Marco A. Campinho

The Molecular and Endocrine Basis of Flatfish Metamorphosis

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.

Disruption of the thyroid system by diethylstilbestrol and ioxynil in the sea bream (Sparus aurata)

Some environmental contaminants are thought to cause disruption of the thyroid system in vertebrates acting as endocrine disrupting chemicals (EDCs). Such chemicals may affect synthesis, transport and metabolism of thyroid hormones (THs). Ioxynil (IOX) and diethylstilbestrol (DES) are potential EDCs with strong affinity in vitro for sea bream transthyretin (TTR), a TH distributor protein (THDP). The aim of the present study was to establish how such chemicals influence the thyroid axis in sea bream (Sparus aurata). DES, IOX and propilthyouracil (PTU, a goitrogen) were administered in the diet to sea bream juveniles at 1 mg/kg fish (n = 14/treatment) for 21 days. After exposure plasma TH levels, quantified by RIA, were similar to those of control fish (p > 0.05) in all treatment groups. Analysis by quantitative PCR revealed that all treatments down-regulated TSH gene transcription (p < 0.05) in the brain and pituitary and deiodinase II and III transcription in the brain (p < 0.001). In contrast, PTU caused DII up-regulation in the liver (p < 0.05). Thyroid receptor beta (TRbeta) transcription was down-regulated in the pituitary by PTU (p < 0.001) and DES (p < 0.05). TTR plasma levels, quantified by ELISA, were elevated by all the chemicals including PTU (p < 0.001) which also increased TTR gene transcription in the liver (p < 0.05). Thyroid histology indicated follicular hyperstimulation in all treatments with marked hyperplasia, hypertrophy and colloid depletion in the PTU group. It appears therefore, that in vitro TTR-binders, IOX and DES, can strongly influence several components of the fish thyroid system in vivo but that the thyroid axis may have the ability to maintain or re-establish plasma TH homeostasis.

Molecular, cellular and histological changes in skin from a larval to an adult phenotype during bony fish metamorphosis

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 1α1 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.

Coordination of deiodinase and thyroid hormone receptor expression during the larval to juvenile transition in sea bream (Sparus aurata, Linnaeus).

To test the hypothesis that THs play an important role in the larval to juvenile transition in the marine teleost model, sea bream (Sparus auratus), key elements of the thyroid axis were analysed during development. Specific RT-PCR and Taqman quantitative RT-PCR were established and used to measure sea bream iodothyronine deiodinases and thyroid hormone receptor (TR) genes, respectively. Expression of deiodinases genes (D1 and D2) which encode enzymes producing T3, TRs and T4 levels start to increase at 20-30 days post-hatch (dph; beginning of metamorphosis), peak at about 45 dph (climax) and decline to early larval levels after 90-100 dph (end of metamorphosis) when fish are fully formed juveniles. The profile of these different TH elements during sea bream development is strikingly similar to that observed during the TH driven metamorphosis of flatfish and suggests that THs play an analogous role in the larval to juvenile transition in this species and probably also in other pelagic teleosts. However, the effect of T3 treatment on deiodinases and TR transcript abundance in sea bream is not as clear cut as in larval flatfish and tadpoles indicating divergence in the responsiveness of TH axis elements and highlighting the need for further studies of this axis during development of fish.

Molecular characterization and transcriptional regulation of the Na +/K+ ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis).

In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills.

Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

BackgroundFlatfish 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.ResultsIn 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.ConclusionMuscle 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.

Maternal Thyroid Hormones Are Essential for Neural Development in Zebrafish

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

Flatfish metamorphosis: A hypothalamic independent process?

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 hybridization (WISH) and quantitative PCR in sole are 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.

Regulation of troponin T expression during muscle development in sea bream Sparus auratus Linnaeus: the potential role of thyroid hormones

SUMMARY In the sea bream Sparus auratus three stage-specific fast troponin T (fTnT) isoforms have been cloned and correspond to embryonic-, larval- and adult-specific isoforms. Characterisation, using database searches, of the putative genomic organisation of Fugu rubripes and Tetraodon nigroviridis fTnT indicates that alternative exon splicing in the 5 region of the gene generates the different isoforms. Moreover, comparison of teleost fTnTs suggests that alternative splicing of fTnT appears to be common in teleosts. A different temporal expression pattern for each fTnT splice varotnt is found during sea bream development and probably relates to differing functional demands, as a highly acidic embryonic form (pI 5.16) is substituted by a basic larval form (pI 9.57). Thyroid hormones (THs), which play an important regulatory role in muscle development in flatfish and tetrapods, appear also to influence TnT gene expression in the sea bream. However, THs have a divergent action on different sea bream TnT genes and although the slow isoform (sTnT1) is TH-responsive, fTnT, sTnT2 and the itronless isoform (iTnT) are unaffected. The present results taken together with those published for flatfish seem to suggest differences may exist in the regulation of larval muscle development in teleosts.

Molecular and cellular changes in skin and muscle during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) are accompanied by changes in deiodinases expression

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.