Maria J. Darias

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

BackgroundVitamin 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.ResultsDuring 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.ConclusionsPresent 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.

Development temperature has persistent effects on muscle growth responses in gilthead sea bream

Initially we characterised growth responses to altered nutritional input at the transcriptional and tissue levels in the fast skeletal muscle of juvenile gilthead sea bream. Fish reared at 21–22°C (range) were fed a commercial diet at 3% body mass d−1 (non-satiation feeding, NSF) for 4 weeks, fasted for 4d (F) and then fed to satiation (SF) for 21d. 13 out of 34 genes investigated showed consistent patterns of regulation between nutritional states. Fasting was associated with a 20-fold increase in MAFbx, and a 5-fold increase in Six1 and WASp expression, which returned to NSF levels within 16h of SF. Refeeding to satiation was associated with a rapid (<24 h) 12 to 17-fold increase in UNC45, Hsp70 and Hsp90α transcripts coding for molecular chaperones associated with unfolded protein response pathways. The growth factors FGF6 and IGF1 increased 6.0 and 4.5-fold within 16 h and 24 h of refeeding respectively. The average growth in diameter of fast muscle fibres was checked with fasting and significant fibre hypertrophy was only observed after 13d and 21d SF. To investigate developmental plasticity in growth responses we used the same experimental protocol with fish reared at either 17.5–18.5°C (range) (LT) or 21–22°C (range) (HT) to metamorphosis and then transferred to 21–22°C. There were persistent effects of development temperature on muscle growth patterns with 20% more fibres of lower average diameter in LT than HT group of similar body size. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups, whereas there were no differences in the expression of MAFbx and Six1. It was concluded that myogenesis and gene expression patterns during growth are not fixed, but can be modified by temperature during the early stages of the life cycle.

Isolipidic diets differing in their essential fatty acid profiles affect the deposition of unsaturated neutral lipids in the intestine, liver and vascular system of Senegalese sole larvae and early juveniles

Abstract How lipid content and composition in the diet is utilized by the various organs and tissues of fish is reflected in their structure, such as the intestine through which dietary lipids are digested and absorbed, the vascular system which is involved in their transport, and the liver where lipids are stored and metabolized. However, no study has been conducted to compare the effect of different diets containing different levels of highly unsaturated fatty acids (HUFA) and essential fatty acids (EFA) on lipid deposition in fish larvae and early juveniles. Thus, we evaluated the effects of six isolipidic diets (enriched Artemia salina ), differing in their fatty acid profile, on the lipid accumulation patterns in selected target tissues (intestine, liver and vascular system) in Senegalese sole ( Solea senegalensis ) larvae and early juveniles. Results showed that the profile of fat accumulation in these three tissues was significantly affected by the dietary treatments, the developmental stage of the fish (premetamorphosis, metamorphosis or postmetamorphosis), as well as by the interaction between these two factors that were responsible for changes in the histological organization of the tissues. Histological results revealed that a slight variation in the EFA levels (e.g. EPA, DHA or ARA) or in their ratios (EPA/DHA, ARA/EPA, ARA/DHA, (n-3)/(n-6) HUFA, OA/PUFA) modified the metabolism of lipids and disrupted the pattern of lipid accumulation in the target tissues, leading to intestinal and hepatic steatosis.

Molecular regulation of both dietary vitamin A and fatty acid absorption and metabolism associated with larval morphogenesis of Senegalese sole (Solea senegalensis)

The present study aimed to deepen the understanding of molecular mechanisms governing the absorption and metabolism of some nutrients, growth and development in larvae of Senegalese sole (Solea senegalensis) fed with Artemia enriched with Easy Selco (ES, INVE) or Aquagrow Gold (AGG, ABN), which mainly differed in their vitamin A (VA) content and fatty acid composition. The expression profile of genes involved in VA metabolism (crbp2, rbp, crabp1), lipid transport (i-fabp, l-fabp), nuclear receptors for VA and fatty acids (rarα1, rxrα, pparβ), growth (igf1, igf2 and their receptor igf1r) and development (bgp) was analyzed at 22, 30 and 38 days post hatching. The main results suggested that the amount of VA absorbed by larvae is controlled at the intestinal level by crbp2 in both groups, preventing excessive accumulation of this vitamin in the target tissues. The stable expression of i-fabp in the ES group with age could cause an excessive fat accumulation in the intestine inducing, in turn, the steatosis found in the liver and vascular system of these specimens. In liver, the regulation of rbp and fabp expression reflected the status of the physiological functions demanding VA and lipids. The findings revealed that dietary composition induced different strategies for VA and lipid absorption and metabolism affecting, in turn, larval development, growth and health.

Coordinated Regulation of Chromatophore Differentiation and Melanogenesis during the Ontogeny of Skin Pigmentation of Solea senegalensis (Kaup, 1858)

Abnormal pigmentation of Senegalese sole has been described as one problem facing the full exploitation of its commercial production. To improve our understanding of flatfish pigmentation of this commercially important species we have evaluated eleven genes related to two different processes of pigmentation: melanophore differentiation, and melanin production. The temporal distribution of gene expression peaks corresponds well with changes in pigmentation patterns and the intensity of skin melanization. Several gene ratios were also examined to put in perspective possible genetic markers for the different stages of normal pigmentation development. Further, the phenotypic changes that occur during morphogenesis correspond well with the main transitions in gene expression that occur. Given the dramatic phenotypic alterations which flatfish undergo, including the asymmetric coloration that occurs between the ocular and the blind side, and the synchrony of the two processes of morphogenesis and pigmentation ontogenesis, these species constitute an interesting model for the study of pigmentation. In this study we present a first approximation towards explaining the genetic mechanisms for regulating pigmentation ontogeny in Senegalese sole, Solea senegalensis.

