Xungang Tan

Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata).

Abstract. Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoD genes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoD expression in adaxial cells.

Gonadal transcriptome analysis of male and female olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is an important commercially cultured marine flatfish in China, Korea, and Japan, of which female grows faster than male. In order to explore the molecular mechanism of flounder sex determination and development, we used RNA-seq technology to investigate transcriptomes of flounder gonads. This produced 22,253,217 and 19,777,841 qualified reads from ovary and testes, which were jointly assembled into 97,233 contigs. Among them, 23,223 contigs were mapped to known genes, of which 2,193 were predicted to be differentially expressed in ovary and 887 in testes. According to annotation information, several sex-related biological pathways including ovarian steroidogenesis and estrogen signaling pathways were firstly found in flounder. The dimorphic expression of overall sex-related genes provides further insights into sex determination and gonadal development. Our study also provides an archive for further studies of molecular mechanism of fish sex determination.

Characterization of Muscle-Regulatory Gene, MyoD, from Flounder (Paralichthys olivaceus) and Analysis of Its Expression Patterns During Embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix–loop–helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Characterization of Muscle-Regulatory Genes, Myf5 and Myogenin, from Striped Bass and Promoter Analysis of Muscle-Specific Expression

Myf5 and Myogenin are basic helix-loop-helix transcription factors that belong to the muscle regulatory factor (MRF) gene family, which plays important roles in regulating skeletal muscle development and growth. Members of the MRF family, including Myf5, MyoD, Myogenin, and MRF4 are specifically expressed in skeletal muscle cells. They have the remarkable property of converting a variety of cells into myoblasts and myotubes when ectopically expressed in other cell types. To better understand their role and regulation of expression in fish muscle cells, Myf5 and myogenin genomic genes were isolated from striped bass (Morone saxatilis). Sequence analysis revealed that these 2 genes shared similar structures. They both contained 3 exons and 2 introns, and a highly conserved basic helix-loop-helix domain. Promoter analysis identified several putative E box sites in both Myf5 and myogenin promoters that might confer muscle-specific expression. To determine if the striped bass Myf5 and myogenin promoters could control muscle-specific expression, the Myf5 or myogenin promoter was linked with the green fluorescent protein (GFP) reporter gene, and their promoter activity was analyzed in zebrafish embryos by transient expression assay. Our data showed that both striped bass Myf5 and myogenin promoters could drive muscle-specific GFP expression in zebrafish. These data demonstrated that a muscle-specific regulatory element or elements were located within the striped bass Myf5 and myogenin promoters, and were conserved between striped bass and zebrafish. Moreover, these data suggested the muscle-specific regulatory element could function across fish species in regulating gene expression.

Characterization of amphioxus GDF8/11 gene, an archetype of vertebrate MSTN and GDF11

MSTN, also known as growth and differentiation factor 8 (GDF8), and GDF11 are members of the transforming growth factor-β (TGF-β) subfamily. They have been thought to be derived from one ancestral gene. In the present study, we report the isolation and characterization of an invertebrate GDF8/11 homolog from the amphioxus (Branchiostoma belcheri tsingtauense). The amphioxus GDF8/11 gene consists of five exons flanked by four introns, which have two more exons and introns than that of other species. In intron III, a possible transposable element was identified. This suggested that this intron might be derived from transposon. The amphioxus GDF8/11 cDNA encodes a polypeptide of 419 amino acid residues. Phologenetic analysis shows that the GDF8/11 is at the base of vertebrate MSTNs and GDF11s. This result might prove that the GDF8/11 derived from one ancestral gene and the amphioxus GDF8/11 may be the common ancestral gene, and also the gene duplication event generating MSTN and GDF11 occurred before the divergence of vertebrates and after or at the divergence of amphioxus from vertebrates. Reverse transcriptase polymerase chain reaction results showed that the GDF8/11 gene was expressed in new fertilized cell, early gastrulation, and knife-shaped embryo, which was different from that in mammals. It suggested that the GDF8/11 gene might possess additional functions other than regulating muscle growth in amphioxus.

Transcriptional responses of olive flounder (Paralichthys olivaceus) to low temperature.

