Genome editing of pufferfish saxitoxin- and tetrodotoxin-binding protein type 2 in Takifugu rubripes.

The pufferfish saxitoxin- and tetrodotoxin-binding protein 2 (PSTBP2), which is involved in toxin accumulation, was knocked out in Takifugu rubripes embryos by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing technology. Treating the embryos with one of two single-guide RNA (sgRNA) resulted in mutation rates of 57.1% and 62.5%, respectively, as estimated using a heteroduplex mobility assay at 3 days postfertilization. Both sgRNAs might induced frameshift mutations that knocked out the T. rubripes PSTBP2.

Promoter analysis of the fish gene of slow/cardiac-type myosin heavy chain implicated in specification of muscle fiber types.

Vertebrate skeletal muscles consist of heterogeneous tissues containing various types of muscle fibers, where specification of the fiber type is crucial for muscle development. Fish are an attractive experimental model to study the mechanisms of such fiber type specification because of the separated localization of slow and fast muscles in the trunk myotome. We examined regulation of expression of the torafugu gene of slow/cardiac-type myosin heavy chain, MYH M5 , and isolated an operational promoter in order to force its tissue-specific expression across different fish species via the transgenic approach in zebrafish and medaka. This promoter activity was observed in adaxial cell-derived superficial slow muscle fibers under the control of a hedgehog signal. We also uncovered coordinated expression of MYH M5 and Sox6b, which is an important transcriptional repressor for specification of muscle fiber types and participates in hedgehog signaling. Sequence comparison in the 5'-flanking region identified three conserved regions, CSR1-CSR3, between torafugu MYH M5 and its zebrafish ortholog. Analysis of deletion mutants showed that CSR1 significantly stimulates gene expression in slow muscle fibers. In contrast, deletion of CSR3 resulted in ectopic expression of a reporter gene in fast muscle fibers. CSR3 was found to contain a putative Sox family protein-binding site. These results indicate that the dual mechanism causing inhibition in fast muscle fibers and activation in slow muscle fibers is essential for slow muscle fiber-specific gene expression in fish.

Developmental transcriptomic analyses for mechanistic insights into critical pathways involved in embryogenesis of pelagic mahi-mahi (Coryphaena hippurus).

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac ß-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

Embryonic development of the grass pufferfish (Takifugu niphobles): From egg to larvae.

Tetraodontidae (pufferfish) family members carry the smallest genomes among vertebrates, and these pocket-sized genomes have directly contributed to our understanding of the structure and evolution of higher animals. The grass pufferfish (Takifugu niphobles) could be considered a potential new model organism for comparative genomics and development due to the potential access to embryos, and availability of sequence data for two similar genomes: that of spotted green pufferfish (Tetraodon nigroviridis) and Fugu (Takifugu rubripes). In this study, we provide the first description of the normal embryonic development of T. niphobles, by drawing comparisons with the closely related species cited above. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored at a constant temperature consistent with natural conditions. T. niphobles development was divided into seven periods of embryogenesis: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods; and stages subdividing these periods are defined based on morphological characteristics. The developmental stage series described in this study aims to provide the utilization of T. niphobles as an experimental model organism for comparative developmental studies.

Role of notochord cells and sclerotome-derived cells in vertebral column development in fugu, Takifugu rubripes: histological and gene expression analyses.

Despite the common structure of vertebrates, the development of the vertebral column differs widely between teleosts and tetrapods in several respects, including the ossification of the centrum and the function of the notochord. In contrast to tetrapods, vertebral development in teleosts is not fully understood, particularly for large fish with highly ossified bones. We therefore examined the histology and gene expression profile of vertebral development in fugu, Takifugu rubripes, a model organism for genomic research. Ossification of the fugu centrum is carried out by outer osteoblasts expressing col1a1, col2a1, and sparc, and the growing centra completely divide the notochord into double cone-shaped segments that function as intercentral joints. In this process, the notochord basal cells produce a thick notochord sheath exhibiting Alcian-blue-reactive cartilaginous properties and composing the intercentral ligament in cooperation with the external ligament connective tissue. Synthesis of the matrix by the basal cells was ascertained by an in vitro test. Expression of twist2 indicates that this connective tissue is descended from the embryonic sclerotome. Notochord basal cells express sox9, ihhb, shh, and col2a1a, suggesting that the signaling system involved in chondrocyte proliferation and matrix production also functions in notochord cells for notochord sheath formation. We further found that the notochord expression of both ntla and shh is maintained in the fugu vertebral column, whereas it is turned off after embryogenesis in zebrafish. Thus, our results demonstrate that, in contrast to zebrafish, a dynamic morphogenesis and molecular network continues to function in fugu until the establishment of the adult vertebral column.

