Nádia Silva currently works at the CEIB group, Centro de Ciências do Mar. Nádia does research in Developmental Biology and Endocrinology. Their most recent publication is 'Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis.'
Research Items (34)
- Jan 2015
Anuran and flatfish metamorphosis are tightly regulated by thyroid hormones that are the necessary and sufficient factors that drive this developmental event. In the present study whole mount in situ hybridisation (WISH) and quantitative PCR in sole is used to explore the central regulation of flatfish metamorphosis. Central regulation of the thyroid in vertebrates is mediated by the hypothalamus-pituitary-thyroid (HPT) axis. Teleosts diverge from other vertebrates as hypothalamic regulation in the HPT axis is proposed to be through hypothalamic inhibition although the regulatory factor remains enigmatic. The dynamics of the HPT axis during sole metamorphosis revealed integration between the activity of the thyrotrophes in the pituitary and the thyroid follicles. No evidence was found supporting a role for thyroid releasing hormone (trh) or corticotrophin releasing hormone (crh) in hypothalamic control of TH production during sole metamorphosis. Intriguingly the results of the present study suggest that neither hypothalamic trh nor crh expression changes during sole metamorphosis and raises questions about the role of these factors and the hypothalamus in regulation of thyrotrophs. Copyright © 2014. Published by Elsevier Ireland Ltd.
- Mar 2007
Developmental models for skin exist in terrestrial and amphibious vertebrates but there is a lack of information in aquatic vertebrates. We have analysed skin epidermal development of a bony fish (teleost), the most successful group of extant vertebrates. A specific epidermal type I keratin cDNA (hhKer1), which may be a bony-fish-specific adaptation associated with the divergence of skin development (scale formation) compared with other vertebrates, has been cloned and characterized. The expression of hhKer1 and collagen 1alpha1 in skin taken together with the presence or absence of keratin bundle-like structures have made it possible to distinguish between larval and adult epidermal cells during skin development. The use of a flatfish with a well-defined larval to juvenile transition as a model of skin development has revealed that epidermal larval basal cells differentiate directly to epidermal adult basal cells at the climax of metamorphosis. Moreover, hhKer1 expression is downregulated at the climax of metamorphosis and is inversely correlated with increasing thyroxin levels. We suggest that, whereas early mechanisms of skin development between aquatic and terrestrial vertebrates are conserved, later mechanisms diverge.
Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.
Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.
- Sep 2017
- EMBO Conference Gene Regulatory Mechanisms in Neural Fate Decisions
Thyroid hormones (THs) are essential for embryonic brain development, but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. Here to begin to understand the underlying genetic mechanisms under MTHs regulation we used an established zebrafish MCT8 knockdown model and performed transcriptome analysis in morphant and control zebrafish embryos. Subsequent mapping of differentially expressed genes in Reactome pathway analysis together with in situ expression analysis and immunohistochemistry allowed to unravel the genetic networks and cells under MTHs regulation in zebrafish embryogenesis. We found 4,343 differentially expressed genes and Reactome pathway analysis revealed TH is involved in 1681 pathways. Notably, MTHs regulate the expression of core developmental pathways, such as WNT and NOTCH in a cell specific context. Cellular distribution of neural MTHs-target genes demonstrated cell specific action for MTHs in neural stem cells maintenance and fate decision of neuron classes. Together our data shows that MTH acts as an integrator by regulating the expression of genes involved in the cross-talk between key pathways in neural development during zebrafish embryogenesis. All in line with observed knockdown phenotype and the phenotype of human AHDS patients our data is able to clarify the genetic origins behind this human congenital condition.
- Sep 2012
Studies on the role of thyroid hormones (THs) in teleost fish physiology have deployed the synthetic goitrogens, methimazol (MMI), propilthiouracil (PTU) and thiourea (TU) that are used to treat human hyperthyroidism. However, the action of the goitrogens, MMI, PTU and TU at different levels of the hypothalamic-pituitary-thyroid (HPT) axis in teleosts is largely unknown. The central importance of the hypothalamus and pituitary in a number of endocrine regulated systems and the cross-talk that occurs between different endocrine axes makes it pertinent to characterize the effects of MMI, PTU and TU, on several endpoints of the thyroid system. The marine teleost, sea bream (Sparus auratus) was exposed to MMI, PTU and TU (1mg/kg wet weight per day), via the diet for 21days. Radioimmunoassays (RIA) of plasma THs and ELISA of the TH carrier transthyretin (TTR) revealed that MMI was the only chemical that significantly reduced plasma TH levels (p<0.05), although both MMI and PTU significantly (p<0.05) reduced plasma levels of circulating TTR (p<0.05). Histological analysis of the thyroid tissue revealed modifications in thyrocyte activity that explain the reduced circulating levels of THs. MMI also significantly (p<0.05) up-regulated transcript abundance of liver deiodinase 1 and 2 while significantly (p<0.05) decreasing TRβ expression in the pituitary, all hallmarks of HPT axis action of goitrogens in vertebrates. The results indicate that in the sea bream MMI is the most effective goitrogen followed by PTU and that TU (1mg/kg wet weight for 21days) failed to have a goitrogenic effect. The study highlights the non-uniform effect of goitrogens on the thyroid axis of sea bream and provides the basis for future studies of thyroid disrupting pollutants.