High dietary arachidonic acid levels affect the process of eye migration and head shape in pseudoalbino Senegalese sole Solea senegalensis early juveniles

The effect of high dietary levels of arachidonic acid (ARA) on the eye migration and cranial bone remodelling processes in Senegalese sole Solea senegalensis early juveniles (age: 50 days post hatch) was evaluated by means of geometric morphometric analysis and alizarin red staining of cranial skeletal elements. The incidence of normally pigmented fish fed the control diet was 99·1 ± 0·3% (mean ± s.e.), whereas it was only 18·7 ± 7·5% for those fed high levels of ARA (ARA-H). The frequency of cranial deformities was significantly higher in fish fed ARA-H (95·1 ± 1·5%) than in those fed the control diet (1·9 ± 1·9%). Cranial deformities were significantly and negatively correlated with the incidence of normally pigmented animals (r² = -0·88, P < 0·001, n = 16). Thus, fish displaying pigmentary disorders differed in the position of their eyes with regard to the vertebral column and mouth axes, and by the interocular distance and head height, which were shorter than in fish not displaying pigmentary disorders. In addition to changes in the positioning of both eyes, pseudoalbino fish showed some ARA-induced osteological differences for some of the skeletal elements from the splanchnocranium (e.g. right premaxillary, dentary, angular, lacrimal, ceratohyal and branchiostegal rays) and neurocranium (e.g. sphenotic, left lateral ethmoid and left frontal) by comparison to normally pigmented specimens. Pseudoalbino fish also had teeth in both lower and upper jaws. This is the first study in Pleuronectiformes that describes impaired metamorphic relocation of the ocular side eye, the right eye in the case of S. senegalensis, whereas the left eye migrated into the ocular side almost normally.

Morphological and Molecular Characterization of Dietary-Induced Pseudo-Albinism during Post-Embryonic Development of Solea senegalensis (Kaup, 1858)

The appearance of the pseudo-albino phenotype was investigated in developing Senegalese sole (Solea senegalensis, Kaup 1858) larvae at morphological and molecular levels. In order to induce the development of pseudo-albinos, Senegalese sole larvae were fed Artemia enriched with high levels of arachidonic acid (ARA). The development of their skin pigmentation was compared to that of a control group fed Artemia enriched with a reference commercial product. The relative amount of skin melanophores, xanthophores and iridophores revealed that larval pigmentation developed similarly in both groups. However, results from different relative proportions, allocation patterns, shapes and sizes of skin chromatophores revealed changes in the pigmentation pattern between ARA and control groups from 33 days post hatching onwards. The new populations of chromatophores that should appear at post-metamorphosis were not formed in the ARA group. Further, spatial patterns of distribution between the already present larval xanthophores and melanophores were suggestive of short-range interaction that seemed to be implicated in the degradation of these chromatophores, leading to the appearance of the pseudo-albino phenotype. The expression profile of several key pigmentation-related genes revealed that melanophore development was promoted in pseudo-albinos without a sufficient degree of terminal differentiation, thus preventing melanogenesis. Present results suggest the potential roles of asip1 and slc24a5 genes on the down-regulation of trp1 expression, leading to defects in melanin production. Moreover, gene expression data supports the involvement of pax3, mitf and asip1 genes in the developmental disruption of the new post-metamorphic populations of melanophores, xanthophores and iridophores.

Vitamin A Affects Flatfish Development in a Thyroid Hormone Signaling and Metamorphic Stage Dependent Manner

Vitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro- or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.

Fat-soluble vitamins in fish: A transcriptional tissue-specific crosstalk that remains to be unveiled and characterized

Fat-soluble vitamins play essential roles in vertebrate’s development and homeostasis, and thus, an optimal, efficient and sustainable fish farming deeply depends on the optimization of their dietary levels provided. Subsequently, nutritional imbalances are considered one of the major causative factors of vertebrate’s abnormal development. Although approaches such as nutritional-dose-response trials, gene knock-down and over-expression studies, have provided valuable knowledge on its metabolism and dietary requirements; this knowledge is still mostly based on studies with mammalian species. Even though nutritional approaches involving different (i) research tools (i.e. NGS, RT-qPCR, proteomics, histology, immunohistochemistry, in situ hybridization, etc.), (ii) experimental approaches (in vivo and in vitro), and (iii) developmental stages (larval, juvenile and adult stages), have been applied to different fish species, the biological roles, underlying mechanisms and nutritional requirements in farmed fish are not fully understood yet. Here, knowledge gained during the last decade for each of the fat-soluble vitamins is compiled, reviewed from a holistic point of view, and the potential points of convergence of fat-soluble vitamins signaling pathways at molecular, cellular and tissue levels identified for the proper development of nutritionally balanced diets, based on integrative, multifactorial and multidisciplinary nutritional studies to be conducted in the nearest future.