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

Fernández I, Darias M, Andree KB, Mazurais D, Zambonino-Infante JL, Gisbert E - BMC Dev. Biol. (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unitat de Cultius Experimentals, IRTA Centre de Sant Carles de la Ràpita (IRTA-SCR), Crta, del Poble Nou s/n, 43540 - Sant Carles de la Ràpita (Spain) ignacio.fernandez@irta.es

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 β-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 α1 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.

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Gilthead sea bream bone mineralization at 60 dph of fishes from the different dietary treatments. Bone mineralization measured as ratios of red pixels (a), blue pixels per larval surface (b), and red pixels over blue pixels (c). Ratios are expressed as mean ± standard deviation. Letters denotes significant differences between dietary groups (ANOVA, P < 0.05; n = 26 larvae per treatment). C, larvae fed with control diet (0.66*108 total VA IU kg-1 DW); 1.5×VA, larvae fed with 1.5 fold increase in dietary VA content (1.00*108 total VA IU kg-1 DW); 10×VA, larvae fed with 10 fold increase in dietary VA content (6.82*108 total VA IU kg-1 DW).
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Figure 2: Gilthead sea bream bone mineralization at 60 dph of fishes from the different dietary treatments. Bone mineralization measured as ratios of red pixels (a), blue pixels per larval surface (b), and red pixels over blue pixels (c). Ratios are expressed as mean ± standard deviation. Letters denotes significant differences between dietary groups (ANOVA, P < 0.05; n = 26 larvae per treatment). C, larvae fed with control diet (0.66*108 total VA IU kg-1 DW); 1.5×VA, larvae fed with 1.5 fold increase in dietary VA content (1.00*108 total VA IU kg-1 DW); 10×VA, larvae fed with 10 fold increase in dietary VA content (6.82*108 total VA IU kg-1 DW).

Mentions: Regarding the bone mineralization of larvae, bone and cartilage staining quantification performed by IMAQ Vision Builder is summarized in Figure 2. Mineralization values were expressed as the ratio of red/blue colour as well as the specific amounts of each colour per larval surface, from stained fish for each experimental group. There were three patterns observed (Figure 3): (i) in control larvae most of the structures were red coloured with the exception of few structures (mainly pterygiophores and sclerotic related structures); (ii) the 1.5×VA larvae had many structures quite blue in colouration (pectoral and caudal fin related structures, pterygiophores, and splanchnocranium related structures such as frontral, pterotic, sphenotic and sclerotic related structures); and (iii) in the 10×VA larvae blue colouration was intermediate with respect to the previously enumerated structures of 1.5×VA larvae (skeletal structure nomenclature was as in [9-11]). Mineralization values in 1.5×VA and 10×VA larvae at 60 dph were higher than in the control group, although there were no significant differences (ANOVA, P > 0.05; Figure 2a). The absence of a statistically significant difference between treatments is likely due to the high variability observed among replicates from each treatment. Interestingly, significantly higher cartilage staining was found in 1.5×VA and 10×VA larvae with respect to the control group (ANOVA; P < 0.05; Figure 2b). In addition, the ratio of red/blue coloration (bone/cartilage mineralization) showed that both larvae fed with VA supplemented diets (1.5×VA and 10×VA) had lower values than the control group; although this tendency was not statistically significant (ANOVA, P > 0.05; Figure 2c).


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

Fernández I, Darias M, Andree KB, Mazurais D, Zambonino-Infante JL, Gisbert E - BMC Dev. Biol. (2011)

Gilthead sea bream bone mineralization at 60 dph of fishes from the different dietary treatments. Bone mineralization measured as ratios of red pixels (a), blue pixels per larval surface (b), and red pixels over blue pixels (c). Ratios are expressed as mean ± standard deviation. Letters denotes significant differences between dietary groups (ANOVA, P < 0.05; n = 26 larvae per treatment). C, larvae fed with control diet (0.66*108 total VA IU kg-1 DW); 1.5×VA, larvae fed with 1.5 fold increase in dietary VA content (1.00*108 total VA IU kg-1 DW); 10×VA, larvae fed with 10 fold increase in dietary VA content (6.82*108 total VA IU kg-1 DW).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045981&req=5

Figure 2: Gilthead sea bream bone mineralization at 60 dph of fishes from the different dietary treatments. Bone mineralization measured as ratios of red pixels (a), blue pixels per larval surface (b), and red pixels over blue pixels (c). Ratios are expressed as mean ± standard deviation. Letters denotes significant differences between dietary groups (ANOVA, P < 0.05; n = 26 larvae per treatment). C, larvae fed with control diet (0.66*108 total VA IU kg-1 DW); 1.5×VA, larvae fed with 1.5 fold increase in dietary VA content (1.00*108 total VA IU kg-1 DW); 10×VA, larvae fed with 10 fold increase in dietary VA content (6.82*108 total VA IU kg-1 DW).
Mentions: Regarding the bone mineralization of larvae, bone and cartilage staining quantification performed by IMAQ Vision Builder is summarized in Figure 2. Mineralization values were expressed as the ratio of red/blue colour as well as the specific amounts of each colour per larval surface, from stained fish for each experimental group. There were three patterns observed (Figure 3): (i) in control larvae most of the structures were red coloured with the exception of few structures (mainly pterygiophores and sclerotic related structures); (ii) the 1.5×VA larvae had many structures quite blue in colouration (pectoral and caudal fin related structures, pterygiophores, and splanchnocranium related structures such as frontral, pterotic, sphenotic and sclerotic related structures); and (iii) in the 10×VA larvae blue colouration was intermediate with respect to the previously enumerated structures of 1.5×VA larvae (skeletal structure nomenclature was as in [9-11]). Mineralization values in 1.5×VA and 10×VA larvae at 60 dph were higher than in the control group, although there were no significant differences (ANOVA, P > 0.05; Figure 2a). The absence of a statistically significant difference between treatments is likely due to the high variability observed among replicates from each treatment. Interestingly, significantly higher cartilage staining was found in 1.5×VA and 10×VA larvae with respect to the control group (ANOVA; P < 0.05; Figure 2b). In addition, the ratio of red/blue coloration (bone/cartilage mineralization) showed that both larvae fed with VA supplemented diets (1.5×VA and 10×VA) had lower values than the control group; although this tendency was not statistically significant (ANOVA, P > 0.05; Figure 2c).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unitat de Cultius Experimentals, IRTA Centre de Sant Carles de la Ràpita (IRTA-SCR), Crta, del Poble Nou s/n, 43540 - Sant Carles de la Ràpita (Spain) ignacio.fernandez@irta.es

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 β-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 α1 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.

Show MeSH
Related in: MedlinePlus