Analyse_de_microarrays_application_poisson.pptx (Lecture seule)

Sophie Depiereux – PhD student Université Namur Faculté des sciences Départment de biologie URBE
Unité de Recherche en Biologie
Environmentale et Evolutive
0 Prof. J.P. Descy Structure, fonc>on et qualité des écosystèmes aqua>ques Ass. Prof. K. Van Doninck Evolu>onary gene>cs and ecology URBE Ass. Prof. F. Silvestre Physiologie environnementale Ecophysiologie Ecotoxicologie Ecotoxicologie systemique Prof. P. Kestemont 1 Prof. P. Kestemont Ecophysiologie Ecotoxicologie Principaux thèmes de recherche en physiologie environnementale   Recherche d’espèces sentinelles en écotoxicologie
  Perturbateurs endocriniens et intersexualité chez les
poissons
  Recherche de biomarqueurs en utilisant une approche in
vitro en toxicologie poisson
  Interaction température – polluants en écotoxicologie
  Ecotoxicologie des amphibiens
  Physiologie du stress et réponse immunitaire chez les
poissons
hGp://webapps.fundp.ac.be/system-­‐ecotox/ 2 Prof. P. Kestemont Ecophysiologie Ecotoxicologie Principaux thèmes de recherche en physiologie environnementale   Recherche d’espèces sentinelles en écotoxicologie
  Perturbateurs endocriniens et intersexualité chez les
poissons
  Recherche de biomarqueurs en utilisant une approche in
vitro en toxicologie poisson
  Interaction température – polluants en écotoxicologie
  Ecotoxicologie des amphibiens
  Physiologie du stress et réponse immunitaire chez les
poissons
hGp://webapps.fundp.ac.be/system-­‐ecotox/ 3 Etude transcriptomique des effets de l’éthynylestradiol sur les gonades de truite arc-­‐en-­‐ciel Sophie Depiereux, Bertrand DeMeulder, Eric Bareke, Michael Pierre, Yann Guiguen, Florence LeGac, Alexis Fos>er, Eric Depiereux, Patrick Kestemont URBM INRA 4 Context Objec>ves Approaches Results Conclusion Gonad : bipoten>al nature Mammals TESTIS Sox9 SRY OVARY Brennan et Capel, 2004 5 Context Objec>ves Approaches Results Conclusion Gonad : bipoten>al nature Teleosts fish TESTIS Sox9 Matsuda et al., 2002 Yano et al, 2012 DMY -­‐ sdY OVARY Brennan et Capel, 2004 6 Context Objec>ves Approaches Results Conclusion Gonad : bipoten>al nature Teleosts fish TESTIS Sox9 Matsuda et al., 2002 Yano et al, 2012 DMY -­‐ sdY Temperature Exogenous hormones Pollutants OVARY Brennan et Capel, 2004 7 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC 8 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC •  Xenoestrogens 9 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC •  Xenoestrogens –  >100 molecules –  Natural E1-­‐E2; synthe>c EE2 –  industrial chemicals NP-­‐OP-­‐BPA –  Surface waters –  ng/L or µg/L 10 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC •  Xenoestrogens •  Impact 11 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC •  Xenoestrogens •  Impact –  1 ng/L for E2 (Purdom et al, 1994) –  0.1 ng/L for EE2 (Hansen et al, 1998) –  Male fish Female oocytes •  Vitellogenin •  Feminisa>on : ovotes>s Male tes>s from a gudgeon sampled downstream of a STP 12 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC •  Xenoestrogens •  Impact –  1 ng/L for E2 (Purdom et al, 1994) –  0.1 ng/L for EE2 (Hansen et al, 1998) –  Male fish –  Industrialised countries 13 Context Objec>ves Approaches Results Conclusion Endocrine disrup>on •  EDC •  Xenoestrogens •  Impact –  1 ng/L for E2 (Purdom et al, 1994) –  0.1 ng/L for EE2 (Hansen et al, 1998) –  Male fish –  Industrialised countries –  Individual (Jobling 1996, 2002) and popula>onal level (Kidd 2009) 14 Objec>ves Context Approaches Results Conclusion Ques>on Spermatogenesis:
Spermatogonia
type A
Spermatogonia
type B
Spermatocyte
2n
Meiosis
Spermatid
How a germ cell with XY genotype can develop into an oocyte under chronic exposure to a potent xenoestrogen (EE2) at environmental doses? n
Spermatozoïd
15 Context Objec>ves Approaches Results Conclusion Objec>ves •  Molecular mechanisms underlying male fish feminisa>on → pathways involved in gonad trans-­‐differen>a>on 16 Context Objec>ves Approaches Results Conclusion MICROARRAY In silico In vivo Objec[ves
New pathways Morpho-­‐func>onal impact of EE2 on juvenile RT tes>s In vitro Direct gene expression response to EE2 Method
Bioinforma>c re-­‐analysis of exis>ng dataset Chronic exposure Cri>cal period of sex determinism -­‐  Histology -­‐  DNA microarray Tes>s culture Short exposure to EE2 Valida>on of target genes by Q-­‐PCR 17 Objec>ves Context Approaches Results Conclusion Objec>ves •  Rainbow trout -­‐ Oncorhynchus mykiss –  Sex gene>cally determined •  Male heterogamecy (XX-­‐XY) –  Genome being sequenced –  Model species •  molecular biology •  Physiology of reproduc>on •  Ecology –  Microarray available –  Sexual reversion by exogenous hormones •  Response to EE2 –  VTG +++ but NO ovotes>s 18 Context Objec>ves Approaches Results Conclusion MICROARRAY In silico In vivo Objec[ves
New pathways Morpho-­‐func>onal impact of EE2 on juvenile RT tes>s In vitro Direct gene expression response to EE2 Method
Bioinforma>c re-­‐analysis of exis>ng dataset Chronic exposure Cri>cal period of sex determinism -­‐  Histology -­‐  DNA microarray Tes>s culture Short exposure to EE2 Valida>on of target genes by Q-­‐PCR 19 Context Objec>ves Methods Results Conclusion In vivo experiment : chronic exposure to EE2 •  EE2 contamina[on from first feeding to juvenile stage in all-­‐male popula[on trout 60 to 136 dpf •  4 concentra>ons used : 0.01 ; 0.1 ; 1 and 10 µg/L (Jobling et al., 1996, Bejerregaard 2008) •  3 tanks per concentra>on 20 Context Objec>ves Methods Results Conclusion In vivo experiment : chronic exposure to EE2 •  EE2 contamina[on from first feeding to juvenile stage in all-­‐male popula[on trout 60 to 136 dpf •  4 concentra>ons used : 0.01 ; 0.1 ; 1 and 10 µg/L (Jobling et al., 1996, Bejerregaard 2008) •  3 tanks per concentra>on • Analysis –  Gonad histology –  Gene expression by microarray on gonads –  Expression of aromatase and ER isoforms by Q-­‐PCR in brain –  Sex steroid (E2, T, 11-­‐KT) quan>fica>on by radio-­‐immuno-­‐assay 21 Context Objec>ves Approaches Results Conclusion In vivo : histological analysis CTL [0.01] Male Intersex Age :14 weeks Ovotes>s Asymmetric Cysts Sertoli cells Leydig cells 22 Context Objec>ves Approaches Results Conclusion In vivo : histological analysis CTL [0.01] Male Intersex Age :14 weeks Ovotes>s [0.1] Intermediate Asymmetric Cysts Sertoli cells Leydig cells 23 Context Objec>ves Approaches Results Conclusion In vivo : histological analysis CTL [0.01] Male Intersex Age :14 weeks Ovotes>s [1] [10] Sex reversed Advanced [0.1] Delayed Asymmetric Cysts Sertoli cells Leydig cells Primary oocytes Primordial follicles Ovigerous lamellae Atypical structures 24 Context Objec>ves Approaches Results Conclusion In vivo : physiological analysis RIA Testosterone Concentra[on (ng/g) 20 15 10 5 0 0,01 0,1 Concentra[on EE2 (µg/l) 35 Concentra[on (ng/g) 0 Estradiol (E2) 8 Concentra[on E2 (ng/g) 25 7 6 5 4 3 2 1 0 0 10 a 25 10 11-­‐KT ab 30 0,01 0,1 Concentra[on d'EE2 (µg/l) b 20 b 15 10 Regression significant R2 = 0.9 5 0 0 0,01 0,1 Concentra[on EE2 (µg/l) 10 25 Objec>ves Context Approaches Results Conclusion In vivo : Q-­‐PCR BRAIN Cyp19 b