- Aug 2012
Flatfish metamorphosis is the most dramatic post-natal developmental event in teleosts. Thyroid hormones (TH), thyroxine (T4) and 3,3'-5'-triiodothyronine (T3) are the necessary and sufficient factors that induce and regulate flatfish metamorphosis. Most of the cellular and molecular action of TH is directed through the binding of T3 to thyroid nuclear receptors bound to promoters with consequent changes in the expression of target genes. The conversion of T4 to T3 and nuclear availability of T3 depends on the expression and activity of a family of 3 selenocysteine deiodinases that activate T4 into T3 or degrade T4 and T3. We have investigated the role of deiodinases in skin and muscle metamorphic changes in halibut. We show that, both at the whole body level and at the cellular level in muscle and skin of the Atlantic halibut (Hippoglossus hippoglossus) during metamorphosis, the coordination between activating (D2) and deactivating (D3) deiodinases expression is strongly correlated with the developmental TH-driven changes. The expression pattern of D2 and D3 in cells of both skin and muscle indicate that TH are necessary for the maintenance of larval metamorphic development and juvenile cell types in these tissues. No break in symmetry occurs in the expression of deiodinases and in metamorphic developmental changes occurring both in trunk skin and muscle. The findings that two of the major tissues in both larvae and juveniles maintain their symmetry throughout metamorphosis suggest that the asymmetric changes occurring during flatfish metamorphosis are restricted to the eye and head region.
Stanniocalcin (STC), first isolated from the corpuscles of Stannius (CS) of teleost fishes and a systemic regulator of mineral metabolism, is present in all vertebrates as two isoforms, STC1 and STC2, encoded by separate genes. Here we show that the genome of Tetraodon nigroviridis, and other teleosts, possess duplicate genes for each STC isoform, designated stc1-a and -b, and stc2-a and -b. Stc1-a was cloned from CS, stc2-a from muscle and the two novel cDNAs, stc1-b and stc2-b, from brain. However, stc2-b was isolated as a conjoined (read-through) transcript with bod1 (bi-orientation defective 1, or FAM44B), and two additional alternative conjoined transcripts were also isolated. The predicted STC products shared the typical vertebrate 10 conserved cysteine residues and N-linked glycosylation motifs, in addition to specific features. Gene structure was generally conserved with four exons and three introns with the exception of stc1-a which gained an extra intron in exon three, originating one extra exon. Gene order and synteny is also maintained across vertebrates and the cpeb4 gene identified in the homologue region of the chordate Ciona was linked to vertebrate stc2 but not stc1. Immunohistochemistry in different species revealed that STC1-A was found only in CS and in a few cells in kidney. STC1-B had a restricted expression and was more prominent in the gills. STC2-A was detected in a variety of tissues, including pituitary, with most abundant immunoreaction in kidney cells and gill rakers and the CS was negative. Expression of stc1-a in CS of Tetraodon was 15-fold (p<0.05) up-regulated 2 h after transfer from 2.9 mM Ca(2+) to 10 mM Ca(2+) water and down-regulated after 12 hours to 11-fold lower than 2.9 mM Ca(2+) fish (p<0.05). With the exception of stc1-a in CS, low expression levels and high individual variation were generally found for the expression of stc transcripts in kidney and gills, with no statistically significant changes in response to the hypercalcemic shock. In conclusion, both stc1 and stc2 genes are represented by paralogues in teleosts genomes and the analysis performed suggests that only stc1-a in the CS is involved in extracellular calcium regulation. The widespread distribution of stcs in fish tissues supports pleiotropic roles.
