The Function Of Non-classical Wnt Signaling Pathway In Naural Regeneration In Planarian

Wnt signaling pathway can be divided into the canonical Wnt signaling pathway and the non-classical Wnt signaling pathway according to the presence or absence of beta-catenin.Wnt5 a is a typical protein in non-classical Wnt signaling pathway playing an important role in the development of various organs.In Xenopus,over expression of Wnt5 a could lead to complex malformations,including incomplete atresia of the anterior neural tube and shortening of the anterior and posterior axes.During zebrafish gastrulation,Wnt5 a regulated the cell morphology and concentration extension,regulating cell elongation in the late gastrulation.In mice,Wnt5 a regulates fission fusion in the mitochondria in hippocampal neurons.As a typical representation of platyhelminthes,planaria,which have many transitional characteristics,is an important species for the study of the biological evolutionary and development history.The cDNA DjWnt5 a,encoding a planarian Wnt5 a was cloned by RT-PCR(reverse transcription PCR)and RACE from Dugesia japoncia.The results showed that the DjWnt5 a was comprised of 1523 bp and contained an open-reading frame corresponding to a deduced protein of 371 amino acids.Phylogenetic analysis using MAGE5.2 revealed that DjWnt5 a and Schmidtea mediterranea Wnt5 a joined together and positioned at the base of bilatera Wnt5 a,suggesting that both planarian DjWnt5 a might share some characteristics of the archetype of the bilatera Wnt5 a proteins.Expression analysis of DjWnt5 a gene indicated that the DjWnt5 a mRNA was first found in stage 3 of the embryonic development,expressing in the embryonic pharynx and germ band.Along with the embryonic development,DjWnt5a-positive embryonic cells were increased and were distributed in the neural primordiums and the central nervous system of the fully hatched larvae.These results suggested that DjWnt5 a is one of the essential genes in the embryonic nervous development,plays a vital role in the formation and differentiation of the neural primordium during the planarians embryonic development.Whole mount in situ hybridyzation of DjWnt5 a were performed in the intact planarian and nerve regeneration.The results showed that DjWnt5 a gene expressed in the brain,ventral nerve cord and pharynx.The signals were detected in the brain nerve cord and pharynx during the regenerating planarians.Loss of function of DjWnt5 a by RNAi,the DjWnt5 a expression level was decreased obviously,which suggested DjWnt5 a have been successfully inhibited.Fluorescent immunohistochemistry with an anti-SYNORF1 antibody against synapsin during regeneration revealed that DjWnt5a-RNAi-treated planarian nervous system and the brain could not be with the corresponding to the normal planarian regeneration in time.We use microinjection RNAi technology to interfer planarians,total RNA of the normal and interference planarians was extracted.The relative expression level of non-canconic Wnt pathway including DjJnk?DjRac?DjRhoA?DjROCK were detected by qPCR after RNAi and normal planarians.The results showed these genes expression was decrassed,indicating that the relationship between DjWnt5 a and DjJnk?DjRac?DjRhoA?DjROCK genes excits in planarian.We had successfully constructed the prokaryotic expression vector of PET28a-DjWnt5 a.DjWnt5a protein expression can give the way for studying the function of DjWnt5 a at the protein level in the planarian homeostasis and regeneration process.