R2=0.7 3,5
b ab 2
1,5
a 1
a log dilution
log dilution
3
2,5
a 0,5
0
0
0,01
0,1
1
ERα 2 ERβ 2 2,5
2
1,5
1
0,5
0
10
0
Concentration EE2 (ug/L)
ERα 1
log dilution
3
R2=0.9 2,5
2
1,5
a a b b c 0,5
0
0,01
0,1
2,5
2
1
0
1
Concentration EE2 (ug/L)
0,1
1
10
Concentration EE2 (ug/L)
log dilution
3,5
0,01
10
1,5
ERβ 1
R2=0.3 c b b 1
ab a 0,5
0
0
0,01
0,1
1
10
Concentration EE2 (ug/L)
26 Objec>ves Context Approaches Results Conclusion Microarray study RT → → mRNA → cDNA Labeled cDNA cDNA = Probe Probe : sequence specific to a gene 27 27 Objec>ves Context Approaches Results Conclusion Microarray study RT → → mRNA → cDNA Labeled cDNA cDNA = Probe Probe : sequence specific to a gene 28 Context Objec>ves Microarray study mRNA Approaches Results Conclusion + EE2 mRNA Marked Marked inhibi>on 29 29 29 Context Objec>ves Approaches Results Conclusion Microarray study •  6 replicates (10 fish/pool) •  Agilent technology (60K) 30 Context Objec>ves Approaches Microarray study Clustering results Results CTL Conclusion 0.01 0.1 1 •  6 replicates (10 fish/pool) •  Agilent technology (60K) •  First results encouraging Single profiles 30 000 DEG (p<0.05)(!!) Filters pathways 31 10 Context Objec>ves Approaches Results Conclusion Microarray study : in silico analysis "g
tg =
Yg
Pierre et al, 2010;2011 Row data DEG FP +++ FN +++ Filters Pathways Genes 32 Context Objec>ves Approaches Results Conclusion Microarray study : in silico analysis •  First FILTER : sta>s>cal analysis –  ANOVA 1 –  Limma (R) –  Sheffe’s post-­‐hoc µg/L 0 0.01 0.1 1 0 0.01 0.1 1 10 1285 7933 12956 20932 4883 8152 10820 218 13673 13 10 Table of contrats, total number of DEG per contrast 33 Context Objec>ves Approaches Results Conclusion Microarray study : in silico analysis •  First FILTER : sta>s>cal analysis –  ANOVA 1 –  Limma (R) –  Sheffé’s contrats µg/L 0 0.01 0.1 0 0.01 0.1 1 10 1285 7933 12956 20932 4883 8152 10820 218 13673 1 13 10 Table of contrats, total number of DEG per contrast 34 Context Objec>ves Approaches Results Conclusion Microarray study : in silico analysis •  First FILTER : sta>s>cal analysis –  ANOVA 1 –  Limma (R) –  Sheffé’s contrats µg/L 0 0 0.01 0.1 0.01 0.1 1 10 1285 7933 12956 20932 4883 8152 10820 218 13673 1 13 10 Table of contrats, total number of DEG per contrast •  2nd FILTER : DAVID (Huang et al., 2009) –  Gene_Ontology (GO terms) –  Pathways (Kegg, Biocarta) –  … 35 Context Objec>ves Approaches Results Conclusion Microarray study : in silico analysis •  First FILTER : sta>s>cal analysis –  ANOVA 1 –  Limma (R) –  Sheffé’s contrats µg/L 0 0 0.01 0.1 0.01 0.1 1 10 1285 7933 12956 20932 4883 8152 10820 218 13673 1 13 10 Table of contrats, total number of DEG per contrast •  2nd FILTER : DAVID (Huang et al., 2009) –  Gene_Ontology (GO terms) –  Pathways (Kegg, Biocarta) –  … 36 Context Objec>ves Approaches Results Conclusion Microarray study : in silico analysis •  First FILTER : sta>s>cal analysis –  ANOVA 1 –  Limma (R) –  Sheffé’s contrats µg/L 0 0 0.01 0.1 0.01 0.1 1 10 1285 7933 12956 20932 4883 8152 10820 218 13673 1 13 10 Table of contrats, total number of DEG per contrast •  2nd FILTER : DAVID (Huang et al., 2009) –  Gene_Ontology (GO terms) –  Pathways (Kegg, Biocarta) –  … 37 Context Objec>ves Approaches Results Microarray study : ontologies Conclusion P-­‐value < 0,05 Specific DEG groups Intersec[on (800 DEG) Contrast CTL-­‐0,01 (1285 DEG) Specific DEG CTL-­‐0,01 (256 DEG) steroid biosynthesis structural molecule ac>vity binding growth factor ac>vity gonad development sex differen>a>on skeletal development female gonad development receptor binding integrin binding nucleus transmembrane receptor protein phosphatase ac>vity C21-­‐steroid hormone metabolism C21-­‐steroid hormone metabolism transmembrane receptor protein tyrosine phosphatase ac>vity C21-­‐steroid hormone biosynthesis C21-­‐steroid hormone biosynthesis cell adhesion neurogenesis neurogenesis extracellular matrix structural cons>tuent extracellular organogenesis development morphogenesis extracellular matrix collagen extracellular matrix structural cons>tuent extracellular organogenesis development morphogenesis extracellular matrix 38 Common structural cons>tuent of cytoskeleton extracellular organogenesis development morphogenesis extracellular matrix Context Objec>ves Approaches Results Conclusion Microarray study : pathways Steroid hormone biosynthesis 39 Context Objec>ves Approaches Results Conclusion Microarray study : pathways Steroid hormone biosynthesis Rainbow trout DEG Annota>on Numerous species Ontologies 40 Context Objec>ves Approaches Results Conclusion Microarray study : pathways Steroid hormone biosynthesis Rainbow trout DEG Annota>on Danio rerio Pathways Numerous species Ontologies 41 Context Objec>ves Approaches Results Conclusion MICROARRAY In silico In vivo Objec[ves
New pathways Morpho-­‐func>onal impact of EE2 on juvenile RT tes>s In vitro Direct gene expression response to EE2 Method
Bioinforma>c re-­‐analysis of exis>ng dataset Chronic exposure Cri>cal period of sex determinism -­‐  Histology -­‐  DNA microarray Tes>s culture Short exposure to EE2 Valida>on of target genes by Q-­‐PCR 42 •  Jeu de données de Yann Guiguen –  Ciné>que de l’effet de EE2 •  Stades précoces •  Doses élevées (20 mg/kg) •  Combinaison avec mes données –  Stade tardif 136 dpf –  Doses faibles (0,01 et 0,1 ug/L) •  557 DEG d’intérêt •  11 pathways significa>fs Gènes clés à tester in vitro 43 Context Objec>ves Approaches Results Conclusion MICROARRAY In silico In vivo Objec[ves
New pathways Morpho-­‐func>onal impact of EE2 on juvenile RT tes>s In vitro Direct gene expression response to EE2 Method
Bioinforma>c re-­‐analysis of exis>ng dataset Chronic exposure Cri>cal period of sex determinism -­‐  Histology -­‐  DNA microarray Tes>s culture Short exposure to EE2 Valida>on of target genes by Q-­‐PCR 44 Context Objec>ves Approaches Results Conclusion In vitro Exposi>on de gonades de truites monosexe mâle immatures -­‐  À plusieurs doses de EE2 -­‐  Durant différents temps 0 ng/ml 0,5 ng/ml 5 ng/ml 50 ng/ml 24H n=8 n=6 n=6 n=6 96H n=8 n=6 n=6 n=6 168H (7J) n=8 n=6 n=6 n=6 Mesure de la réponse de gènes clés par Q-­‐PCR 7th ISFE – Buenos Aires 2012 45 Sophie Depiereux Context Objec>ves Approaches Results Conclusion Discussion -­‐ Conclusions •  Histology –  Intersex in rainbow trout at environmental concentra>ons •  Steroids –   11-­‐KT – no effect on E2 and T levels •  Gene a expression BRAIN Cyp19   ER isoforms •  Microarray (gonads) GONADS   –  Adapta>on of a method –  Limita>ons with RT genomic resources 7th ISFE – Buenos Aires 2012 46 Sophie Depiereux Merci pour votre awen>on! 47