Characterisation and expression during sex differentiation of Sox19 from the sea bass Dicentrarchus labrax.

The Sox family of transcription factors are involved in a variety of developmental processes including sex determination and gonadal differentiation. Sox19 is a particularly interesting member of this family that has been found only in fish, though mammals have a very diverged orthologue that is designated Sox15 and assigned to a different Sox family subgroup. Here we describe the cloning and characterisation of sox19 from the European sea bass (Dicentrarchus labrax), an important aquaculture species in which sex ratios skewed in favour of males are frequently encountered. The sea bass sox19 gene contains a single intron, encodes a protein of 309 amino acids, has multiple transcription start sites and may produce a truncated splice variant. Sox19 mRNA is present in many adult tissues, with the highest expression in the brain and gonads. Interestingly, the gene is strongly upregulated in the differentiation of the ovary but not the testis, suggesting a role in ovarian differentiation.

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.

Differential gene expression during smoltification of Atlantic salmon (Salmo salar L.): a first large-scale microarray study.

The life cycle of the Atlantic salmon (Salmo salar) involves a period of 1 to 3 years in freshwater followed by migration to the sea where the salmon undergoes rapid growth. In preparation for the marine environment, while still in freshwater, the salmon undergo a transformation from a freshwater dwelling parr to a saltwater adapted smolt, a process known as smoltification. The Atlantic salmon Transcriptome Analysis of Important Traits of Salmon/Salmon Genome Project (TRAITS/SGP) cDNA microarray was used to investigate how gene expression alters during smoltification. Genes differentially expressed during smoltification were identified by comparing gene expression profiles in smolt brain, gill, and kidney tissue samples with those of parr. Of the three tissues investigated, the number of differentially expressed genes was the greatest in gill. Many of the differentially expressed genes could be assigned to one of four main categories: growth, metabolism, oxygen transport, and osmoregulation. Quantitative polymerase chain reaction successfully confirmed the differential expression of seven of the upregulated genes. The TRAITS/SGP cDNA microarray was used to successfully demonstrate for the first time how gene expression mediates smoltification in the Atlantic salmon. Changes in gene expression observed in this study reflected the physiological and biochemical changes recorded by previous studies describing the parr-smolt transformation. This study significantly increases our knowledge of smoltification and will benefit future studies in this area of research.

Different sox17 transcripts during sex differentiation in sea bass, Dicentrarchus labrax.

Sox genes participate in several developmental processes, including sex determination and differentiation. In this study, the genomic structure of sox17 was characterized in the sea bass (sb). Two transcripts, one producing a normal protein (sb Sox17) and another producing a truncated protein (sb t-Sox17) were detected. A third, novel transcript, originated by intron retention (sb i-sox17) was also observed. Sb sox17 was widely distributed, whereas sb i-sox17 was predominantly found in skin and brain. In gonads, sb sox17 expression first increased at 150 days of age, coinciding with the onset of sex differentiation. At 250 days and onwards, sb sox17 expression was significantly higher in females, and mRNA levels correlated with those of gonadal aromatase. Thus, this study provides the first evidence for the presence of alternative splicing by intron retention in a Sox17 gene, and for sex-related differences in expression, implicating sox17 in ovarian development and function in fish.

Thyroid hormone receptor expression during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus).

Flatfish such as the Atlantic halibut (Hippoglossus hippoglossus) undergo a dramatic metamorphosis that transforms the pelagic, symmetric larva into a benthic, cranially asymmetric juvenile. In common with amphibian metamorphosis, flatfish metamorphosis is under endocrine control with thyroid hormones being particularly important. In this report we confirm that tri-iodothyronine (T(3)) levels peak at metamorphic climax during halibut metamorphosis. Moreover, we have isolated cDNA clones of TRalpha and TRbeta genes and confirmed the presence in halibut of two TRalpha isoforms (representing the products of distinct genes) and two TRbeta isoforms (generated from a single gene by alternative splicing). Real-time PCR was used to assess expression of these genes during metamorphosis. TRbeta shows the most dramatic expression profile, with a peak occurring during metamorphic climax.

