Analysis Of β-catenin Gene Function In Amphioxus During Embryonic Development And Activity Of Two Vertebrate Neural Crest Related Gene Promoters In Amphioxus

As the transitional group from invertebrates to vertebrates,amphioxus is an ideal animal model to study the functional evolution of genes related to vertebrate development.Nucleus-cytoplasmic shuttle protein β-catenin is a key component of the canonical Wnt signaling pathway,and its function has been reported in many species,but the researches on the function of β-catenin gene in amphioxus embryogenesis has just started,which is limited to the pattern analysis of some members involved in Wnt signaling and effects of some chemical reagents treaments.In this paper,the spatial-temporal pattern of fi-catenin in amphioxus during early development stage was detected firstly,and then the function ofβ-catenin in early axial development of amphioxus was studied by Talen knockout and over-expression of Wnt signaling related gene.Results tell that β-catenin gene expresssed in a uniform pattern throughout embryogenesis process,consistent with the data from transcriptome.Observing with the optical microscope,we found the body structure of the posterior trunk and tail was significantly absent in β-catenin knockout mutants,consistent with a shorten body length.Obviously,there is a defect in somitogenesis at tailbud,while at the anterior end complete pharyngeal oral and gill structure could not form.In subsequent observation we found that all the β-catenin/-/larvae died at about 5 days past fertilization.The results above concludes that β-catenin in amphioxus is indeedly involved in the establishment of the anterior-posterior axis of embryo and the formation of specific organs.In order to detailedly identify the changes on gene expression in β-catenin knockout,the expression of some tissue marker genes were detected in β-catenin/-/embryos by in situ hybridization.As the results show,foxQ2 expanded at the anterior ectoderm;Wnt antagonist sFRP2 remain expression of high level at the anterior ectoderm while the unexpressed domain shrink at the posterior end.Consistently,Anterior marker Fzl5/8 and Wnt antagonists Six3/6 also expanded at the anterior end,the expression domain of Pax4/6 in the forebrain expanded,then disappeared in the preoral pit in the late neurala stage.At the posterior end,the expression of Wnt family member Wnt3、Wnt8、and tail bud marker Brachyury、Evx and Cdx genes in the mesoderm at the posterior end were significantly decreased,in fact,the expression of Brachyury and Cdx in the tailbud of the posterior end basically disappeared from the mid-neurula stasge.Moreover,the expression domain of ventral]marker FoxAa shifted to the posterior end However,the expression of Nodal(dorsal maker),Evx(ventral maker)at gastrula stage were basically not affected.At the domain of oral and gills,gills maker Sixl/2,Eya and Paxl/9 all expand slightly,noticing that Paxl/9 was activated in the preoral domain.Accordingly,oral maker Dkkl/2/4 and Pou4 disappeared at the oral site.These results indicated that,after the knockout of β-catenin gene function in the zygote,while dorsal-ventral axis forms normally,the tail growth,somitogenesis and the regionalization of oral and gills primordia were seriously affected.Moreover,in the over-expression assay of Wnt signaling related genes,we found that the endoderm and ectoderm were no longer closely adhered to each other in the embryos injected of Dkkl/2/4 mRNA at the late gastrula stage,the gastrula parietal plate formed normally,but the posterior growth was blocked at neurula stage.Confusingly,the injection of stable form of β-catenin mRNA seems cause no abnormal phenotype,which may attribute to the translation fault of(△P)△-catenin.These results show that Wnt/△-catenin signaling participates in the modulation of ectoderm and endoderm regionalization but not the dorsal-ventral axis establishment during early development period,and then the anteri or-posterior extension at neurula stage.The transgenic vector system mediated by Tol2 transposon is able to carry interest fragments larger than 10 kb to insert into the host genome.Recently,it has been successfully applicated into the establishment of transgenic amphioxus strains using this system.We noticed that amphioxus lack neural crest structure compared with vertebrates,to investigate the evolution relationship of genes related with neural crest induction and expression environment in amphioxus,we apply Tol2 system to analyse the activities of some special functional promoters,and ultimately established the Snail-mCherry and Sox9-mCherry transgenic amphioxus strains.For the successfully transgenic individuals,the in situ detected specifically expression of mCherry at the most anterior neural border.As the result,this two promoter tools from vertebrates will provide valid support for subsequent function researches of neural crest related genes.