Microbubbles are used to improve ultrasound imaging of the vascular bed. Optical microscopy has shown microbubbles in different size tubes which have different responses to ultrasound. The acoustic scatter associated with such differences has not been previously measured. Echoes from two types of microbubbles, in narrow tubes, were collected at incident ultrasound parameters relevant to diagnostic imaging. Microbubbles were found to have increased second harmonic signatures in 50 μm diameter tubes compared to 200 μm. There was decreased survival of lipid microbubbles in the smaller tube. Understanding scatter mechanisms in narrow tubes is useful for signal processing optimisation for imaging applications.
- Mar 2011
Modifications have been characterised in terms of cellular organisation and the extracellular matrix (ECM) during bone ontogeny in the sea bream (Sparus auratus). During endochondral development, the agglomeration of matrix-secreting cells gives rise to chondrones; these chondrones frequently contain proliferating-cell-nuclear-antigen-positive cells, which subsequently become large collagen-II-positive cells with the characteristics of chondrocytes. Moreover, the matrix:cell ratio within the perichondrium increases, accompanied by a modification in ECM composition. Mineralisation of cartilage ECM is marked by a rapid fall in cell number, the switching off of collagen II transcription and the switching on of collagen X transcription, followed by collagen I transcription and bone mineralisation. The formation of dermal structures initiated upon the condensation of mesenchyme cells defines the future location of the dermal bone. Subsequent cellular differentiation gives rise to cells on the bone surface; these cells are positive for collagen I and osteonectin transcripts. The fish skeleton, with the exception of vertebrae, tends to comprise flattened bones that are covered by a monolayer of cells, the periosteum. A third type of tissue, present in gills, consists of chondrocyte-like cells embedded in a mineralised matrix resembling chondroid bone in mammals. The results suggest that the cellular organisation and ontogeny of endochondral and dermal bone in the sea bream are similar to those described in other vertebrates.
- Jan 2011
Ultrasound contrast agents have been the subject of microvascular imaging research. The sheep corpus luteum (CL) is a microvascular tissue that provides a natural angiogenic and antiangiogenic process, which changes during the luteal phase of the estrous cycle of the ewe. It can also be controlled and monitored endocrinologically, providing a very attractive in vivo model for the study and development of microvascular measurement. The perfusion of the fully developed CL between days 8 and 12 of the estrous cycle was studied in six ewes. A Philips iU22 ultrasound scanner (Bothell, WA, USA) with the linear array probe L9-3 was used to capture contrast-enhanced images after an intravenous bolus injection of 2.4 mL SonoVue (Bracco S.P.A., Milan, Italy). Time-intensity curves of a region of interest inside the CL were formed from linearized image data. A lagged-normal model to simulate the compartmental kinetics of the microvascular flow was used to fit the data, and the wash-in time was measured. Good contrast enhancement was observed in the CLs of all animals and the wash-in time averaged at 5.5 s with 9% uncertainty. The regression coefficient was highly significant for all fits. These data correlated with stained endothelial area in the histology performed postmortem. Two ewes were injected with prostaglandin F2alpha to induce CL regression, which resulted in an increase of wash-in time after a few hours. The CL of the ewe is thus proposed as an ideal model for the study and development of microvascular measurements using contrast ultrasound. Our initial results demonstrate a highly reproducible model for the study of the microvascular hemodynamics in a range of tissues and organs.
- Dec 2010
Proopiomelanocorticotrophin (POMC) in vertebrates is produced in the pituitary gland and undergoes post-translational processing to give rise to a range of biologically active peptides. Teleosts possess 2-3 different POMC transcripts which have been proposed to have originated from a whole or partial genome duplication. In the present study 2 transcripts of gilthead sea bream POMC (sbPOMC-α1 and α2) were cloned and characterised. sbPOMC-α1 is expressed principally in the melanotroph cells of the pars intermedia (PI) and sbPOMC-α2 is expressed in the corticotroph cells of the rostral pars distalis and probably also in the PI. The 2 sbPOMC transcripts have a differential tissue distribution in extra-pituitary sites. An appraisal of POMC evolution indicates sbPOMCs belong to one of the two main clades that exist in teleosts and that overall a non conservative process of gene loss occurred in this infraclass.
