The Function Of Amphioxus Lefty Gene In Embryonic Development

The organizer is an essential signaling center required for axial formation and germ layer patterning during vertebrate embryo development.Accurate formation of the organizer requires regionalized activation of Wnt/?-catenin and Nodal signaling as well as Nodal signaling antagonist Lefty.In the basal chordate amphioxus,the dorsal lip of gastrula has been proposed to be an analogous structure to vertebrate organizer,but its development mechanism and function remain largely uninvestigated.Amphioxus is fertilized in vitro,the process of its embryo development is simple,and it has similar body axis with vertebrate,but its structure is simpler than that of vertebrate,so it is helpful for gene functional researches during the embryonic development.Lefty is the earliest gene expressed in primitive organizer region,and so the analysis of amphioxus lefty function in embryonic development can help to study the development mechanism and function of the organizer.In the present study,the spatial expression pattern of amphioxus Lefty gene at different development stages was systematically studied.The result showed that Lefty was not maternally expressed,and the expression of zygotic transcription was restricted to a subset of blastomeres at one side of the vegetal pole at 32-cell stage,as well as 64-cell and 128-cell stages.During the gastrula stage,Lefty was expressed in the presumptive dorsal cells.At the early neural stage,Lefty was mainly expressed in the dorsal ectoderm and mesodermal tissues,and not expressed in the posterior of the embryo.At the early-mid neural stage,Lefty was detected in the left dorsal part with asymmetric distribution,and it was expressed in the anterior and middle dorsal neural plate,somatic mesoderm and pharyngeal endoderm.At the mid-neural,the dorsal expression of Lefty was weakened,and was mainly limited in the left pharyngeal region and the tailbud.By the late neural stage,the gene dorsal expression was completely disappeared,and was mainly detected in the endostyle,branchial anlage and posterior tailbud,however the expression of Lefty in the anterior structure was completely disappeared at larva stage.To further investigate the role of Lefty in amphioxus,we overexpressed or knocked out Lefty gene in the embryos.First,we overexpressed Lefty by injecting Lefty mRNA into unfertilized eggs,and the phenotype of injected embryos and controls were indistinguishable before late-gastrula,but with the development of embryos,the injected embryos failed to form flatting dorsal lip during late-gastrula stage,and subsequently,lacked anterior organs and dorsal nerve cord,notochord and somites.The larval could not form mouth,gill slits and pharynx,and then died because of serious deformity.To more understanding of this phenotype,we examined the changes of early genes expression which were specific for embryonic body axis and germ layers.The results indicted that the expression of organize genes Nodal and Vg1,dorsal related genes Gsc and Chordin,anterior marker genes FoxQ2 and Cer were down-regulated or disappeared during gastrulae stage,while the ventral marker EvxA and posterior marker Wnt8 were upregulated and radialized,the neuronectoderm marker Sox1/2/3 and somatic mesoderm marker Six1/2 were both absent,while the epidermal ectoderm marker AP2 was expanded.Furthermore,we knocked out Lefty gene by TALEN,Lefty mutation resulted in opposite phenotype of Lefty overexpression,during neural stage,Lefty mutation resulted in an enlarged anterior and dorsal structures,and the disappearce of posterior gut,during larval stage,mutants formed mouth and expanded preoral pit both on the left and right sides,but the anterior rostral coelom was not formed,and the endostyle and the club shaped gland protruded ventrally.Several abnormalities lead to gradual death of mutants,and the mutant could only survive for a week.In gastrulae,the domain of two organizer genes(Nodal and Vgl)were expanded ventrally,as well as two dorsal related genes(Gsc and Chordin),and the anterior marker(Cer)was expanded,but the ventral marker(EvxA)and posterior marker(Wnt8)were severely reduced,the expression of neuronectoderm marker Sox1/2/3 and somatic mesoderm marker Six1/2 were both expanded,while the epidermal ectoderm marker AP2 was eliminated.In addition,in order to detect if the function of Lefty was realized by antagonizing Nodal signaling mediated by Vgl protein,we tested whether Vgl could overcome the effect of overexpressing Lefty.Compared with injecting Lefty mRNA,co-injection of Vgl mRNA and Lefty mRNA in amphioxus showed recoverd dorsal and anterior structures,and the expression of organizer gene(Nodal)and dorsal gene(Gsc)were normal in some gastrulaes,and the Brachuyury was normally expressed in the notochord during mid-neural satge.These results indicated that the effect of Lefty overexpression could be overcome by Vgl.And all these suggested an essential role for Lefty in the organizer specification and axial patterning of amphioxus embryos by limiting Nodal signaling activity.Furthermore,to examined the zygotic expression of amphioxus Lefty was induced by Nodal signaling pathway,we tested the expression of Lefty after blocking or over activating Nodal signaling.The result showed that the expression of Lefty was failed to express after blocking Nodal signaling,in contrast,the expression of Lefty was up-regulated and expanded by over activating Nodal signaling.Taken together,amphioxus Lefty was induced by Nodal signaling,and Lefty regulted the organizer and axis development by feedback inhibition of Nodal signaling.