Analyses of fugu hoxa2 genes provide evidence for subfunctionalization of neural crest cell and rhombomere cis-regulatory modules during vertebrate evolution.

Hoxa2 gene is a primary player in regulation of craniofacial programs of head development in vertebrates. Here we investigate the evolution of a Hoxa2 neural crest enhancer identified originally in mouse by comparing and contrasting the fugu hoxa2a and hoxa2b genes with their orthologous teleost and mammalian sequences. Using sequence analyses in combination with transgenic regulatory assays in zebrafish and mouse embryos we demonstrate subfunctionalization of regulatory activity for expression in hindbrain segments and neural crest cells between these two fugu co-orthologs. hoxa2a regulatory sequences have retained the ability to mediate expression in neural crest cells while those of hoxa2b include cis-elements that direct expression in rhombomeres. Functional dissection of the neural crest regulatory potential of the fugu hoxa2a and hoxa2b genes identify the previously unknown cis-element NC5, which is implicated in generating the differential activity of the enhancers from these genes. The NC5 region plays a similar role in the ability of this enhancer to mediate reporter expression in mice, suggesting it is a conserved component involved in control of neural crest expression of Hoxa2 in vertebrate craniofacial development.

Responses of Takifugu obscurus fertilized eggs and larvae to increased ammonia exposure.

Ammonia is a common toxicant in aquatic systems; this substance has become a critical threat to fish, especially in early life stages. This study aimed to evaluate the effects of unionized ammonia (NH3-N: 0, 0.068, 0.138, 0.206, 0.275, 0.343, 0.412, and 0.481 mg L(-1)) on fertilized eggs and larvae of obscure puffer Takifugu obscurus, a fish species with potential economic value. Results showed that hatch time was significantly retarded and hatch rate was significantly decreased as NH3-N concentrations increased; newly hatched larvae exhibited high rate of abnormalities and low viability. The survival rate of larvae also decreased significantly as NH3-N concentrations increased; larvae could tolerate NH3-N to a less extent than embryos. NH3-N also caused a significant decrease in superoxide dismutase (SOD) and Na(+)/K(+) ATPase activities but not in malondialdehyde (MDA) levels of larvae. Two-way ANOVA indicated that there was a statistically significant interaction between NH3-N concentrations and exposure times on SOD activity but not on Na(+)/K(+) ATPase activity. Such responses indicated that an increase in ammonia concentration in surface water may negatively affect the early development of T. obscurus and thus likely impair population recruitment and persistence of this fish species.

Novel sex-determining genes in fish and sex chromosome evolution.

Although the molecular mechanisms underlying many developmental events are conserved across vertebrate taxa, the lability at the top of the sex-determining (SD) cascade has been evident from the fact that four master SD genes have been identified: mammalian Sry; chicken DMRT1; medaka Dmy; and Xenopus laevis DM-W. This diversity is thought to be associated with the turnover of sex chromosomes, which is likely to be more frequent in fishes and other poikilotherms than in therian mammals and birds. Recently, four novel candidates for vertebrate SD genes were reported, all of them in fishes. These include amhy in the Patagonian pejerrey, Gsdf in Oryzias luzonensis, Amhr2 in fugu and sdY in rainbow trout. These studies provide a good opportunity to infer patterns from the seemingly chaotic picture of sex determination systems. Here, we review recent advances in our understanding of the master SD genes in fishes.

Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: transient and transgenic analysis of torafugu MYH(M86-2) promoter in zebrafish embryos.

The myosin heavy chain gene, MYHM86-2, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYHM86-2 promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614bp 5'-flanking sequences of MYHM86-2 contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYHM86-2 expression in the fast muscle fibers. The transcriptional mechanism that prevents MYHM86-2 expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYHM86-2 expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYHM86-2 expression.

The expression of multiple myosin heavy chain genes during skeletal muscle development of torafugu Takifugu rubripes embryos and larvae.