Previous studies on the histochemistry and immunoreactivity of fibres in lateral muscle of blackspot seabream indicated that there is a developmental transition in the composition of myofibrillar proteins, which presumably reflects changes in contractile function as the fish grows. We hypothesize that the phenomenon underscores age and spatial differences in the expression of myosin light chains (MLC), not studied yet in this species. In this study, we examined selected stages in the post-hatching development of the muscle of blackspot seabream: hatching (0 days), mouth opening (5 days), weaning (40 days) and juveniles (70 days). The spatial expression of embryonic MLC 1 (MLC1), 2 (MLC2) and 3 (MLC3) was studied by in situ hybridization. Overall, MLC expression patterns were overlapping and restricted to the fast muscle. At hatching and mouth opening, all MLC types were highly expressed throughout the musculature in fast muscle. The expression levels in fast muscle remained high until weaning when germinal zones appeared on the dorsal and ventral areas. The germinal zones were characterized by small-diameter fast fibres with high levels of MLC expression. This pattern persisted up to day 70, when the germinal zones disappeared and expression of MLCs was observed only in the smaller cells of the fast muscle mosaic. These results support our hypothesis and, together with previous imuno- and histochemistry results, allow a better understanding of the mechanism of muscle differentiation and growth in fish beyond larval stages, and form- the basis for further comparative and experimental studies with this economically relevant species.
- Feb 2010
Cartilage acidic protein 1 (CRTAC1) gene expression is used as a marker for chondrocyte differentiation in stem cell-based tissue engineering. It is also transcribed outside the skeleton where at least two different transcripts are expressed in lung and brain. In the pituitary gland of the teleost fish sea bream Sparus auratus, we have found a transcript with a high degree of sequence identity to CRTAC1 family members but lacking the EGF-like calcium-binding domain encoding sequence of CRTAC1 and designated it as CRTAC2. Database searches revealed many previously unidentified members of the CRTAC1 and CRTAC2 in phylogenetically distant organisms, such as cyanobacteria, bryophyta, lancelets, and diverse representatives of vertebrates. Phylogenetic analyses showed that the genes encoding CRTAC1 and CRTAC2 proteins coexist in teleost fish genomes. Structural prediction analysis identified the N-terminal region of the CRTAC1/CRTAC2 family members as a potential seven-bladed beta-propeller structure, closely related to those of integrin alpha chains and glycosylphosphatidylinositol-specific phospholipase D1 protein families. This relationship is confirmed by phylogenetic analysis with the N-terminal domain of sea bream CRTAC2 as the most divergent sequence. Because teleost fishes are the only phylogenetic group where both CRTAC1 and CRTAC2 genes are present, they occupy a pivotal position in studies of the mechanisms governing the specific expression patterns of each gene/protein subfamily. This will be essential to elucidate their respective biological roles.
- Dec 2009
The effects of dietary levels of phosphorus (P) and calcium (Ca) on skeletal development and mineral deposition in rainbow trout (Oncorhynchus mykiss) fry were studied. Six semi-purified diets were formulated with graded levels of P and Ca. The basal diet A contained only P supplied by casein at 0.5% of dry matter. Other diets B, C, D and E were supplemented with 0.4, 0.8, 1.2 and 1.6% P supplied as a 1:1 mixture of NaH2PO4/KH2PO4 resulting in 0.8, 1.2, 1.6 and 2.2% total P, respectively. These five diets were supplemented with 1% Ca supplied as CaCO3 whereas another diet F, supplemented with 0.8% P, was Ca-free. Each diet was distributed to 3 replicate tanks of 600 swim-up fry (initial mean weight: 0.1 g) at a water temperature of 17 °C over a 12-week growth trial. Fish were hand-fed 6 times a day to visual satiety.
A significant component of aquaculture is the production of good quality larvae, and, in the case of flatfish, this is tied up with the change from a symmetric larva to an asymmetric juvenile. Despite the pioneering work carried out on the metamorphosis of the Japanese flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus), the underlying molecular basis of flatfish metamorphosis is still relatively poorly characterized. It is a thyroid hormone (TH) driven process, and the role of other hormones in the regulation of the process along with the interplay of abiotic factors are still relatively poorly characterized as is the extent of tissue and organ remodeling, which underlie the profound structural and functional modifications that accompany the larval/juvenile transition. The isolation of genes for hormones, receptors, binding proteins, and other accessory factors has provided powerful tools with which to pursue this question. The application of molecular methodologies such as candidate gene approaches and microarray analysis coupled to functional genomics has started to contribute to understanding the complexity of tissue and organ modifications that accompany, flatfish metamorphosis. A better understanding of the biology, of normal metamorphosis is essential to identify factors contributing to abnormal metamorphosis.