Troponin T isoform expression is modulated during Atlantic halibut metamorphosis.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Molecular characterization and seasonal changes in gonadal expression of a thyrotropin receptor in the European sea bass.

The thyroid stimulating hormone (TSH) is a glycoprotein synthesized and secreted from thyrotrophs of the anterior pituitary gland. It acts by binding to and activating its specific receptor, the TSHR, to induce the synthesis and secretion of thyroid hormones. Recent studies conducted in diverse fish species suggest a direct role of TSH on gonadal physiology. In this work, we describe the cloning of a cDNA encoding a TSHR which was isolated from the gonads of the European sea bass (Dicentrarchus labrax). The mature protein displays typical features of the members of the glycoprotein hormone receptor family and shows the highest amino acid sequence identity with the TSHRs of other fish species. An insertion of approximately 50 amino acids, specific for the TSHR subfamily is also present in the carboxyl end of the extracellular domain of the sbsTSHR. By RT-PCR analysis, we demonstrate the extrathyroidal expression of sbsTSHR in numerous tissues of the sea bass. Also, two transcripts that differ in the length of their 3' untranslated regions were found. They reflect the use of alternative polyadenylation cleavage sites. Seasonal changes in sbsTSHR mRNA levels in female and male sea bass during the first ovarian and testicular recrudescence suggest that in females the TSHR could participate in active vitellogenesis and in the regulation of gamete maturation and ovulation, whereas in males, the TSHR would be involved in the regulation of processes that occur during the early stages of the gonadal development and also of gamete maturation and spermiation. The results of this work indicate that a sbsTSHR has been cloned from the testis of the European sea bass and they provide the basis for future studies concerning the function of TSHR in this species.

Identification of two isoforms of Atlantic halibut insulin-like growth factor-I receptor genes and quantitative gene expression during metamorphosis.

Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (>or=95%) and similarity (>or=97%) with other fish IGF-IR and lower identity (>or=77%) and similarity (>or=83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut.

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 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.

Cloning of Atlantic halibut growth hormone receptor genes and quantitative gene expression during metamorphosis.

To gain insight into the possible regulatory role of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system in flatfish metamorphosis, body GHR gene expression as well as IGF-I protein content was quantified in larval Atlantic halibut throughout metamorphosis (developmental stages 5-10). The cDNA of the full-length GH receptor (hhGHR) was cloned from adult liver and characterized. The hhGHR shows common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and an intracellular domain containing a Box 1 and Box 2. Additionally, a truncated GHR (hhGHRtr), similar to turbot and Japanese flounder GHRtr, was cloned and sequenced. These sequences are highly similar to the full-length and truncated GHRs in turbot (89%/86%) and Japanese flounder (93%/91%) with lower identity with other fish type I GHR (81%) and type II GHRs (58%). A quantitative real-time RT-PCR assay was used to measure hhGHR and hhGHRtr mRNA content in normally and abnormally metamorphosed individuals at six developmental stages, from early pre-metamorphosis to post-metamorphosis, when the fish is considered a juvenile. The level of hhGHR gene expression was highest at pre-metamorphic stage 6 and at stage 8 at the onset of metamorphosis, and then decreased during metamorphic climax and post-metamorphosis. Expression of hhGHRtr reached highest levels at stage 6 and then decreased to post-metamorphosis. The ratio of expression between the full-length and the truncated GHR (hhGHR:hhGHRtr) varied among stages and was highest at the onset of metamorphosis and at metamorphic climax. A radioimmunoassay was used to measure halibut IGF-I body content throughout metamorphosis. IGF-I increases from early metamorphosis to the onset of metamorphosis and then decreases towards post-metamorphosis. In comparison between normally and abnormally metamorphosing larvae, IGF-I content, hhGHR and hhGHRtr mRNA levels were reduced in the abnormal fish. These data indicate that the GH-IGF-I system either has a regulatory role in metamorphosis, or is being affected as a consequence of the abnormal metamorphosis.

Cloning of the promoter from the gonadal aromatase gene of the European sea bass and identification of single nucleotide polymorphisms.

