Vg1 Gene In Amphioxus Body Axis Establishment

Nodal signaling plays an important role in the establishment of body axes during vertebrate embryonic development.As ligands of Nodal signaling,Vgl,Nodal and Activin,members of TGF-? superfamily,participate in the body axis establishment of vertebrates.In amphixous,Nodal signaling is also involved in body axis establishment,but it has not been determined which ligand is involved in this process.In this study,we focused on Vgl gene,and studied its function from the following aspects.1.We first analyzed the spatiotemporal expression pattern of Vg1 gene in early stages of amphioxus embryos,and found that Vgl gene was not expressed in unfertilized eggs and fertilized eggs.At 32-cell stage,zygotic Vgl expression was first detected in some vegetal cells,like Nodal signaling antagonist Lefty.As devekopment proceeds,Vgl transcripts was still concentrated in some of the vegetal cells.At the late blastrula stage,Vgl gene was mainly expressed in the region which would develop into the dorsal blastoporal lip in the future.At the late gastrula stage,Vgl gene was expressed in the dorsal mesendoderm.2.In order to study the function of Vgl gene in the development of amphioxus embryos,we generated amphioxus Vg1 homozygous mutants using TALEN method.We found that Vgl mutation led to severe deformity with the embryo showing no anterior and dorsal structures,and enlarged posterior end.The ventral structures of the mutant were however overdeveloped.Consistent with these,expression of the anteroir and dorsal marker genes was disappeared and the ventral marker gene was extended dorsally in the mutant.These results indicated that Vgl gene is required for the establishment of dorsal-ventral axis in amphioxus.3.In order to determine whether zygotic activationof Vgl expression depends on Nodal signaling,were spectively treated the embryos with Nodal signaling activator and inhibitor.It was found while over-activation of Nodal signaling at early blastrula stage up-regulated Vgl gene expression,inhibition of Nodal signaling abolished Vgl expression.This result indicates Nodal signaling is necessary and sufficient for initial zygotic expression of Vg1 gene at the early cleavage stages.4.Among embryos from cross of Vg1 heterozygotes,we noticed that some of them showed two-right phenotype which were late proved all heterozygotes.In these embryos,markers of right organs became bilaterally symmetrically expressed while Nodal,Pitx and those of left organs was disappeared.These results indicated that Vg1 gene had a dosage-dependant effect on amphioxus left-right axis establishment.To address this further we injected amphioxus embryos with Hsp70::Vg1 plasmids and heat-shocked them at mid-gastrula stage to overexpress VgI After the treatment,some of the embryos showed two-left phenotype.In these embryos,expression of left-side genes Nodal and Pitx,and that of marker genes of left organs were symmetrically expressed,while expression of markers of right ogans was lost.These results demonstrate that Vg1 is essential for the development of left identity in amphioxus.5.We finally compared the expression pattern of Vg1 and those of other genes related to left-right asymmetry,and found asymmetric expression of Vg1 is turned on later than that of Cer,but earlier than those of Nodal and Pitx,This result,together those shown above,suggested Vg1 acts downstream of Cer but upstream of Nodal and Pitx during amphioxus left-right asymmetry development.