Calcium regulation in sturgeon is of special interest because they are a representative of the ancient fishes possessing mainly cartilaginous skeletons and a supposedly low calcium demand. The present study aimed to characterize the effect of a chronic absence of dietary calcium and the effect of parathyroid hormone-related protein (PTHrPA) (1-34) (7) on calcium balance in juvenile sturgeon (Acipenser naccarii). At rest, sturgeon juveniles are in net positive calcium balance, since whole body calcium uptake is significantly higher than efflux and calcium accumulates in the body. To study the importance of dietary calcium, the sturgeon were kept on a calcium-free diet for 8 wk. This manipulation impaired growth as measured by failure to gain weight or increase in length and indicates that dietary calcium is important for growth in sturgeon. An increased whole body calcium uptake partially compensated dietary calcium deficiency and was associated with increased gill chloride cell number in lamellae and filaments in parallel with increased gill Na(+)K(+)-ATPase activity. In addition, a single injection of piscine PTHrP(1-34) significantly increased whole body calcium uptake and decreased whole body calcium efflux. Administration of PTHrP significantly increased circulating plasma calcium 4-24 h postinjection. The increase in net calcium transport and increased plasma levels of calcium is consistent with the actions of a hypercalcemic factor. It would appear that the sturgeon rely on calcium for growth and tightly regulate calcium transport. The action in calcium balance is consistent with PTHrP acting as a hypercalcemic factor in sturgeon.
Flatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT) expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.
- Jan 2007
The expression of fTnT genes in a flatfish which undergoes an overt TH driven metamorphosis and a roundfish which does not was studied throughout development and in adult tissue by means of Northen blot, semi-quantitative RT-PCR and in-situ hybridization. In both halibut (Hippoglossus hippoglossus) and sea bream (Sparus auratus) three alternative spliced forms of fTnT were identified which are generated by alternative exon splicing in the 5' region. In both halibut and sea bream the isoforms appear to be stage specific and correspond in sea bream to embryonic (efTnTsb), larval (LfTnTsb) and adult (afTnTsb) specific isoforms, while in halibut they correspond to embryonic/larval (efTnThh) and juvenile/adult (fTnThh-1 and -2) isoforms. In pre-metamorphic halibut larvae all three fTnThh isoforms are present although the most acidic form, efTnThh, is most abundant up until metamorphosis after which it is downregulated to almost undetectable levels. At metamorphosis and in subsequent stages fTnThh-2 is upregulated ∼3-fold and becomes the most abundant isoform in halibut muscle. In contrast, fTnThh-1 expression is constant throughout development. Thyroxine treatment of pre-metamorphic halibut larvae leads to the precocious downregulation of efTnThh. In contrast, in the sea bream each isoform is characteristic of the principal developmental stages and the embryonic-acidic isoform (efTnTsb) is downregulated immediately after hatching and is replaced by a larval isoform, LfTnTsb. T3-treatment of sea bream juveniles had no effect on fTnT isoform expression. The results from this comparative analysis of fTnTs in two teleosts suggests molecular processing is common but that regulation of their expression during muscle ontogeny may be species-specific and adapted to their specific ecologies.
The process of eye migration in bilaterally symmetrical flatfish larvae starts with asymmetrical growth of the dorsomedial parts of the ethmoid plate together with the frontal bones, structures initially found in a symmetrical position between the eyes. The movement of these structures in the future ocular direction exerts a stretch on the fibroblasts in the connective tissue found between the moving structures and the eye that is to migrate. Secondarily, a dense cell population of fibroblasts ventral to the eye starts to proliferate, possibly cued by the pulling forces exerted by the eye. The increased growth ventral to the eye pushes the eye dorsally. Osteoblasts are deposited in the dense cell layer, forming the dermal part of the lateral ethmoid, and at full eye migration this will cover the area vacated by the migrated eye. When the migrating eye catches up with the previous migrated dermal bones, the frontals, these bones will be remodelled to accommodate the eye. Our findings suggest that a combination of extremely localized signals and more distant factors may impinge upon the outcome of the tissue remodelling. Early normal asymmetry of signalling factors may cascade on a series of events.
