Two Activin Type 2B Receptors from Sea Bream Function Similarly in vitro.

Myostatin (MSTN) and activin are members of the transforming growth factor-ß superfamily. Both signal through the activin type II receptors (ActRII and ActRIIB). In a previous report, we demonstrated that fish possess at least 2 genes for ActRIIB: ActRIIB-1 and ActRIIB-2, which differ in their amino acid sequence. We also showed that affinity-purified, fish-soluble ActRIIB-1 (extracellular domain; ECD), produced in the yeast Pichia pastoris, inhibited recombinant mouse/rat/human mature MSTN activity in vitro using a reporter gene assay in the mammalian A204 cell line. In the present study, we produced soluble ActRIIB-2a in P. pastoris, and showed that it is N-glycosylated, similar to soluble ActRIIB-1. Inhibition of MSTN and activin A activities by affinity-purified ActRIIB-2a was compared with that of soluble ActRIIB-1 using the CAGA-luciferase assay in A204 cells. The findings of this study provide evidence that both paralogs, which probably resulted from gene duplication, did not diversify in their functionality (neofunctionalization), but rather retained a similar function. Both ActRIIB isoforms are equally potent in the mammalian system, and both exhibited an inhibitory effect on mammalian MSTN and activin A. Moreover--in spite of the amino acid differences in ECD between the two paralogs--it appears that the residues important for ligand binding are conserved, and that they recognize the mammalian ligands activin A and MSTN to the same extent.

Genomic cloning and promoter functional analysis of myostatin-2 in shi drum, Umbrina cirrosa: conservation of muscle-specific promoter activity.

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily, known as a negative regulator of skeletal muscle development and growth in mammals. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. Here we describe the cloning and sequence analysis of spliced and precursor (unspliced) transcripts as well as the 5' flanking region of MSTN-2 from the marine fish Umbrina cirrosa (ucMSTN-2). In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed high transcriptional activity in muscle cells and in differentiated neural cells, in accordance with our previous findings in MSTN-2 promoter from Sparus aurata. Comparative informatics analysis of MSTN-2 from several fish species revealed high conservation of the predicted amino acid sequence as well as the gene structure (exon length) although intron length varied between species. The proximal promoter of MSTN-2 gene was found to be conserved among Perciforms. In conclusion, this study reinforces our conclusion that MSTN-2 promoter is a very strong promoter, especially in muscle cells. In addition, we show that the MSTN-2 gene structure is highly conserved among fishes as is the predicted amino acid sequence of the peptide.

Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish.

Myostatin (MSTN), a negative regulator of muscle growth and a member of the transforming growth factor-ß superfamily, can bind the two activin type 2 receptors (ACVR2). It has been previously shown that WT mice injected with ACVR2B extracellular domain (ACVR2B-ECD) had higher muscle mass. Likewise, fish larvae immersed in Pichia pastoris culture supernatant, containing goldfish Acvr2b-ECD, showed enhanced larval growth. However, it is not clear whether fish Mstn1 and Mstn2 signal through the same receptor and whether fish express more than one acvr2b gene. In the current study, three cDNAs encoding acvr2b (saacvr2b-1, saacvr2b-2a, and saacvr2b-2b) were cloned from gilthead sea bream. All three contain the short extracellular binding domain, a short transmembrane region, and a conserved catalytic domain of serine/threonine protein kinase. Bioinformatics analysis provided evidence for the existence of two acvr2b genes (acvr2b-1 and acvr2b-2) in several other fish species as well, probably as a result of gene or genome duplication. The two isoforms differ in their amino acid sequences. The direct inhibitory effect of Acvr2b-ECD on Mstn activity was tested in vitro. The saAcvr2b-1-ECD was expressed in the yeast P. pastoris. Evidence is provided for N-glycosylation of Acvr2b-1-ECD. The affinity-purified Acvr2b-1-ECD inhibited recombinant mouse/rat/human mature MSTN activity when determined in vitro using the CAGA-luciferase assay in A204 cells. A lower inhibitory activity was obtained when unprocessed purified, furin-digested, and activated saMstn1 was used. Results of this study demonstrate for the first time the existence of two acvr2b genes in fish. In addition, the study shows that bioactive fish Acvr2b-ECD can be produced from P. pastoris.

