The Role Of Djpi3k In The Regulation Of Planarian Regeneration And Cilia Maintenance

Background:Animal regeneration has a complex regulatory mechanism,which requires precise integration of processes such as cell proliferation,differentiation and tissue reorganization.The remarkable capacity for epimorphic regeneration of planarian is derived from its plenty adult stem cells(called neoblasts),and therefore it also known as the "living culture dishes".Planarian has become an ideal model to stem cell and regenerative medicine research.As a bridge molecule,phosphatidylinositol 3-kinase(PI3K)interacts with extracellular signals and cell responses.It plays an important role in cell proliferation,apoptosis,differentiation and migration.In addition,PI3K is a proto-oncogene whose abnormal expression is closely related to tumorigenesis.Thus,it has been used as a target for tumor therapy.However,the study of PI3K gene in planarian regeneration and tissue maintenance has not been reported yet.Methods and Results:In this study,we compared the amino acid sequences of Schmidtea mediterranea,Schistosoma mansoni,Caenorhabditis elegans and other species.According to their conservative region,we designed the specific primers and then obtained the core sequence of Dugesia japonica PI3K gene,about 777 bp.Firstly named it Djpi3k.Then we amputated the planarian in front of their pharynx.The samples of planarian head or tail regeneration were collected at 0 hpa,2 hpa,4 hpa,8 hpa,12 hpa,1 dpa,3 dpa,5 dpa and 7 dpa,respectively.Analyses of Djpi3k mRNA expression level and distribution were performed by real-time quantitative PCR(RT-PCR)and whole-mount in situ hybridization(WISH).Real-time quantitative PCR results revealed that the mRNA expression level of Djpi3k peaked at 1 day post amputation(dpa)and finally returned back to the initial level at 7 dpa.The results of WISH indicated that Djpi3k mainly distributes in the blastema during regeneration,and the trend of Djpi3k transcription level was consistent with the results of real-time quantitative PCR.Next,we used the PI3K specific inhibitor LY294002 to investigate to the effect of Djpi3k on the planarian regeneration.Finally,we compared the inhibitor group with normal regeneration group by H3P staining and TUNEL detection,The results demonstrated that Djpi3k plays the vital part in the cell proliferation and apoptosis in planarian.Interestingly,we discovered that LY294002 led to planarian movement disorder owing to the disruption of cilia maintance,which was confrimed by immunofluorescence staining with the ciliated specific antibodies.In conclusion,this study confirmed that inhibition of Djpi3k activity affects planarian tissue maintenance,also damages its movement.Conclusions:Our study demonstrated that Djpi3k plays a vital role in the early regeneration and tissue maintenance of planarian.Inhibition of Djpi3k activities decreases the level of cell proliferation and apoptosis,which finally could result in its tissue regeneration defect.Our results lay the foundation for the further study of the role of PI3K gene in tissue regeneration.