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Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

ABSTRACT: BACKGROUND: Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor ?-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen ?1 chain (COL1A1) have been studied in gilthead sea bream. RESULTS: During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. CONCLUSIONS: Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages.

SUBMITTER: Fernandez I

PROVIDER: S-EPMC3045981 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

Fernández Ignacio I Darias Maria M Andree Karl B KB Mazurais David D Zambonino-Infante Jose Luís JL Gisbert Enric E

BMC developmental biology 20110209

<h4>Background</h4>Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in m ...[more]

PMID: 21306609

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Project description:BACKGROUND: Aquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved. The application of genomic technologies offers much promise to rapidly increase our knowledge on biological processes in farmed species and overcome such bottlenecks. Here we present an integrated platform for mRNA expression profiling in the gilthead sea bream (Sparus aurata), a marine teleost of great importance for aquaculture. RESULTS: A public data base was constructed, consisting of 19,734 unique clusters (3,563 contigs and 16,171 singletons). Functional annotation was obtained for 8,021 clusters. Over 4,000 sequences were also associated with a GO entry. Two 60mer probes were designed for each gene and in-situ synthesized on glass slides using Agilent SurePrint technology. Platform reproducibility and accuracy were assessed on two early stages of sea bream development (one-day and four days old larvae). Correlation between technical replicates was always > 0.99, with strong positive correlation between paired probes. A two class SAM test identified 1,050 differentially expressed genes between the two developmental stages. Functional analysis suggested that down-regulated transcripts (407) in older larvae are mostly essential/housekeeping genes, whereas tissue-specific genes are up-regulated in parallel with the formation of key organs (eye, digestive system). Cross-validation of microarray data was carried out using quantitative qRT-PCR on 11 target genes, selected to reflect the whole range of fold-change and both up-regulated and down-regulated genes. A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates. Good concordance between qRT-PCR and microarray data was observed between 2- and 7-fold change, while fold-change compression in the microarray was present for differences greater than 10-fold in the qRT-PCR. CONCLUSION: A highly reliable oligo-microarray platform was developed and validated for the gilthead sea bream despite the presently limited knowledge of the species transcriptome. Because of the flexible design this array will be able to accommodate additional probes as soon as novel unique transcripts are available.

Project description:Proteolytic systems exert an important role in vertebrate muscle controlling protein turnover, recycling of amino acids (AA) or its use for energy production, as well as other functions like myogenesis. In fish, proteolytic systems are crucial for the relatively high muscle somatic index they possess, and because protein is the most important dietary component. Thus in this study, the molecular profile of proteolytic markers (calpains, cathepsins and ubiquitin-proteasome system (UbP) members) were analyzed during gilthead sea bream (Sparus aurata) myogenesis in vitro and under different AA treatments. The gene expression of calpains (capn1, capn3 and capns1b) decreased progressively during myogenesis together with the proteasome member n3; whereas capn2, capns1a, capns1b and ubiquitin (ub) remained stable. Contrarily, the cathepsin D (ctsd) paralogs and E3 ubiquitin ligases mafbx and murf1, showed a significant peak in gene expression at day 8 of culture that slightly decreased afterwards. Moreover, the protein expression analyzed for selected molecules presented in general the same profile of the mRNA levels, which was confirmed by correlation analysis. These data suggest that calpains seem to be more important during proliferation, while cathepsins and the UbP system appear to be required for myogenic differentiation. Concerning the transcriptional regulation by AA, the recovery of their levels after a short starvation period did not show effects on cathepsins expression, whereas it down-regulated the expression of capn3, capns1b, mafbx, murf1 and up-regulated n3. With regards to AA deficiencies, the major changes occurred at day 2, when leucine limitation suppressed ctsb and ctsl expression. Besides at the same time, both leucine and lysine deficiencies increased the expression of mafbx and murf1 and decreased that of n3. Overall, the opposite nutritional regulation observed, especially for the UbP members, points out an efficient and complementary role of these factors that could be useful in gilthead sea bream diets optimization.

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Dataset Information

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

Publications

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

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