Cloning, Expression And Functional Analysis Of Genes Regulating Muscle Development From Zebrafish

This study focuses on the genes relating to muscle development during embrogenesis in zebrafish. Full lengths of four genes are cloned and their expession patterns are analyzed by in situ hybridization and RT-PCR. Overexpession experiments are done in two genes to analyze their function on somite and muscle during embyo development in zebrafish.Thyroid hormone receptor associated protein 150 (TRAP150) is cloned from zebrafish cDNA library. Results of in situ hybridizaton show that TRAP150 is detected in slow muscle of adaxial cells and in fast muscle of paraxial mesoderm at early stage of embyogenesis. The expression pattern of TRAP150 is similar to MyoD and exhibits the specific character related to muscle. The high level of expession of TRAP150 in somites and muscle indicates that TRAP150 probably plays an important role in muscle development. Furthermore, TRAP150 is detected in cardiac muscle. Overexpession analysis of TRAP150 results in overexpression of MyoD in paraxial mesoderm. Since fast muscle is induced by MyoD in paraxial mesoderm, overexpression of TRAP150 can up-regulates fast muscle development. It is indicated that TRAP150 might be a positive upstream regulator in regulating MyoD expression and functions in fast muscle development..Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 isoform 2 (DYRK2) is cloned from zebrafish cDNA library. The result of RT-PCR shows this gene is maternal expression and can be expressed in every stage before 36h. Results of in situ hybridizaton showed that DYRK2 is detected in adaxial cells at early stage of embyogenesis, and detected in fast muscle at 15 somites stage. The expession pattern of DYRK2 performs muscle specific character. In 18 hours stage, DYRK2 is also detected in brains and eyes. Overexpession of DYRK2 results in overexpression of MyoD in adaxial cells and paraxial mesoderm in the injected side at early stage of embyogenesis, paticularly in presomite mesoderm. Overexpession analysis of DYRK2 showed that MyoD expression in slow muscle is up-regulated, indicating that DYRK2 is involved in slow muscle development by the way of regulating expression of MyoD.Hematopoietic Pre-B-cell leukemia transcription factor interacting protein 1 (HPIP1) is cloned from zebrafish cDNA library.Results of RT-PCR indicated that Pbxip1 has maternal resourse, but HPIP1 is firstly detected at 10 somites stage of embyogenesisby in situi hybridization, which indicates that HPIP1 is expressed after muscle begin to differentiate and HPIP1 plays an important role in muscle differentiation and maturation. Furthermore, HPIP1 is detected in eyes, indicateing that HPIP1 probably is involved in eye muscle development.CHORDC1 is cloned from zebrafish cDNA library. At 3 somites stage, CHORDC1 is detected in adaxial cells and is found in paraxial mesoderm at 5 somites stage. The expression character of CHORDC1 is similar to MyoD. The difference is that CHORDC1 is also detected in presomitic mesoderm which is similar to Myf5. CHORDC1 expression followed the expression of MyoD,Myf5, which indicates that CHORDC1 might play an important role in muscle differentiation and maturation. The expression of CHORDC1 differs from MyoD and Myf5 in the presomitic mesoderm.The differences display not only in the slow muscle and fast muscle; but also CHORDC1 expression dislays a gradual expansion from posterior to anterior. Furthermore, CHORDC1 is detected in heart muscle as well. Therefore, CHORDC1 performs the specific expression in muscle but not specific muscle type.