Structural and functional analysis of myostatin-2 promoter alleles from the marine fish Sparus aurata: evidence for strong muscle-specific promoter activity and post-transcriptional regulation.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. In this study, we analyzed the structural-functional features of the four variants of Sparus aurata MSTN-2 5'-flanking region: saMSTN-2a, saMSTN-2as, saMSTN-2b and saMSTN-2c. In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed surprisingly high transcriptional activity in muscle cells, suggesting the presence of regulatory elements unique to differentiated myotubes. These observations were confirmed by in situ intramuscular injections of promoter DNA followed by reporter gene assays. Moreover, high promoter activity was found in differentiated neural cell, in agreement with MSTN-2 expression in brain. Progressive 5'-deletion analysis, using reporter gene assays, showed that the core promoter is located within the first -127 bp upstream of the ATG, and suggested the presence of regulatory elements that either repress or induce transcriptional activity. Transient transgenic zebrafish provided evidence for saMSTN-2 promoter ability to direct GFP expression to myofibers. Finally, our data shows that although no mature saMSTN-2 mRNA is observed in muscle; unspliced forms accumulate, confirming high level of transcription. In conclusion, our study shows for the first time that MSTN-2 promoter is a very robust promoter, especially in muscle cells.

Myostatin-2 gene structure and polymorphism of the promoter and first intron in the marine fish Sparus aurata: evidence for DNA duplications and/or translocations.

BACKGROUND: Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Fish express at least two genes for MSTN: MSTN-1 and MSTN-2. To date, MSTN-2 promoters have been cloned only from salmonids and zebrafish. RESULTS: Here we described the cloning and sequence analysis of MSTN-2 gene and its 5' flanking region in the marine fish Sparus aurata (saMSTN-2). We demonstrate the existence of three alleles of the promoter and three alleles of the first intron. Sequence comparison of the promoter region in the three alleles revealed that although the sequences of the first 1050 bp upstream of the translation start site are almost identical in the three alleles, a substantial sequence divergence is seen further upstream. Careful sequence analysis of the region upstream of the first 1050 bp in the three alleles identified several elements that appear to be repeated in some or all sequences, at different positions. This suggests that the promoter region of saMSTN-2 has been subjected to various chromosomal rearrangements during the course of evolution, reflecting either insertion or deletion events. Screening of several genomic DNA collections indicated differences in allele frequency, with allele 'b' being the most abundant, followed by allele 'c', whereas allele 'a' is relatively rare. Sequence analysis of saMSTN-2 gene also revealed polymorphism in the first intron, identifying three alleles. The length difference in alleles '1R' and '2R' of the first intron is due to the presence of one or two copies of a repeated block of approximately 150 bp, located at the 5' end of the first intron. The third allele, '4R', has an additional insertion of 323 bp located 116 bp upstream of the 3' end of the first intron. Analysis of several DNA collections showed that the '2R' allele is the most common, followed by the '4R' allele, whereas the '1R' allele is relatively rare. Progeny analysis of a full-sib family showed a Mendelian mode of inheritance of the two genetic loci. No clear association was found between the two genetic markers and growth rate. CONCLUSION: These results show for the first time a substantial degree of polymorphism in both the promoter and first intron of MSTN-2 gene in a perciform fish species which points to chromosomal rearrangements that took place during evolution.

Growth/differentiation factor-11: an evolutionary conserved growth factor in vertebrates.

Growth and differentiation factor-11 (GDF-11) is a member of the transforming growth factor-ß superfamily and is thought to be derived together with myostatin (known also as GDF-8) from an ancestral gene. In the present study, we report the isolation and characterization of GDF-11 homolog from a marine teleost, the gilthead sea bream Sparus aurata, and show that this growth factor is highly conserved throughout vertebrates. Using bioinformatics, we identified GDF-11 in Tetraodon, Takifugu, medaka, and stickleback and found that they are highly conserved at the amino acid sequence as well as gene organization. Moreover, we found conservation of syntenic relationships among vertebrates in the GDF-11 locus. Transcripts for GDF-11 can be found in eggs and early embryos, albeit at low levels, while in post-hatching larvae expression levels are high and decreases as development progresses, suggesting that GDF-11 might have a role during early development of fish as found in tetrapods and zebrafish. Finally, GDF-11 is expressed in various tissues in the adult fish including muscle, brain, and eye.

Identification of a novel transforming growth factor-beta (TGF-beta6) gene in fish: regulation in skeletal muscle by nutritional state.

