Instituto de Acuiculturade Torre de la Sal (CSIC), Castellón, Spain. Laboratoire de Physiologie et Génomique des Poissons (INRA), Rennes, France
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1 Mitochondria as key responders of metabolic and hormonal disturbances in fish. Meta-analysis flowchart of transcriptomicdata in gilthead sea bream (Sparus aurata) Josep Calduch-Giner 1, YannEchasseriau 2, Patrick Prunet 2 & Jaume Pérez-Sánchez 1 1 Instituto de Acuiculturade Torre de la Sal (CSIC), Castellón, Spain 2 Laboratoire de Physiologie et Génomique des Poissons (INRA), Rennes, France Barcelona, July 2013
2 Muscle Growth Hyperplasia and hypertrophy determine: Growthpotential Final weight Barcelona, July
3 Key targeted genes of muscle growth Myogenic regulatory factors myod, mf5, myogenin, MRF4 Insulin& somatotropic axes Fastgrowthphenotypefora given nutritionaland developmental condition in sea bream: Low circulating GH levels Enhanced hepatic GHR-I & IGF-I expression Highcirculatinglevelsof IGF-I Enhanced skeletal muscle expression of IGF-II & GHR-II Pérez-Sánchez et al., Fish Physiol. Biochem Saera-Vila et al., J. Endocrinol Benedito-Palos et al., Aquaculture 2007 Saera-Vila et al., Comp. Biochem. Physiol. A 2009
4 Genomic tools: new targeted genes and processes Sea bream microarrays Array type Target tissue Challenge Reference cdna microarray Kidney Cortisol administration Sarropoulou et al., Physiol. Genomics 2005 Oligo-array Liver Cortisol administration Teles et al., Mar. Biotechnol Oligo-array Whole larva Ontogeny Ferraresso et al., BMC Genomics 2008 Oligo-array Skin/scales Tissue damage Vieira et al., BMC Genomics 2011 cdna microarray Liver Crowding stress Calduch-Giner et al., BMC Genomics 2010 cdna microarray Intestine Parasite infection Davey et al., Mol. Immunol Oligo-array Intestine Parasite x nutritional Calduch-Giner et al., BMC Genomics 2012 Barcelona, July
5 Genomic tools: new targeted genes and processes Next-generationsequencing(sea bream) NGS Target tissue Reference 454 Whole larvae Yúfera et al., Mar. Biotechnol Skeletal muscle García de la Serrana et al., BMC Genomics SSH Skeletalmuscle,intestine, head kidney, blood, liver, adiposetissue, brain, gills Calduch-Giner et al., BMC Genomics 2013 ( High coverage of the codifying sea bream transcriptome
6 Gene expresssionprofilingof cardiacand skeletal muscle tissues Experimental design& setup: Sea breamjuveniles (15 g initialbw) Restricted nutrientavailability (11 wks) Two experimental groups: Full ration size(r100) 70% full ration + finishingphase at themaintenanceratio (R70-20) Barcelona, July
7 Gene expresssionprofilingof cardiacand skeletal muscle tissues Analytical tools: Oligo-array: 4 x 44 K designformat 15,845 sequences with 2-3 probes each Fish and Chips Tool ( 334 datasets from zebrafish, European Sea bass, Atlantic cod, longjaw mudsucker, rainbow trout and Atlantic salmon microarray experiments 21 datasets from sea bream microarray experiments Hierarchical gene clustering and comparison of clusters among datasets Barcelona, July
8 Gene expresssionprofilingof cardiacand skeletal muscle tissues FOTO doradas Principal component analysis(3499 differentially expressed genes) CM-R WM-R 100 WM-R White Muscle Cardiac Muscle Red Muscle CM-R 100 RM-R RM-R % of variation is explained by the tissue component Tissue distribution along X-axis follows tissue oxidative capacity: WM < RM < CM Barcelona, July
9 Gene expression profiling: K-means clustering Highly expressed genes Definitionof 3 clusters according to transcript abundance in a giventissue 0 White muscle Cluster Red muscle Cluster Cardiac muscle Cluster Barcelona, July
10 Cluster phenotyping White muscle cluster Red muscle cluster Cardiac muscle cluster Relative expression Relative expression Relative expression WM RM HEART WM RM HEART WM RM HEART Nutritionally-regulated genes WM RM HEART Nutritionally-regulated genes WM RM HEART Nutritionally-regulated genes WM RM HEART WM is poorly transcriptionally regulated Heart is highly transcriptionally regulated Different metabolic plasticity & survival functions Barcelona, July
11 Meta-analysis flowchart Sub-cluster definition First round of meta-analysis comparison within experiment Second round of meta-analysis comparison among experiments Barcelona, July
12 Meta-analysisflowchart: Sub-cluster definition Sub-cluster definition First round of meta-analysis comparison within experiment Second round of meta-analysis comparison among experiments Nutritionally-regulated genes WM RM HEART Selection of a well-defined cluster according to experimental condition Barcelona, July
13 Meta-analysisflowchart: Sub-cluster definition R R 100 White musclecluster17 (224 genes) Barcelona, July
14 Meta-analysis flowchart within experiment White muscle cluster 17 (224 genes) Profile comparison among dataset clusters Cardiac muscle cluster 17 (501 genes) 61 overlapping genes Red muscle cluster 40 (398 genes) 52 overlapping genes Barcelona, July
15 Meta-analysis flowchart among independent experiments Cardiac muscle cluster 17 (501 genes) Profile comparison among dataset clusters Early development cluster 4 (1464 genes) 146 overlapping genes Liver Cortisol administration cluster 40 (139 genes) 41 overlapping genes Barcelona, July
16 Concluding remarks Sea bream has divergent muscle transcriptomes Sea bream muscle transcriptomes are differentially regulated by nutrient availability Highly expressed and nutritionally regulated genes of WM are enriched in genes related to proteolysis and protein ubiquitination Highly expressed and nutritionally regulated genes of heart and RM are enriched in the GO term mitochondria Microarray meta-analysis evidences that mitochondria-related genes are a common link within and among tissue transcriptomes in challenged fish Barcelona, July
17 Mitochondria, a subcellular marker of metabolic stress phenotyping 1000 proteins 13 mitochondria-encoded genes 990 nuclear-encoded genes 926 nuclear-encoded mitochondrial genes are present in the CSIC sea bream reference transcriptome database( Development of two PCR-arrays for 150 selected mitochondrial markers Mito-Chip I (Mitochondria activity, 60 genes) Mitochondrial biogenesis Antioxidant defence Protein import, folding and assembly Mitochondrial dynamics and apoptosis Mito-Chip II (OXPHOS, 90 genes) Complex I-V of mitochondria respiratory chain Barcelona, July
18 Poster
19 ACKNOWLEDGMENTS Nutrigenomics and Fish Growth Endocrinology Jaume Pérez Sánchez Josep Calduch Giner Azucena Bermejo Nogales Laura Benedito Palos Gabriel Ballester Lozano M. Ángeles González Albaladejo Patricia Cabrera Martínez Laboratoire de Physiologie et Génomique des Poissons Patrick Prunet Yann Echasseriau Funding: Barcelona, July 2013
20 Thank you for your attention!! Barcelona, July 2013
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