Marine fish species undergo major changes during early developmental stages

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D VD R R XR N ucleu s VDR E target genes Recent results on influence

Cahu, B. Petton, C. Falguière, E. Gasset, J. Zambonino, D.

Gueguen Marine fish species undergo major changes during early

changes are controled by changes in the expression of a lot of genes

1 D VD R R XR N ucleu s VDR E target genes Recent results on influence of nutrients on fish larvae development: a transcriptomic study C. Cahu, B. Petton, C. Falguière, E. Gasset, J. Zambonino, D. Mazurais, D. Coves presented by Y. Gueguen Marine fish species undergo major changes during early developmental stages D4 D9 D22 These morphological and functional changes are controled by changes in the expression of a lot of genes gilthead sea bream: Sarropoulou et al., 2005 Atlantic halibut :Douglas et al., 2008 European sea bass: Darias et al.,

sea bass larvae development 6626 genes included on the arrays 450 genes differentially expressed throughout development (> 900 biological

2 Gene expression pattern during sea bass larvae development by transcriptomic D7 D9 D13 D17 D23 D25 D27 D31 D35 D43 sea bass larvae sampled at 10 development stages inter-species hybridizations Rainbow trout microarray (AGENAE, INRA) > 6000 genes Gene expression pattern during sea bass larvae development 6626 genes included on the arrays 450 genes differentially expressed throughout development (> 900 biological functions) D Gene Ontology Ossification Muscular development Metabolism Digestion proteic synthesis genes 2

800 700 600 500 400 300 200 100 0 Expression pattern of genes involved in ossification, muscular development and glycolysis Expressions of genes involved in ossification and associated to muscular

surface, after double staining and image analysis.

3 Expression pattern of genes involved in ossification, muscular development and glycolysis Expressions of genes involved in ossification and associated to muscular development process follow an exponential kinetic D23 Glycolysis Ossification level D23 D1 D5 D9 D13 D17 D21 D25 D29 D33 D37 D20 Ossification assessed by surface of mineralized bones per larval surface, after double staining and image analysis. Gene expression pattern during sea bass larvae development Transcriptomic patterns allow to characterize two main physiological status during larvae development - before 23 dph, coresponding to larval metabolism - after 23 dph, coresponding to more mature functions These period, around d23, corresponds to metamorphosis in sea bass larvae. This study allowed to identify what is the normal gene pattern of expression during normal development. Based on these data, we are now able to assess the impact of changes in larval environment, including nutrionnal factors, on genes governing fish development. 3

Impact of vitamin D deficiency on larvae development Vitamin D plays a crucial role in the development of vertebrates: proliferation and differentiation processes, calcium homeostasis playing a role

4 Impact of vitamin D deficiency on larvae development Vitamin D plays a crucial role in the development of vertebrates: proliferation and differentiation processes, calcium homeostasis playing a role in skeletal formation 2 diets: Control & DVD D15 D22 D45 24 pools of European sea bass larvae at 3 stages of development (4 pools / stage) clones originated from 26 cdna libraries obtained from different sea bass tissues (Rex MGE, Aquafirst) - Representing 6990 annotated genes involved in 2627 different biological processes At day 15: effect of vit D on Mitotic cell cycle/differentiation processes At 15 dph: C DVD - + relative expression level POLD4: positive regulation of cell proliferation ANAPC7: RAN: required for normal mitosis progression BUB1B: KIF23: localizes to the interzone of mitotic spindles API5: antiapoptotic factor... CD33: RARES3: negative regulation of cell cycle CUL2: ABL1: signal transduction resulting in apoptosis GADD45G: growth arrest and induction of cell differentiation MLF1: SYNE1: muscle cells differentiation Vitamin D deficiency is associated with activation of proliferation process to the detriment of differentiation pathways 4

At day 22: effect of vit D on genes involved in skeletal larvae development - 939 clones representing 243 annotated genes differentially expressed at 22 dph - 409 biological processes DVD C In case

Osteocalcin: osteoblastic differentiation, associated with mineralization Experiment conducted by feeding sea bass larvae with different Vit D levels - Sea bass larvae fed from mouth opening with

