Functional Analysis Of Dmrt2a And Igf2bp1 During Zebrafish Embryo Development

MicroRNAs(miRNAs)play an important role in muscle development,such as the process of muscle prolifeiation,regeneration and repair.Also,they regulate muscle mass and quality.Dmrt2b(doublesex and mab-3 related transcription factor 2b)has been revealed to be involved in zebrafish slow muscle development.However,the function of dmrt2 a,a paralogue gene of dmrt2 b,remains unclear during zebrafish muscle development.Here,we demonstrated that knockdown of dmrt2 a resulted in severe developmental defects,and caused downregulation of fast muscle marker myhz-2 and upregulation of slow muscle marker myhz-5,respectively.Dmrt2 a was predicted to be a target gene of mi R-203,which contain mi R-203 a and mi R-203 b,there is only one nucleotide difference between them,and their seed sequences are identical,we took mi R-203 a for future studies.Luciferase reporter assay verified that mi R-203 a can directly reduce the expression of dmrt2 a by binding to the seed sequence of its 3?UTR.After mi R-203 a injection into zebrafish embryos,the expression of dmrt2 a was significantly inhibited.Similar to the effect of dmrt2 a knockdown,mi R-203 a overexpression led to downregulation of myhz-2 and upregulation of myhz-5.Our studies indicated that mi R-203 a directly regulated dmrt2 a expression to control fast and slow muscle differentiation,while overexpression of mi R-203 a or knockdown of dmrt2 a will impair fast muscle development and promote slow muscle development.Igf2bp1(insulin-like growth factor-2 m RNA-binding protein 1)was predicted to be a target gene of mi R-203.Here we demonstrated that overexpression of mi R-203 a can downregulate igf2bp1 expression in zebrafish embryos,suggesting that mi R-203 may potentially target igf2bp1.However,knockdown of igf2bp1 had no affect on muscle development in zebrafish embryos,indicating that igf2bp1 has no functional relationship in muscle development.Further studies showed that knockdown of igf2bp1 led to a smaller size liver and caused downregulation of the hepatocyte maker fabp10 a,whereas the expression of the endoderm makers gata6 and foxa3 was not changed in zebrafish embryos.Gene chip technology was used to analyze the gene expression profile between Cont-MO injected embryos and igf2bp1-MO injected embryos.The results showed that the expression of fabp10 a in igf2bp1-MO injected embryos was significantly downregulated compared to Cont-MO injected embryos.Moreover,the expression of gata6 and foxa3 has no obvious change between these two groups.There were no obvious phenotype difference between igf2bp1-/-zebrafish and WT(wild type)zebrafish,except that the liver size was smaller and the expression of fabp10 a was downregulated in igf2bp1-/-zebrafish embryos,while the expression of gata6 and foxa3 were not changed.These data suggest igf2bp1 is required for hepatic outgrowth during early liver development in zebrafish.In conclusion,dmrt2 a and igf2bp1 play an important role in muscle development and liver development,respectively.