| Vertebrates possess the most complex central nervous system than other animals by having billions of cells with hundreds of types in the brain and spinal cord.Illustrating how vertebrate central nerves system has evolved is one of the most important issues in the field of developmental biology,and the issue remains elusive.Sonic hedgehog(Shh)plays essential role in dorsoventral(DV)patterning of vertebrate neural tube.Concentration gradient of diffused morphogen Shh secrete from notochord and floor plate further drives a gene regulatory network of cross-repressive transcription factors,including Nkx6.1,Nkx2.2,Olig2,Pax6,Irx3,Pax3 and Pax7,leading to the differentiation of proliferative neural progenitors within the respective regulatory areas of the neural tube.As a basal chordate lineage,amphioxus(Cephalochordata)and its dorsal neural tube structure,with the similar expression position of genes(Hh,Nkx6,OligA,Pax4/6,Irx,and Pax3/7)between amphioxus and vertebrates neural tube,these features together make amphioxus an ideal model for studying how evolution has resulted in such a robust yet adaptable patterning system in vertebrates neural tube.In this study,Hh(ligand)and Ptch(receptor)mutant embryos were used to investigate the function of Hh signal in the formation of DV patterning of amphioxus neural tube.Further more,the differentiation mechanism of motor neurons(induced by Hh signal)in the ventral neural tube of amphioxus was further analyzed.The results are list as follows:1.In Hh-/-embryos,Hh signaling pathway was inhibited,and the expression of ventral neural tube marker gene Netrin,Nkx6 and OligA were reduced in Hh-/embryos,while the intermediate CNS marker gene Pax4/6,IrxA,IrxB and IrxC along with the ventral CNS marker gene Nkx2.2 were all expressed normally in Hh-/embryos,and Pax3/7 was overexpressed in Hh-/-embryos dorsal cerebral vesicle.In Ptch-/-embryos,Hh signaling pathway was over activated,the expression changes of Netrin,OligA and Pax3/7 gene in Ptch-/-embryos were opposite to those in Hh-/embryos,and the other marker genes were all expressed normally in Ptch-/-embryos.These results indicated that Hh only regulates some ventral neural tube marker genes,but not intermediate or dorsal neural tube marker genes(except for Pax3/7 in dorsal cerebral vesicle).2.In vertebrates,motor neurons are located in the ventral region of neural tube,and regulated by Nkx6.1,Olig2,Mnr2,Lim3 and Isletl genes.The reduced expression of Nkx6 and OligA in amphioxus Hh-/-embryos suggested that Hh signal could be involved in the induction of amphioxus motor neurons development.Consistent with this hypothesis,the expression of Mnxa,Lhx3 and Islet(Mnr2,Lim3 and Islet1 homologous genes)were all reduced in amphioxus Hh-/-embryos neural tube.In situ hybridization results indicated that Hh signaling activity initially affected the expression of Nkx6 and OligA,followed by Mnxa,Lhx3 and Islet.Besides,OligA,Nkx6 and Mnxa are coexpressed in amphioxus neural tube from the position of somiteⅠ to somite Ⅳ,and the expression of Lhx3 and Islet were located in the same position.These results suggested that Nkx6,OligA,Mnxa,Lhx3 and Islet are playing important roles in the differentiation of amphioxus MNs which under the induction of Hh and located in the ventral region of neural tube.3.There are three types of motor neurons in amphioxus:Dorsal compartment motor neurons(DC MN),ventral compartment motor neurons(VC MN),and visceral motor neurons(vMN).Previous studies believed that Err was specifically expressed in DC MN,while Vacht(acetylcholine transporter encoding gene)was expressed in the all three motor neurons.In amphioxus Hh-/-embryos,the numbers of Vacht and Err positive cells were both decreased.While in Ptch-/-embryos,only the number of Err positive cells increased but not Vacht positive cells.We used double ISH methods and found that the expression of Vacht and Err were not colocalized,and the expression of Vacht was not affected in Err-/-embryos.These results indicated that:1)Hh signal participates in the regulation of some MNs formation;2)Err positive cells are not motor neurons,and therefore are not DC MN either;3)but the differentiation of some Err positive cells are also regulated by Hh signal.4.In order to clarify the regulatory relationship of Nkx6,OligA,Mnxa,Lhx3 and Islet in the differentiation of MNs in amphioxus,we performed knockout and overexpression experiment of Nkx6,OligA and Mnxa genes.From my reseach results,Nkx6 expression disappeared in Nkx6-/-embryo,and there were no significant expression changes of OligA,Mnxa,Lhx3 and Islet gene in Nkx6-/-embryo.In OligA-/embryo,there were no significant expression changes of OligA or other above genes.In Mnxa-/-embryo,the expression of Mnxa,Lhx3 and Islet gene reduced,while the expression of Nkx6 and OligA gene were not affected.We also found the number of Err positive cells increased in Mnxa-/-embryos.Overexpression of Nkx6 and OligA had no significant effect on Mnxa gene expression,and Mnxa overexpression had no significant effect on Lhx3 gene expression,but overexpresion of Mnxa result in a reduced number of Err positive cells.These results suggest that:1)in the regulation network of amphioxus MNs differentiation,Mnxa gene may be located downstream of Nkx6 and OligA,and upstream of Lhx3 and Islet;2)there could be compensatory pathways in Nkx6 and OligA regulatory levels,which was consistent with the results that multiple Olig genes in amphioxus with a similar expression pattern;3)Mnxa can inhibit the formation of Err positive cells.Although the expression position of Hh gene in amphioxus and vertebrate embryos is similar(both expressed in the notochord and ventral region of neural tube),there are significant differences between these two groups of species in the function of Hh signal during the formation of neural tube:in amphioxus embryo,Hh is only been recruited in the regulation of some MNs and Err positive neurons which located in the ventral part of the neural tube,while in vertebrate embryo,the function of Shh is evolved to regulate the formation of dorsoventral patterning of the entire neural tube.In addition,transcription factors in amphioxus embryos lacks significant cross repression gene regulatory network(except Mnxa has an inhibitory effect on Err).We speculate these differences may be the reason for the high complexity of vertebrate central nervous system. |