BACKGROUND: The transforming growth factor-beta (TGF-beta) family constitutes of dimeric proteins that regulate the growth, differentiation and metabolism of many cell types, including that of skeletal muscle in mammals. The potential role of TGF-betas in fish muscle growth is not known. RESULTS: Here we report the molecular characterization, developmental and tissue expression and regulation by nutritional state of a novel TGF-beta gene from a marine fish, the gilthead sea bream Sparus aurata. S. aurata TGF-beta6 is encoded by seven exons 361, 164, 133, 111, 181, 154, and 156 bp in length and is translated into a 420-amino acid peptide. The exons are separated by six introns: >643, 415, 93, 1250, 425 and >287 bp in length. Although the gene organization is most similar to mouse and chicken TGF-beta2, the deduced amino acid sequence represents a novel TGF-beta that is unique to fish that we have named TGF-beta6. The molecule has conserved putative functional residues, including a cleavage motif (RXXR) and nine cysteine residues that are characteristic of TGF-beta. Semi-quantitative analysis of TGF-beta6 expression revealed differential expression in various tissues of adult fish with high levels in skin and muscle, very low levels in liver, and moderate levels in other tissues including brain, eye and pituitary. TGF-beta6 is expressed in larvae on day of hatching and increases as development progresses. A fasting period of five days of juvenile fish resulted in increased levels of TGF-beta6 expression in white skeletal muscle compared to that in fed fish, which was slightly attenuated by one injection of growth hormone. CONCLUSION: Our findings provide valuable insights about genomic information and nutritional regulation of TGF-beta6 which will aid the further investigation of the S. aurata TGF-beta6 gene in association with muscle growth. The finding of a novel TGF-beta6 molecule, unique to fish, will contribute to the understanding of the evolution of the TGF-beta family of cytokines in vertebrates.

Molecular cloning and characterization of follistatin in the gilthead sea bream, Sparus aurata.

Follistatin (FST) is an activin-binding protein that neutralizes the activity of activin. FST also binds other members of the transforming growth factor-beta (TGF-beta) superfamily, including myostatin (MSTN). We report herein on the isolation and characterization of a full-length cDNA sequence predicted to encode FST in a marine fish, the gilthead sea bream Sparus aurata. The deduced amino acid sequence of sea bream FST (saFST) is highly conserved to the counterpart sequences in other vertebrates and contains the N-terminal domain and three FST domains. The deduced mature saFST shows 81-86% identity with FSTs from other vertebrates. It is 290 amino acids long, similar to other fish FSTs and the short isoform of Xenopus FST but longer by two residues than mammalian FST288. Ontogeny of MSTN (a TGF-beta superfamily member and a negative growth regulator of skeletal muscle in mammals), and FST (known to bind MSTN) gene expression revealed the presence of both transcripts throughout larval development. However, a different expression pattern was found in earlier developmental stages; while MSTN could not be detected prior to the day of hatching, FST transcript was detected in embryos 12 h post-fertilization, confirming its role during vertebrate embryonic development. Both FST and MSTN were expressed in many adult tissues, with variable levels of expression, including muscle. Recombinant saFST inhibited saMSTN activity in a reporter gene assay, indicating a similar effect to that reported in mammals.

Characterization and functional analysis of the 5' flanking region of Sparus aurata myostatin-1 gene.

Myostatin (MSTN) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Although several MSTN promoters were described in fish, no functional analysis was reported so far. Here, the 5' flanking region (1372 bp) of the MSTN-1 gene of the marine fish Sparus aurata (saMSTN-1) was cloned, sequenced and characterized. It contains two consensus sequences for TATA box (TATAA), a CAAT box, ten putative E-boxes known as binding sites to myogenic basic helix-loop-helix transcription factors (TFs) and two putative binding sites to TF Myocyte enhancing factor-2 (MEF2). In addition, it has several putative binding sites to TF Pit-1a and several response elements to nuclear receptors (GRE, ERE, PRE, ARE, TRE, RARE and PPARRE) and cAMP-response elements. Transcriptional activity of five genomic fragments (truncated at their upstream region) of 372, 941, 972, 1113 and 1355 bp was studied in vitro, using transient transfection in A204 cells. All constructs directed luciferase activity, with the highest activity obtained by the 1113 bp fragment. These experiments show that all five genomic fragments are functional MSTN promoters and differences in promoter activity might be due to presence of enhancers and/or repressor sequences, regulating MSTN promoter activity.