Fish larval development, not least the spectacular process of flatfish metamorphosis, appears to be under complex endocrine control, many aspects of which are still not fully elucidated. In order to obtain data on the functional development of two major endocrine glands, the pituitary and the thyroid, during flatfish metamorphosis, histology, immunohistochemistry and in situ hybridization techniques were applied on larvae of the Atlantic halibut (Hippoglossus hippoglossus), a large, marine flatfish species, from hatching through metamorphosis. The material was obtained from a commercial hatchery. Larval age is defined as day-degrees (D degrees =accumulated daily temperature from hatching). Sporadic thyroid follicles are first detected in larvae at 142 D degrees (27 days post-hatch), prior to the completion of yolk sack absorption. Both the number and activity of the follicles increase markedly after yolk sack absorption and continue to do so during subsequent development. The larval triiodothyronine (T(3)) and thyroxine (T(4)) content increases, subsequent to yolk absorption, and coincides with the proliferation of thyroid follicles. A second increase of both T(3) and T(4) occurs around the start of metamorphosis and the T(3) content further increases at the metamorphic climax. Overall, the T(3) content is lower than T(4). The pituitary gland can first be distinguished as a separate organ at the yolk sack stage. During subsequent development, the gland becomes more elongated and differentiates into neurohypophysis (NH), pars distalis (PD) and pars intermedia (PI). The first sporadic endocrine pituitary cells are observed at the yolk sack stage, somatotrophs (growth hormone producing cells) and somatolactotrophs (somatolactin producing cells) are first observed at 121 D degrees (23 days post-hatch), and lactotrophs (prolactin producing cells) at 134 D degrees (25 days post-hatch). Scarce thyrotrophs are evident after detection of the first thyroid follicles (142 D degrees ), but coincident with a phase in which follicle number and activity increase (260 D degrees ). The somatotrophs are clustered in the medium ventral region of the PD, lactotrophs in the anterior part of the PD and somatolactotrophs are scattered in the mid and posterior region of the pituitary. At around 600 D degrees , coinciding with the start of metamorphosis, somatolactotrophs are restricted to the interdigitating tissue of the NH. During larval development, the pituitary endocrine cells become more numerous. The present data on thyroid development support the notion that thyroid hormones may play a significant role in Atlantic halibut metamorphosis. The time of appearance and the subsequent proliferation of pituitary somatotrophs, lactotrophs, somatolactotrophs and thyrotrophs indicate at which stages of larval development and metamorphosis these endocrine cells may start to play active regulatory roles.
Atlantic halibut is an important commercial fish in the countries of the North Atlantic and is emerging as a promising species for marine cold-water aquaculture. The axial musculature of the developing larvae is the largest and most rapidly growing tissue and during the transition from larval to adult muscle fibre types significant changes in fibre morphology and gene transcription occur. In fact the change in myotome height correlates well with different larval halibut stages. In the present study the spatial and temporal expression of myosin light chain 1 (MLC1), 2 (MLC2) and 3 (MLC3) was studied in metamorphosing halibut by in situ hybridization. As a first step to establishing a role for the thyroid axis in halibut muscle development whole body thyroid hormone (TH) concentrations were also determined. In first feeding larvae MLC1, MLC2A and MLC3 transcripts had a similar distribution and were confined to the muscle fibres of the germinal zones. In pre- metamorphic larvae transcripts were highly expressed throughout the epaxial and hypaxial musculature and expression levels reached a maximum in larvae starting metamorphosis, this change coincided with a significant increase in the concentration of thyroid hormones. By the time larvae reached the metamorphic climax, MLC1, MLC2A and MLC3 expression was still high throughout the musculature but expression was confined to fibres adjacent to the myosepts and to small cells scattered in the musculature, possibly satellite cells. MLC2A was also expressed in the red muscle fibres; no transition between larval and adult MLC isoforms was detected.
Myosin light chain 2 (MLC2) is an essential component of the myosin molecule, with a regulatory role in binding Ca2+. In gilthead sea bream, a MLC2 clone has been isolated and characterized, that encodes for a 170 aa peptide and contains three potential polyadenylation signals in the 3′ UTR. In this study, the isolation of three alternative transcripts of the already known MLC2 (isoform A) is reported, along with the isolation and characterization of a second MLC2 isoform (B). All three isoform A transcripts encode the same peptide but differ in the length of their 3′ UTRs (284bp, 788bp and 876bp respectively) and are generated by alternative polyadenylation site selection. Transcripts of isoform A were detected both in white and red muscle. MLC2 isoform B encodes also for a 170 aa protein. Isoform B was detected in all tissues examined (red, white, smooth and cardiac muscle, kidney, liver, spleen, brain, gills, epidermis). The differential expression of the two isoforms and of the alternative transcripts of isoform A during development is currently under study, in order to investigate the functional significance and regulation of 3′ UTR length in transcription and mRNA turnover rate.