutation is rescued by the expression of an inhibitor of caspase + GMR-p35 Fatp Name l(3)06536 j2e5 CG9523 l(2)k09828 fatp function unknown Adénylyltransferase unknown Fatty acid tranport protein
33 Part II Characterization of Fatp mutation-induced Neurodegeneration
34 Fatp : Fatty acid transport protein - Unknown in drosophila - Mammalian homologs ( 50% identity): * Fatp1 : obesity, thermogenesis * Fatp4 : Ichthyosis syndrom -Molecular activity : * Fatty acid transporter * Acyl coa synthetase
35 Fatp expression pattern PlacW insertion in Fatp gene brain medulla lamina retina lacz Fatp Horizontal cryosection 100µm
36 en>gfp en>fatp Fatp is expressed in the adult retina A D Fatp Actin D Fatp Fatp 72kDa Tubulin 55kDa D D B Cs C GMR>fatp * * E E
37 Histological analysis of fatp mutant retina
38 percent of missing PRs Fatp mutation affects fatp expression RT-PCR Ectopic fatp expression rescues fatp mutation +/+ -/- -/- rh1>fatp +/+ -/- 15 days fatp RP *** +/+ -/- -/- rh1>fatp
39 percent of day1 Fatp mutation induces progressive photoreceptor degeneration during adulthood 1d 4d 8d 14d +/- -/- +/ /+ or +/- -/ days Confirmation of the progressive neurodegeneration: - Resin-embedded section - RNAi
40 d8 Mutant PR loss (%) d5 Photoreceptor degeneration is light dependent in fatp mutant retina ctrl mosaic 350lux fatp mosaic 350lux fatp mosaic 0lux C D E F G H I control mosaic 350lux fatp mosaic 350lux fatp mosaic 0lux p=0.018 * d5 d8
41 Amplitude (mv) Visual response is increased in fatp mutant retina A light ctrl fatp -/- B 0,0-2,0-4,0-6,0-8,0 ctrl fatp -/- -10,0 5mV -12,0 500ms -14,0-16,0 ** p= *
42 fatp mosaic Mutnat PR loss (%) ctrl ctrl Rh1/Tubullin (arbitrary units) ctrl ctrl + VitA - VitA Rh1 expression is increased in fatp mutant retina A 260 kda 60 kda 30 kda fatp -/- rh1 G69D/+ fatp -/- Rh1 oligomer dimer monomer Tubulin B 3 2,5 2 1,5 1 0,5 0 p=0.04 * fatp -/- fatp -/- C 260 kda 60 kda 30 kda fatp mosaic Rh1 Tubulin rh1 G69D/+ D + VitA E - VitA F p=5.5 E-06 ***** +VitA -VitA
43 Rh1/Tubulin (arbitrary unit) lacz/tubulin (arbitrary unit) ctrl Rh1 degradation is compromised in fatp mutant retina A ctrl fatp -/- light Rh1 C rh1-lacz fatp -/- Tubulin p=0.029 B 4 3,5 3 2,5 2 1,5 1 0,5 0 light ctrl fatp -/- lacz Tubulin D 2 1,5 1 0,5 0 ctrl fatp -/-
44 Conclusions Part II - Fatp mutation: affect fatp expression in the retina Is responsible for PR degeneration - Fatp mutation induced degeneration : adult onset progressive caspase dependent, rescued by caspase inhibitors light independent PR degeneration due to an increased Rh1 and increased visual response
45 Lab members Nathalie Davoust Nataf Ph. D Stéphane Vincent Ph.D Dali Ma Ph.D Francesco Napoletano, Ph. D Gilles Chatelain Clémence Levet Marion Robin Matthieu Querenet Maxime Dureau Antoine Ducuing Colleagues for fly stocks and reagents DROSO-TOOLS, PLATIM, UMS3444 CTμ, Univ. Lyon 1 Collaborators M. Roux, H. Steller, C. Desplan, P. Brabet, C. Hamel. Past lab members Pierre Dourlen Ph.D Antoine Fouillet Ph. D Marie Laure-Dichtel Danjoy Ph.D César Mendes Ph.D Pedro Domingos PhD Alexis Gambis PhD Fundings: ATIP-CNRS and Equipe Fondation pour la Recherche Médicale.
46
47 Phototransduction in Drosophila
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