Real-time polymerase chain reaction, in situ hybridization and immunohistochemical localization of insulin-like growth factor-I and myostatin during development of Dicentrarchus labrax (Pisces: Osteichthyes).

The distribution of insulin-like growth factor-I (IGF-I) and myostatin (MSTN) was investigated in sea bass (Dicentrarchus labrax) by real-time polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry. Real-time PCR indicated that IGF-I mRNA increased from the second day post-hatching and that this trend became significant from day 4. ISH confirmed a strong IGF-I mRNA expression from the first week post-hatching, with the most abundant expression being detected in the liver of larvae and adults. Real-time PCR also showed that the level of MSTN mRNA increased significantly from day 25. The expression of MSTN mRNA was higher in muscle and almost absent in other anatomical regions in both larvae and adults. Interestingly, the lateral muscle showed a quantitative differential expression of IGF-I and MSTN mRNAs in red and white muscle, depending on the developmental stage examined. IGF-I immunoreactivity was detected in developing intestine at hatching and in skeletal muscle, skin and yolk sac. MSTN immunostaining was evident in several tissues and organs in both larvae and adults. Both IGF-I and MSTN proteins were detected in the liver from day 4 post-hatching and, subsequently, in the kidney and heart muscle from day 10. Our results suggest, on the basis of a combined methodological approach, that IGF-I and MSTN are involved in the regulation of somatic growth in the sea bass.

Regulatory regions in the promoter and first intron of Sparus aurata growth hormone gene: Repression of gene activity by a polymorphic minisatellite.

Transcriptional activity of the gilthead sea bream (Sparus aurata) growth hormone (saGH) gene promoter has been investigated using transient transfection assays in GH3 cells. Analysis of two fragments (1.7 kb and a 5'-deleted 0.9 kb) of saGH gene promoter directed luciferase reporter gene activity, indicating transcriptional activity of both fragments in vitro. The shorter fragment, containing five potential binding sites for the pituitary-specific transcription factor GHF-1/Pit-1, two cAMP-response elements and two glucocorticoid-response elements conferred higher reporter gene activity than the longer fragment. This result suggests presence of an inhibitory region upstream of the saGH promoter. Transient transfection assays were also used to investigate the effect of the polymorphic minisatellite saGHFIM found in the first intron of saGH gene on gene expression in four cell lines: fish pituitary (RTP-2) and non-pituitary (RTH-149) and mammalian pituitary (GH3) and non-pituitary (HEK293T). Luciferase activity was repressed by saGHFIM containing a high number of tandem repeats compared to a Control construct and to a construct with a smaller number of tandem repeats. Moreover, this repression was dependent on the orientation of the repeats relative to the viral promoter. These in vitro results imply that long GH introns might influence GH gene expression in vivo.

Expression, purification, renaturation and activation of fish myostatin expressed in Escherichia coli: facilitation of refolding and activity inhibition by myostatin prodomain.

Myostatin (growth and differentiation factor-8) is a member of the transforming growth factor-beta superfamily, is expressed mainly in skeletal muscle and acts as a negative growth regulator. Mature myostatin (C-terminal) is a homodimer that is cleaved post-translationally from the precursor myostatin, also yielding the N-terminal prodomain. We expressed in Escherichia coli three forms of fish myostatin: precursor, prodomain and mature. The three forms were over-expressed as inclusion bodies. Highly purified inclusion bodies were solubilized in a solution containing guanidine hydrochloride and the reducing agent DTT. Refolding (indicated by a dimer formation) of precursor myostatin, mature myostatin or a mixture of prodomain and mature myostatin was compared under identical refolding conditions, performed in a solution containing sodium chloride, arginine, a low concentration of guanidine hydrochloride and reduced and oxidized glutathione at 4 degrees C for 14 days. While precursor myostatin formed a reversible disulfide bond with no apparent precipitation, mature myostatin precipitated in the same refolding solution, unless CHAPS was included, and only a small proportion formed a disulfide bond. The trans presence of the prodomain in the refolding solution prevented precipitation of mature myostatin but did not promote formation of a dimer. Proteolytic cleavage of purified, refolded precursor myostatin with furin yielded a monomeric prodomain and a disulfide-linked, homodimeric mature myostatin, which remained as a latent complex. Activation of the latent complex was achieved by acidic or thermal treatments. These results demonstrate that the cis presence of the prodomain is essential for the proper refolding of fish myostatin and that the cleaved mature dimer exists as a latent form.

