Yuqing Zhang

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 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.

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.

Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus)

During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month-old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

Characterization of flounder ( Paralichthys olivaceus ) FoxD5 and its function in regulating myogenic regulatory factor

As one member of winged helix domain transcription factors, FoxD5 was reported to be a trunk organizer. Recent study showed that zebrafish foxd5 is expressed in the somites. To further understand the function of FoxD5 in fish muscle development, the FoxD5 gene was isolated from flounder. Its expression pattern was analyzed by in situ hybridization, while its function in regulating myogenic regulatory factor, MyoD, was analyzed by ectopic expression. It showed that flounder FoxD5 was firstly expressed in the tailbud, adaxial cells, and neural plate of the head. In flounder embryo, FoxD5 is expressed not only in forebrain but also in somite cells that will form muscle in the future. When flounder FoxD5 was over-expressed in zebrafish by microinjection, the expression of zebrafish MyoD in the somites was reduced, suggesting that FoxD5 is involved in myogenesis by regulating the expression of MyoD.

Flounder (Paralichthys olivaceus) FoxD1 and its regulation on the expression of myogenic regulatory factor, MyoD

It has been reported that FoxD1 plays important roles in formation of several different tissues, such as retina and kidney in vertebrates. The function of FoxD1 in muscle development is, however, unclear although it is expressed in muscle cells in zebrafish. Muscles are the major tissue in fish, which serves as a rich protein source in our diet. To further understand the function of FoxD1 in fish muscle development, here we isolated and characterized the FoxD1 gene from flounder (Paralichthys olivaceus), a valuable sea food and an important fish species in aquaculture in Asia. We analyzed its expression pattern and function in regulating myogenic regulatory factor, MyoD, one of the earliest marker of myogenic commitment. In situ hybridization revealed that FoxD1 was expressed in the tailbud, adaxial cells, posterior intestine, forebrain, midbrain and half of the retina in flounder embryos. Functional studies demonstrated that when flounder FoxD1 was over-expressed in zebrafish by microinjection, MyoD expression was decreased, suggesting that FoxD1 may be involved in myogenesis by regulating the expression of MyoD.

Characterization of DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) from Zebrafish (Dario rerio)

Proteins of the DYRK (dual-specificity tyrosine-phosphorylation-regulated kinase) family are characterized by the presence of a conserved kinase domain and N-terminal DH box. DYRK2 is involved in regulating key developmental and cellular processes, such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation. Herein, we report that the ortholog of DYRK2 found in zebrafish shares about 70% identity with that of human, mouse, and chick. RT-PCR showed that DYRK2 is expressed maternally and zygotically. In-situ hybridization results show that DYRK2 is expressed in somite cells that will develop into muscles. Our results provide preliminary evidence for investigating the in-vivo function of DYRK2 in zebrafish muscle development.