The Functions Of Hedgehog Signal In Amphioxus Left-Right Asymmetry Development

Correct patterning of left-right(L-R)asymmetry is essential during the embryonic development of bilaterians.Over the past decade,the molecular mechanisms underlying the establishment of such L-R asymmetry have been revealed in many vertebrate or invertebrate model organisms.Previous studies have shown that the Cer-Nodal-Lefty-Pitx cascade in amphioxus and vertebrate controls the L-R asymmetry development.In addition to aforementioned cascade,Hedgehog(Hh)signal also plays roles in L-R asymmetry development.In chick,mouse and sea urchin Hh signal participad in L-R asymmetry development by controlling Nodal expression.In amphioxus previous study showed that the onset of Hh asymmetric expression occurs long after Nodal expression,therefore if Hh signal involved in amphioxus L-R asymmetry development is still a matter of debate.In our recent study,we found that Hh knockout in amhioxus disturbed the normal placement of preoral pit and gill slits along the L-R axis,showing that Hh is important for amphioxus L-R asymmetry development,but the molecular mechinisam underlining this prosess is unkown yet.In this paper,we carefully examined the phenotypes of Hh-/-mutant amphioxus using situ hybridization,tissue section and scanning electron microscopy methods.The results showed that Hh-/-mutant duplicated left-side structures and lost right-side characters,displaying an abnormal bilaterally symmetric body plan.Corresponding to the symmetric phenotype,the organ-specific genes expression became symmegric after Hh knockout,showing that Hh is critical for amphioxus L-R asymmetric development.In order to detect the mechanism of Hh signal in L-R development,we exemined the L-R regulatory genes,Cer,Nodal,Lefty and Pitx,the results showed that Hh-/-embryos abolished Cer transcription with bilaterally symmetric expression of Nodal,Lefty and Pitx.These LR defects in morphology and gene expression could be rescued byHh mRNA injection.Based on these results,we conclude that Hh participate in L-R asymmetry development by controlling Cer expression.To further detect the Hh regulating mechanism for Cer expression and if Hh also contribute to breaking the initial symmetry,we identified a canonical Gli-binding site in Cer promoter sequence,but the luciferase assay showed that the Gli-binding site mutation had no effect on reporter gene expression,indicating that this canonical Gli binding sequence is not required for Cer expression.Next,we compared the expression patterns of Cer,Hh and Ptch in carefully staged embryos.The results showed that Hh and Ptch exhibite in an L=R manner at the stage when Cer expression exhibite in an L<R manner,showing that Hh may not regulated Cer asymmetric expression.In additon,we also showed that Hh singal upregulation or ectopic activation of Hh did not change the expression pattern of Cer.All these results showed that Hh is necessary for Cer expression,but it might not involve in Cer symmetry breakage.To research the relationship of Nodal signal and Cer expression,we examined Cer expressions after up-regulating/down-regulating Nodal signal activity.The results indicated that up-regulation of Nodal cativity led to the increase of Cer expression and down-regulattion of Nodal activity decrease the Cer expression,showing that Nodal signal could regulate Cer expression.Interestingly,blocking Nodal signal at mid-gastrula stage resulted in the bilateral expression of Cer,showing that Nodal signal was aslo involved in Cer asymmetric expression.In this study,we also found no mouth formation in Hh-/-and Smo-/-mutants,indicating that Hh signal also participate in amphioxus mouth formation.To detect the function of Hh in amphioxus mouth formation,we observed the mouth development after Hh mRNA injection and Ptch knockout.The results showed that up-regulation of Hh signal enlarged the mouth opening,which is compatible with previous finding that Nodal-Pitx regulates amphioxus mouth formation.To figure out the relationship of Nodal and Hh signal in amphioxus mouth formation,we examined the expressions of mouth specific genes Pou4 and Pax2/5/8.The results showed that Hh knockout did not affect oral primordium formation but affect mouth opening.All these results shows that Hh and Nodal work together to regulate amphioxus mouth formation,and Nodal signal regulates the mouth primordium formation while Hh signal regulates the mouth opening prosess.