Spatiotemporal Expression Of Pax Genes Throughout Amphioxus Life Cycle And Evolutionary Analysis Of Alternative Splicing And Duplication Of VapA/B Gene

Amphioxus,one of the closest living invertebrate relatives to the vertebrate,plays an important role in understanding the evolution of chordate body plan and organogenesis. To date,previous researches usually focused only on the embryo and larva stages of amphioxus,whereas the gene expression pattems at later developmental stages have rarely been reported.With our recent progress in continuous culture of amphioxus in the laboratory,we investigated the temporal expression pattems(16 developmental stages) and the spatial expression pattems(four areas at metamorphosis and eight adult tissues) of four amphioxus Pax genes throughout the life cycle of amphioxus(Branchiostoma belcheri) including embryogenesis,metamorphosis and gonad development.We found AmphiPax genes were expressed throughout amphioxus life cycle and presented relatively high expression patterns during metamorphosis.And their dynamic expression patterns are related to the definite developmental processes or organogenesis. Furthermore,comparing orthologous gene expression pattems between cephalochordate and vertebraters,we found AmphiPax genes respectively presented the ancestral expression patterns of four vertebrate Pax subfamilies and displayed both sequential and expression pattem homologues:Pax1/9 subfamily displayed conserved role in pharynx, mesenchyme cell and intestine.Pax2/5/8 subfamily showed its homologous expression in pharynx(endostyle),tail and testis.And Pax3/7 subfamily played roles in muscle and intestine in amphioxus,while Pax6 subfamily was expressed similarly in liver and intestine.Theoretically,gene duplication and alternative splicing are distinct evolutionary mechanisms that provide the raw material for new biological functions.As one of the closest living invertebrate to vertebrate,amphioxus has a simple genome without undergoing genome duplications,so it provides a good model to investigate the evolutionary mechanisms of gene duplication and alterative splicing.In the present study, we cloned the Vap genes(AmphiVapA/B) from amphioxus(B.belcheri),and found this gene had four alterative splicing(AS) types.By analysis of the amphioxus,human,fugu and zebrafish genomes,we found VapA/B genes had undergone gene duplication with the linked genes including Spir1/2,RBM12,RAB22A/B and servral other genes.On the other hand,both of human VAPA and VAPB are contained alterative splicing types which are similar to those of AmphiVapA/B.We therefore described an evolution history of gene duplication and alterative splicing models of VapA/B through amphioxus,human,fugu and zebrafish.Furthermore,we investigated the spatiotemporal expression patterns of four AS types of AmphiVapA/B,and found they were divergent in gene expression spatially and temporally.It is probably that they play different roles in specific developmental stages and tissues.We also predicted the coiled-coil domain and a transmembrane domain four AS types of AmphiVapA/B,and found the AS2 had the coiled-coil domain.Based on the above observations,we discussed the fates of AS models and assumed possible evolutionary mechanisms of gene duplication and alterative splicing in VapA/B gene family:a.To fulfill the function of Vap-A/B,amphioxus gains four AS types,human get four types through both gene duplication and AS,while fugu and zebrafish have three copys through gene duplication,b.The more important alterative splicing(AS2) having coiled-coil domain became exon in duplicated VapA/B genes,whereas the less important one(AS1) is still as alterative splicing or even lost totally,c.The AS type with higher average expression level (more important in function) became a duplicated gene,while the AS type with lower average expression level(less important in function) remained as AS type or lost in evolution.