In vertebrates, the development-dependent and tissue-specific expression of myosin heavy chain (MYH) genes (MYHs) contributes to the formation of diverged muscle fiber types. The expression patterns of developmentally regulated MYHs have been investigated in certain species of fish. However, the expression profiles of MYHs during torafugu Takifugu rubripes development, although extensively studied in adult tissues, have not been sufficiently studied, and also the expression orders of MYHs during development have remained unclear. In the present study, we comprehensively cloned four MYHs (MYH(M743-2), MYH(M86-2), MYH(M5) and MYH(M2126-1)) from embryos, and two MYHs (MYH(M2528-1) and MYH(M1034)) from larvae, and characterized their expression pattern in relation to developmental stages of torafugu by reverse transcription (RT)-PCR and in situ hybridization. The expression of MYHs from torafugu embryos and larvae appeared sequentially and varied largely in relation to the developmental stage-dependent and fibers-type-specific manners. The transcripts of MYH(M743-2) appeared first in embryos at 3 days post fertilization (dpf) and were localized in the epaxial and hypaxial domains of fast muscle fibers of larval myotome, whereas those of MYH(M5) and MYH(M86-2) in 3 dpf and 4 dpf, respectively, and both were localized in superficial slow and horizontal myoseptum regions. The expression of MYH(M1034) and MYH(M2126-1) was quite low and mostly undetectable. Different MYHs from torafugu embryos and larvae have also been found to be expressed differentially in pectoral fin and craniofacial muscles. Interestingly, the transcripts of MYH(M2528-1) first appeared at 6 dpf and were distinctly expressed at the dorsal and ventral extremes of larval myotome, suggesting its involvement in stratified hyperplasia. The novel involvement of MYH(M2528-1) in mosaic hyperplasia was further confirmed in juvenile torafugu, where the transcripts were expressed in fast fibers with small diameters as well as the inner part of superficial slow fibers.

A lineage specific enhancer drives Otx2 expression in teleost organizer tissues.

In mouse Otx2 plays essential roles in anterior-posterior axis formation and head development in anterior visceral endoderm and anterior mesendoderm. The Otx2 expression in these sites is regulated by VE and CM enhancers at the 5' proximal to the translation start site, and we proposed that these enhancers would have been established in ancestral sarcoptergians after divergence from actinopterigians for the use of Otx2 as the head organizer gene (Kurokawa et al., 2010). This would make doubtful an earlier proposal of ours that a 1.1 kb fragment located at +14.4 to +15.5 kb 3' (3'En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (Kimura-Yoshida et al., 2007). In the present study, we demonstrate that fugu Otx2a is not expressed in the dorsal margin of blastoderm, shield and early anterior mesendoderm, and that the fugu Otx2a 3'En do not exhibit activities at these sites of fugu embryos. We conclude that the fugu Otx2a 3'En does not harbor an organizer enhancer, but encodes an enhancer for the expression in later anterior mesendodermal tissues. Instead, in fugu embryos Otx2b is expressed in the dorsal margin of blastoderm at blastula stage and shield at 50% epiboly, and this expression is directed by an enhancer, 5'En, located at -1000 to -800 bp, which is uniquely conserved among teleost Otx2b orthologues.

Multiple cis-elements in the 5'-flanking region of embryonic/larval fast-type of the myosin heavy chain gene of torafugu, MYH(M743-2), function in the transcriptional regulation of its expression.

The myosin heavy chain gene, MYH(M743-2), is highly expressed in fast muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional regulatory mechanism involved in its expression remained unknown. Here, we analyzed the 2075bp 5'-flanking region of torafugu MYH(M743-2) to examine the spatial and temporal regulation by using transgenic and transient expression techniques in zebrafish embryos. Combining both transient and transgenic analyses, we demonstrated that the 2075bp 5'-flanking sequences was sufficient for its expression in skeletal, craniofacial and pectoral fin muscles. The immunohistochemical observation revealed that the zebrafish larvae from the stable transgenic line consistently expressed enhanced green fluorescent protein (EGFP) in fast muscle fibers. Promoter deletion analyses demonstrated that the minimum 468bp promoter region could direct MYH(M743-2) expression in zebrafish larvae. We discovered that the serum response factor (SRF)-like binding sites are required for promoting MYH(M743-2) expression and myoblast determining factor (MyoD) and myocyte enhancer factor-2 (MEF2) binding sites participate in the transcriptional control of MYH(M743-2) expression in fast skeletal muscles. We further discovered that MyoD binding sites, but not MEF2, participate in the transcriptional regulation of MYH(M743-2) expression in pectoral fin and craniofacial muscles. These results clearly demonstrated that multiple cis-elements in the 5'-flanking region of MYH(M743-2) function in the transcriptional control of its expression.