Cloning and expression of insulin-like growth factors I and II in the shi drum (Umbrina cirrosa).

Insulin-like growth factors (IGFs) are evolutionarily ancient polypeptides, with potent metabolic actions, affecting cell development and growth. The IGF system consists of two ligands: IGF-I and IGF-II, several binding proteins and high-affinity transmembrane receptors. To understand growth regulation in the teleost shi drum, Umbrina cirrosa, we cloned IGF-I and IGF-II cDNAs, studied their expression and determined the cellular localization of IGF-II peptide by immunohistochemistry. A fragment of 1110 nucleotides, coding for U. cirrosa IGF-I (ucIGF-I), was cloned from liver by PCR. It includes an open reading frame of 561 nucleotides, encoding a 187 amino acid preproIGF-I. A fragment of 938 nucleotides that includes part of the coding sequence and the 3' UTR of IGF-II (ucIGF-II) was cloned as well. Sequence analysis of ucIGF-I and ucIGF-II showed a high degree of homology with known fish IGF-I and IGF-II. Real-Time PCR showed a higher expression of IGF-I and IGF-II in liver, compared to all other tissues analysed. IGF-II peptide was detected in larval liver, intestine, gills and heart musculature. After metamorphosis, reactivity was particularly evident in the kidney and in red fibres of skeletal muscle. These results add novel information on the nucleotide sequence of IGF-I and IGF-II in a marine teleost, the shi drum, that was recently introduced to the mariculture industry in southern Europe and emphasizes the conservation in the 5' UTR of IGF-I among teleosts. Furthermore, this study suggests, on the basis of a combined approach of RT-PCR, Real-Time PCR and immunohistochemistry that IGF-I and IGF-II are involved in the regulation of somatic growth in the shi drum.

alpha2-Macroglobulin in the marine fish Sparus aurata.

The alpha2-macroglobulin proteinase inhibitors (alpha2Ms) are a family of plasma proteins with the unique ability to inhibit a broad spectrum of proteinases, but are also known as binding proteins for many growth factors and cytokines, including growth hormone and members of the transforming growth factor-beta superfamily. A partial cDNA (475 amino acids) encoding the C-terminus of alpha2M was cloned from the liver of the marine teleostean fish Sparus aurata. The deduced amino acid sequence of the cloned fragment showed 58-60% similarity to carp alpha2Ms. Northern blot analysis of hepatic alpha2M revealed a transcript of about 5 kb. A transcript of a similar size was detected in 1-day larvae. Steady state levels of alpha2M in larvae increased gradually on subsequent days post-hatching. alpha2M expression in embryos was determined by RT-PCR and started in embryos aged 8 h post-fertilization, but not earlier. RT-PCR of muscle RNA detected alpha2M also in fish muscle, albeit with a lower expression than in the liver. Immunoreactive-alpha2M was found in yolk syncytial layer of 3-day larvae and in livers from larvae and adults. Immunoreactive-alpha2M was also identified in soluble total proteins from young larvae with a pattern resembling that of plasma. These data demonstrate that the alpha2M gene is expressed early in fish development. Moreover, in addition to its major expression in liver, alpha2M is expressed also in fish muscle.

Hormonal regulation of hepatic IGF-I and IGF-II gene expression in the marine teleost Sparus aurata.

The present work aimed to determine whether GnRH potentiates the effect of growth hormone (GH) on insulin-like growth factors (IGF-I and IGF-II) hepatic gene expression in Sparus aurata liver. Since several hepatic genes were shown to underlie direct regulation via the hepatic estrogen receptor, another aim was to extend our understanding of direct estrogen effects on liver IGFs gene expression. Pre-reproductive sea bream females were treated with GH, GnRH, estradiol-17beta, GH plus GnRH, and estradiol-17beta plus GH. After 72 hr, all treatment induced an increase of plasma estradiol well correlated with the increase of plasma vitellogenin (VTG) levels. IGF-I and IGF-II expression in the liver of treated females was determined by semi-quantitative RT-PCR, using beta-actin as internal standard. The results reported here show that GH significantly stimulates hepatic transcription of IGF-I and IGF-II genes. Surprisingly, E2 and GnRH treatments decreased both IGF-I and IGF-II mRNA levels. In fishes treated with GH plus GnRH, the GnRH contrasted the GH effect: the IGF-I mRNA levels were still significantly higher than in controls, while the effect of GH on IGF-II gene expression was totally abolished. At the same time, in the combined treatment with GH plus E2, the E2 counteracted the stimulatory effect of GH on both IGF-I and IGF-II genes expression.