The olive flounder (Paralichthys olivaceus) is an economically important flatfish in marine aquaculture with a broad thermal tolerance ranging from 14 to 23°C. Cold-tolerant flounder that can survive during the winter season at a temperature of less than 14°C might facilitate the understanding of the mechanisms underlying the response to cold stress. In this study, the transcriptional response of flounder to cold stress (0.7±0.05°C) was characterized using RNA sequencing. Transcriptome sequencing was performed using the Illumina MiSeq platform for the cold-tolerant (CT) group, which survived under the cold stress; the cold-sensitive (CS) group, which could barely survive at the low temperature; and control group, which was not subjected to cold treatment. In all, 29,021 unigenes were generated. Compared with the unigene expression profile of the control group, 410 unigenes were up-regulated and 255 unigenes were down-regulated in the CT group, whereas 593 unigenes were up-regulated and 289 unigenes were down-regulated in the CS group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that signal transduction, lipid metabolism, digestive system, and signaling molecules and interaction were the most highly enriched pathways for the genes that were differentially expressed under cold stress. All these pathways could be assigned to the following four biological functions for flounder that can survive under cold stress: signal response to cold stress, cell repair/regeneration, energy production, and cell membrane construction and fluidity.

Analyzing Cold Tolerance Mechanism in Transgenic Zebrafish (Danio rerio)

Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13°C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28°C. After 2 h at 13°C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P<0.05). At 13°C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28°C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish.

A new pattern of primordial germ cell migration in olive flounder (Paralichthys olivaceus) identified using nanos3

The olive flounder (Paralichthys olivaceus) is an important cultured marine fish. However, little information is available on primordial germ cell (PGC) development and migration in this species; such information is vital for applications in artificial reproduction and for the preservation of genetic resources. Here, we sought to remedy this information deficit by isolating the germline-specific gene nanos3 and analyzing its expression in olive flounder. Sequencing analysis showed that olive flounder nanos3 contained a typical RNA-binding zinc finger domain. A phylogenetic analysis demonstrated that nanos3 of the olive flounder grouped with that of the barfin flounder (Verasper moseri). In the olive flounder, nanos3 was consistently expressed during embryogenesis. Whole-mount in situ hybridization showed that a new pattern of PGC migration was present in olive flounder, which combined elements of the PGC migration patterns of medaka, herring, and goby. In olive flounder, PGCs aligned along the lateral plate mesoderm in two loose, elongated lines at early embryogenesis, aggregated into a single loose cluster at mid-embryogenesis, then re-aligned into two tight clusters at late somitogenesis, and finally migrated to the genital ridge as two clusters. Furthermore, whole-mount in situ hybridization revealed that expression of stromal derived factor 1 (Sdf1) was important for guiding of PGC migration during somitogenesis. In particular, Sdf1 directed aggregation of PGCs into a single loose cluster from the two elongated lines during mid-embryogenesis. Additionally, PGCs in zebrafish were successfully visualized by injection of chimeric RNA containing the green fluorescent protein (GFP) and 3′ untranslated region of olive flounder nanos3. These findings provide new insights into PGC migration and development in olive flounder and will also facilitate germ cell manipulation in this species.

The olive flounder (Paralichthys olivaceus) Pax3 homologues are highly conserved, encode multiple isoforms and show unique expression patterns.

Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. Pax3 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest and skeletal muscle. Here, we identified two spliced isoforms of Pax3a and three spliced isoforms of Pax3b and characterized their expression patterns. Both of flounder Pax3a-1 and Pax3b-1 contain the conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). But the PD domain in Pax3a-2 and Pax3b-3 is not intact and there is no HD in Pax3b-2 and Pax3b-3. Pax3a and Pax3b show distinct temporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that Pax3a and Pax3b are expressed in overlapping patterns in the dorsal central nervous system, with some subtle regional differences between the two genes. In addition, Pax3a is scattered in the somites while Pax3b is specifically expressed in the newly forming somites. RT-PCR results have shown that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated Pax3 genes. The duplicated Pax3 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis.

Characterization of flounder (Paralichthys olivaceus) FoxD3 and its function in regulating myogenic regulatory factors

As one member of winged helix domain transcription factors, FoxD3 plays an important role in the regulation of neural crest development and maintenance of mammalian stem cell lineages. A recent study showed that zebrafish FoxD3 is a downstream gene of Pax3 and can mediate the expression of Myf5. To further understand the function of FoxD3 in fish muscle development, we isolated the FoxD3 gene from flounder, and analyzed its expression pattern and function in regulating myogenic regulatory factors, MyoD and Myf5. In situ hybridization showed that flounder FoxD3 was firstly detected in the premigratory neural crest cells at 90% epiboly stage. The FoxD3 was expressed not only in neural crest cells but also in somite cells that will form muscle in the future. When flounder FoxD3 was over-expressed in zebrafish by microinjection, the expressions of zebrafish Myf5 and MyoD were both affected. It is possible that FoxD3 is involved in myogenesis by regulating the expression of Myf5 and MyoD. Also, over-expression of flounder FoxD3 in zebrafish inhibits the expression of zebrafish endogenic FoxD3.