Acquisition of glial cells missing 2 enhancers contributes to a diversity of ionocytes in zebrafish.

Glial cells missing 2 (gcm2) encoding a GCM-motif transcription factor is expressed in the parathyroid in amniotes. In contrast, gcm2 is expressed in pharyngeal pouches (a homologous site of the parathyroid), gills, and H(+)-ATPase-rich cells (HRCs), a subset of ionocytes on the skin surface of the teleost fish zebrafish. Ionocytes are specialized cells that are involved in osmotic homeostasis in aquatic vertebrates. Here, we showed that gcm2 is essential for the development of HRCs and Na(+)-Cl(-) co-transporter-rich cells (NCCCs), another subset of ionocytes in zebrafish. We also identified gcm2 enhancer regions that control gcm2 expression in ionocytes of zebrafish. Comparisons of the gcm2 locus with its neighboring regions revealed no conserved elements between zebrafish and tetrapods. Furthermore, We observed gcm2 expression patterns in embryos of the teleost fishes Medaka (Oryzias latipes) and fugu (Fugu niphobles), the extant primitive ray-finned fishes Polypterus (Polypterus senegalus) and sturgeon (a hybrid of Huso huso × Acipenser ruhenus), and the amphibian Xenopus (Xenopus laevis). Although gcm2-expressing cells were observed on the skin surface of Medaka and fugu, they were not found in Polypterus, sturgeon, or Xenopus. Our results suggest that an acquisition of enhancers for the expression of gcm2 contributes to a diversity of ionocytes in zebrafish during evolution.

Embryogenic staging of fugu, Takifugu rubripes, and expression profiles of aldh1a2, aldh1a3 and cyp26a1.

Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis - the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.

Identification of a distant cis-regulatory element controlling pharyngeal arch-specific expression of zebrafish gdf6a/radar.

Skeletal formation is an essential and intricately regulated part of vertebrate development. Humans and mice deficient in growth and differentiation factor 6 (Gdf6) have numerous skeletal abnormalities, including joint fusions and cartilage reductions. The expression of Gdf6 is dynamic and in part regulated by distant evolutionarily conserved cis-regulatory elements. radar/gdf6a is a zebrafish ortholog of Gdf6 and has an essential role in embryonic patterning. Here, we show that radar is transcribed in the cells surrounding and between the developing cartilages of the ventral pharyngeal arches, similar to mouse Gdf6. A 312 bp evolutionarily conserved region (ECR5), 122 kilobases downstream, drives expression in a pharyngeal arch-specific manner similar to endogenous radar/gdf6a. Deletion analysis identified a 78 bp region within ECR5 that is essential for transgene activity. This work illustrates that radar is regulated in the pharyngeal arches by a distant conserved element and suggests radar has similar functions in skeletal development in fish and mammals.

Change in tetrodotoxin content of the puffer fish Takifugu rubripes during seed production from fertilized eggs to juveniles.

Changes in tetrodotoxin (TTX) content of the puffer fish Takifugu rubripes during seed production were examined. Two mature female puffer fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of puffer fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.

A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals.

Spinal interneurons are key components of locomotor circuits, driving such diverse behaviors as swimming in fish and walking in mammals. Recent work has linked the expression of evolutionarily conserved transcription factors to key features of interneurons in diverse species, raising the possibility that these interneurons are functionally related. Consequently, the determinants of interneuron subtypes are predicted to share conserved cis-regulation in vertebrates with very different spinal cords. Here, we establish a link between cis-regulation and morphology of spinal interneurons that express the Evx1 homeodomain transcription factor from fish to mammals. Using comparative genomics, and complementary transgenic approaches, we have identified a novel enhancer of evx1, that includes two non-coding elements conserved in vertebrates. We show that pufferfish evx1 transgenes containing this enhancer direct reporter expression to a subset of spinal commissural interneurons in zebrafish embryos. Pufferfish, zebrafish and mouse evx1 downstream genomic enhancers label selectively Evx1(+) V0 commissural interneurons in chick and rat embryos. By dissecting the zebrafish evx1 enhancer, we identify a role for a 25 bp conserved cis-element in V0-specific gene expression. Our findings support the notion that spinal interneurons shared between distantly related vertebrates, have been maintained in part via the preservation of highly conserved cis-regulatory modules.

Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning.