Aromatase contributes to sex differentiation by catalysing the conversion of androgens into estrogens. We have cloned the promoter of the gonadal aromatase gene from the sea bass, Dicentrarchus labrax, a species whose farming is complicated by male-skewed sex ratios. The promoter shows a conserved binding site for SF-1 as well as two cAMP response elements (CREs) and putative binding sites for transcription factors belonging to the Sox and forkhead families. Analysis of promoter sequences from individual fish suggests the presence of three promoter alleles that arise due to three single nucleotide polymorphisms (SNPs) in linkage disequilibrium.

Analysis of the Sox gene family in the European sea bass (Dicentrarchus labrax).

Sox (SRY-related genes containing a HMG box) genes encode a family of transcription factors that are involved in a variety of developmental processes including sex determination. Twenty Sox genes are present in the genomes of humans and mice, but far less is known about the Sox gene family in other vertebrate types. We have obtained clones representing the HMG boxes of twelve Sox genes from European sea bass (Dicentrarchus labrax), a fish species whose farming is complicated by a heavily skewed sex ratio, with between 70% and 99% of offspring typically being male. The cloned Sox genes are members of the SoxB, SoxC, SoxE and SoxF groups. Sequence analysis shows that some of the clones represent genes duplicated in sea bass with respect to the mammalian Sox gene family.

Molecular cloning and expression of thyroid hormone receptor alpha during salmonid development.

Thyroid hormones have been implicated as important regulators of teleost development. To gain a better understanding of the potential roles of the thyroid system in salmonids a genomic clone which encoded rainbow trout TR-alpha was isolated. This clone exhibited highest amino acid identity to Japanese flounder TR-alphaB (94%) and zebrafish TR-alpha1 (94%). Oligonucleotides were designed against the rainbow trout sequence and the complete coding region of Atlantic salmon TR-alpha was isolated by RACE-PCR. The Atlantic salmon sequence exhibited highest amino acid identity to rainbow trout TR-alpha (98%), Japanese flounder TR-alphaB (93%), and zebrafish TR-alpha1 (90%). Atlantic salmon TR-alpha exhibited the classic modular structure associated with members of the nuclear receptor superfamily and consisted of a divergent A/B domain while the DNA and ligand-binding domains were highly conserved to other teleost TR proteins. Temporal expression from the rainbow trout TR-alpha gene was monitored by semiquantitative RT-PCR at selected stages during rainbow trout embryonic and larval development. High levels of maternal transcripts were present at cleavage (Stage 6) which were rapidly degraded by gastrulation (Stage 13). Low levels of TR-alpha expression were then detected during organogenesis (Stages 20, 24, 26, 29, and 31). A peak in mRNA levels was observed at hatch (Stage 32) after which levels rose in a gradual manner during larval development (Stages 33, 34, 35, and 36) to reach maximal values at first feeding (Stage 37). These results suggest that the thyroid axis is functional and that embryonic and larval rainbow trout are at least capable of responding to thyroid hormones. These observations implicate the thyroid system as being an important regulator of salmonid development.

Precocious synthesis of a thyroid hormone receptor inXenopus embryos causes hormone-dependent developmental abnormalities.

We have investigated the effects of aXenopus alpha thyroid hormone receptor (TR) upon early development ofX. laevis. After deleting the 5'-untranslated region of a cloned TR cDNA, we synthesised TR transcripts that can be translated in oocytes and embryos. When embryos are supplied with this RNA by direct injection, functional TR can be detected through gastrula and neurula stage embryos, considerably in advance of the normal onset of TR expression in larval development. TR mRNA-injected embryos are precociously responsive to thyroid hormone (T3). In the absence of T3 such embryos develop completely normally, but addition of T3 to the medium bathing the embryos results in abnormal embryos with graded anterior (head) deficiencies, losing forehead, eyes and cement gland, progressively. The degree of abnormality is dependent upon the dose of T3 and the stage of development at which it is applied, embryos treated at stage 6 (32 cell) becoming more abnormal than those treated at stage 121/2 (late gastrula). Embryos injected with TR mRNA and treated with T3 are similar, but not identical, to embryos treated with retinoic acid in early development. As is the case with retinoic acid treatment, we show that the T3-dependent effects are due, at least in part, to effects on gastrulation movements.