Cellular localisation of insulin-like growth factor binding protein-2 (IGFBP-2) during development of the marine fish, Sparus aurata.

The spatial localisation of insulin-like growth-factor-binding protein-2 (IGFBP-2) and its mRNA was investigated during larval and post-larval developmental stages of the gilthead seabream ( Sparus aurata) by immunohistochemistry and in situ hybridisation with specific antisera and riboprobes. During larval development, immunoreactivity was found in skin, muscle, gills, pharynx, intestine, liver and olfactory epithelium. After metamorphosis, immunoreactivity was found in the oesophageal epithelium (the strongest reaction) and in red skeletal muscle, heart muscle, the thymus and the epithelium of renal tubules. In the adult, immunostaining with IGFBP-2 antibody was also found in the saccus vasculosus, ovary and testis. IGFBP-2 mRNA was detected by in situ hybridisation mainly in the intestine, skeletal musculature and ovary. These results show that IGFBP-2 protein and mRNA are expressed in a variety of seabream tissues, suggesting that IGFBP-2 regulates the actions of IGFs on these tissues during development and growth.

Application of real-time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped sea bream, Lithognathus mormyrus.

The striped sea bream (Lithognathus mormyrus) has been recently introduced as a bioindicator fish species in Mediterranean coastal habitats. The purpose of this study was to apply a real-time PCR assay for the determination of absolute levels of hepatic VTG transcript in this fish, identifying minimal and maximal levels and establishing the relationship between VTG RNA levels and ovarian development. A partial VTG cDNA was cloned from hepatic RNA of a striped sea bream female. Specific primers were designed based on its sequence and used for PCR and also for in vitro synthesis of partial VTG RNA standard. Hepatic VTG transcript levels were quantified by real-time PCR, using serially diluted VTG RNA standards for construction of a calibration curve and equation. VTG RNA levels were normalized to total RNA or 18S ribosomal RNA (determined by real-time PCR). VTG RNA was hardly detected in the liver of males, or females with small oocytes (diameter < 100 microm). A linear correlation was found between these two parameters at larger oocyte diameter (> 150 microm). VTG level reached a maximum of 204 fmol/pmol 18S RNA or 49 fmol/microg RNA. The results demonstrate the wide dynamic range of the established real-time PCR assay.

Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study.

In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata(sea bream), Solea solea(sole) and Brachydanio rerio(zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).

Differential expression of insulin-like growth factor I and II mRNAs during embryogenesis and early larval development in rabbitfish, Siganus guttatus.

In rodents, the expression of insulin-like growth factor II (IGF-II) is higher than that of insulin-like growth factor I (IGF-I) during fetal life while the reverse is true after birth. We wanted to examine whether this is also true in fish and whether IGF-I and IGF-II are differentially regulated during different stages of embryogenesis and early larval development in rabbitfish. We first cloned the cDNAs of rabbitfish IGF-I and IGF-II from the liver. Rabbitfish IGF-I has an open reading frame of 558 bp that codes for a signal peptide of 44 amino acids (aa), a mature protein of 68 aa, and a single form of E domain of 74 aa. Rabbitfish IGF-II, on the other hand, has an open reading frame of 645 bp that codes for a signal peptide of 47 aa, a mature protein of 70 aa, and an E domain of 98 aa. On the amino acid level, rabbitfish IGF-I shares 68% similarity with IGF-II. We then examined the relative expression of the two IGFs in unfertilized eggs, during different stages of embryogenesis, and in early larval stages of rabbitfish by a semiquantitative reverse transcription-polymerase chain reaction. Primers that amplify the mature peptide region of both IGFs were used and PCR for both peptides was done simultaneously, with identical PCR conditions for both. The identity of the PCR products was confirmed by direct sequencing. Contrary to published reports for seabream and rainbow trout, IGF-I mRNA was not detected in rabbitfish unfertilized eggs; it was first expressed in larvae soon after hatching. IGF-II mRNA, however, was expressed in unfertilized eggs, albeit weakly, and was already strongly expressed during the cleavage stage. mRNAs for both peptides were strongly expressed in the larvae, although IGF-II mRNA expression was higher than IGF-I expression.