The Mesp bHLH genes play a conserved role during segmental patterning of the mesoderm in the vertebrate embryo by specifying segmental boundaries and anteroposterior (A-P) segmental polarity. Here we use a xenotransgenic approach to compare the transcriptional enhancers that drive expression of the Mesp genes within segments of the presomitic mesoderm (PSM) of different vertebrate species. We find that the genomic sequences upstream of the mespb gene in the pufferfish Takifugu rubripes (Tr-mespb) are able to drive segmental expression in transgenic Xenopus embryos while those from the Xenopus laevis mespb (Xl-mespb) gene drive segmental expression in transgenic zebrafish. In both cases, the anterior segmental boundary of transgene expression closely matches the expression of the endogenous Mesp genes, indicating that many inputs into segmental gene expression are highly conserved. By contrast, we find that direct retinoic acid (RA) regulation of endogenous Mesp gene expression is variable among vertebrate species. Both Tr-mespb and Xl-mespb are directly upregulated by RA, through a complex, distal element. By contrast, RA represses the zebrafish Mesp genes. We show that this repression is mediated, in part, by RA-mediated activation of the Ripply genes, which together with Mesp genes form an RA-responsive negative feedback loop. These observations suggest that variations in a direct response to RA input may allow for changes in A-P patterning of the segments in different vertebrate species.

Differential regulation of multiple alternatively spliced transcripts of MyoD.

Splice variants of the basic helix-loop-helix myoblast determination factor (myoD) have not been previously found in vertebrates. Here we report the identification and characterization of three alternative transcripts of a myoD paralogue from the tiger pufferfish (Takifugu rubripes). The T. rubripes myoD1 gene (TmyoD1) has 3 exons and 2 introns and it is present on scaffold 104, in a region of conserved synteny with zebrafish. The isoform TMyoD1-alpha is a putative protein of 281 residues that contains the basic, helix-loop-helix and helix III domains and shares 61%, 56%, 51%, 49% and 56% overall identity with zebrafish, Xenopus, mouse, human and chicken MyoD1, respectively. TMyoD1-beta arises from an alternative 3' splice site and differs from TMyoD1-alpha by a 26-residue insertion adjacent to helix III, which is one of the functional domains required for chromatin remodelling. The third alternative transcript, TmyoD1-gamma, retains intron I and has two premature termination codons far from the 3'-most exon-exon junction. TmyoD1-gamma is therefore likely to be degraded by nonsense-mediated decay, an important widespread post-transcriptional mechanism that regulates transcript levels. Analysis of gene expression by qPCR revealed that TmyoD1-alpha was the most abundant transcript in fast and slow myotomal muscle. TmyoD1-alpha expression was 2-fold higher in fast muscle of juvenile fish that were actively producing new myotubes compared to adult stages that had stopped recruiting fast muscle fibres. A similar expression pattern was observed for TmyoD1-alpha in slow muscle but the differences were not significant. Transcript levels of TmyoD1-gamma only varied significantly in fast muscle and were 5-fold higher in adult compared to juvenile stages. Significant differences in expression of TmyoD1 splice variants were also observed during embryonic development. The differential expression of three alternative transcripts of myoD1 in developing and adult myotomal muscle of T. rubripes supports the hypothesis that diversity generated by alternative splicing may be of functional significance in muscle development in this species.

Expression of fugu TIMP-3 and -4 genes in adult tissues and embryos.

The tissue inhibitors of metalloproteinases (TIMPs) are involved in various processes of extra-cellular matrix (ECM) metabolism by inhibiting matrix metalloproteinases (MMPs). However, the fundamental information for these genes is little known in fish. Previously, we report cDNA cloning and gene expressions of two fugu (Takifugu rubripes) TIMP-2s. Here, we cloned cDNA of fugu TIMP-3 and performed an expression analysis of TIMP-3 and -4 mRNA in fugu adult tissues using a quantitative real-time PCR. The expression level of TIMP-3 mRNA was constitutive in all tissues, while TIMP-4 was significantly higher in the brain (P=0.05). Further, we performed a whole mount in situ hybridization in fugu embryos at different stages. In early stages, TIMP-3 mRNA was abundant in the somites and the caudal end of the notochord. At hatching larvae, the TIMP-3 mRNA was abundant in the pectoral fin, dorsal and ventral fin fold along the entire antero-posterior axis. TIMP-3 may be involved in axis elongation and somitogenesis. TIMP-4 mRNA was expressed in the tail bud, at the midbrain-hindbrain boundary and in the diencephalon from 72 to 104 hpf. This indicates TIMP-4 is highly expressed in the brain matrix in vivo.