Expression Patterns And Functional Analysis Of MHB Related Genes In Amphioxus

In the vertebrate central nervous system(CNS),mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain-hindbrain boundary(MHB),and then a series of signal molecules(such as Wntl and Fgf8)and transcription factors(such as Pax2,5,8 and Engrailed)are expressed in this area.Although MHB related genes have been well studied in the Drosophila,zebrafish,Xenopus and mouse species concerning its involvement in early embryonic development,it remains poorly characterized in other lineages.The cephalchordate amphioxus is the closest living relative to the vertebrate,and has been widely known as the most important animal to study the origin and evolution of vertebrates.Studies on gene expression patterns and function in amphioxus will contribute to understand the origin and evolution of the MHB,and further illuminate the molecular mechanism of the development of amphioxus nervous system.In this research,we cloned amphioxus Otx,Gbx,En,Hoxl,Hox3,FGF8/17/18 and Wnts,and analyzed their expression patterns.The expression patterns of these genes revealed several results.Firstly,division of neurogenic ectoderm into anterior Otx and posterior Gbx domains was evolutionarily conserved.Secondly,these genes early expression patterns were basically the same between Branchiostoma belcheri and B.floridae.Thirdly,there was a apparent boundary between the expression of Otx and Gbx in the neural tube and this area was most likely the homologous of vertebrate MHB.Although expression patterns of Otx and Gbx suggested that they may be mutual inhibition,MHB markers such as Wnt1 and En were not expressed at high levels in this area.To further demonstrate the role of Otx and Gbx during embryogenesis,we designed two siRNAs to knockdown Otx expression,three siRNAs to knockdown Gbx expression and constructed overexpression vectors to study their function.RT-qPCR and ISH between the experimental group and the control group which were injected with Otx-siRNA1,Gbx-siRNA3 and Control siRNA respectively showed that siRNAs could knockdown Otx and Gbx expression remarkably.Through the normal developmental ratio analysis and phenotypic observation,we found that the normal developmental ratio of experimental group had some decline,but there was no significant difference between the two groups,supposing that small difference be not easy to tell or the amount of knockdown effect can't cause phenotypic difference.Through the phenotypic observation,we found high concentration of Otx-mRNA and Gbx-mRNA injection could lead to obvious defects in the experimental embryonic front when embryos developed to 3-day larva,which indicated that the two genes play an important role in the development of amphioxus nervous system.In addition,we detect the MHB related genes expression in embryos after injection,found that,the expression level of FGF8/17/18 was obvious up(down)regulated,Gbx expression region in neural tube slightly forward former when Otx was knockdown,and Otx expression area was slightly larger and border backwards in neural tube when Gbx was knockdown.After Otx overexpression,Gbx,Hoxl and Pax2/5/8 were up-regulated.After Gbx overexpression,Otx expression region in neural tube moved forward obviously,Hoxl gene expression slight up.So we inferred Otx and Gbx can regulate MHB related genes expression.