5 At day 22: effect of vit D on genes involved in skeletal larvae development clones representing 243 annotated genes differentially expressed at 22 dph biological processes DVD C In case of vit D deficiency, negative regulation of genes involved in skeletal development COL10A1; COL1A1: formation of tendons ligaments and bones MATN1; MGP; SOX9; TUFT1 BMP-4: osteoblast maturation Osteocalcin: osteoblastic differentiation, associated with mineralization Experiment conducted by feeding sea bass larvae with different Vit D levels - Sea bass larvae fed from mouth opening with compound diet conteaining diffferent vit D3 (calciferol) levels - Source of vitamin D3: Rovimix D Dietary concentrations after analysis: 11, 28, 42, 120 IU VD3 per gram of diet -Assesment of growth, ossification level, skeletal deformities, gene expression at day 11, 22 and 45 posthatching 5

6 Gene expression in 45 dph larvae fed diet with different vit D levels: Verification with qpcr Control diet Control diet 0, Relative expression of BMP-4 0,5 0,4 0,3 0,2 0, Relative expression of Osteocalcin diet VD level (IU VD3 per gram of diet) diet VD level (IU VD3 per gram of diet) BMP-4 involved in the differentiation of the bipotent adipocyte-osteoblast cells Osteocalcin associated with the mineralization of the extracellular matrix Control diet 4 3,5 Ossification level 3 2,5 2 1,5 1 0, diet VD level (IU VD3 per gram of diet) Vit D induces expression of genes involved in cell differentiation processes including osteoblast formation Vitamin D 1,25(OH)D 3 D Nucleus D VDR RXR VDRE Cell differentiation GADD45G MLF1 SYNE1 Skeletal formation COL BMP-4 Osteocalcin MGP; SOX9; TUFT1 Cell proliferation POLD4 ANAPC7 RAN BUB1B KIF23 6

Conclusions Transcriptomic data allows a better understanding of the impact of vitamin D deficiency on different biological processes involved in ontogenesis (cell differentiation, neurogenesis,

Sevrage précoce : remplacement de la proie vivante Artémia salina par des microparticules d aliment composé lors de la phase précoce d élevage Ombrine Sciaenops ocellatus : Une formule nutritionnelle

7 Conclusions Transcriptomic data allows a better understanding of the impact of vitamin D deficiency on different biological processes involved in ontogenesis (cell differentiation, neurogenesis, ossification ) More generally, the transcriptomic approach will be interesting to have a global understanding of the effect of one or several environmental parameters on fish larval development. Sevrage précoce : remplacement de la proie vivante Artémia salina par des microparticules d aliment composé lors de la phase précoce d élevage Ombrine Sciaenops ocellatus : Une formule nutritionnelle adaptée Facteurs testés Rotifères+ A0 +A1 Séquence standard Rotifères + A1 Rotifères + micro Particules Sevrage précoce Platax orbicularis Poids (g) Témoin Micoparticules t J0 R.A.T J15 J25 J30 J40 J50 J60 100% 80% 60% 40% 20% 0% % R.A.T Performances Rapportées Aux Témoins proies vivantes Survie équivalente en fin de phase larvaire (75 à 95 %) Croissance inférieure en fin de phase larvaire (j20) Mais compensation en fin d alevinage (j60) Population plus homogène en taille ( longueur) Absence de prédation intra spécifique Résultats : Survie plus faible mais 3 entre 1 er et 2 ème essai Croissance plus faible en larvaire mais croissance compensatrice et rattrapage en alevinage (J40) Larves plus homogènes en fin de larvaire Alevins de qualité morphologique identique aux autres régimes 14 7

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Instituto de Acuiculturade Torre de la Sal (CSIC), Castellón, Spain. Laboratoire de Physiologie et Génomique des Poissons (INRA), Rennes, France

Instituto de Acuiculturade Torre de la Sal (CSIC), Castellón, Spain. Laboratoire de Physiologie et Génomique des Poissons (INRA), Rennes, France 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