Identify Transcription Factors Required For Planarian Regeneration And Homeostasis By Genome Wide RNAi Screening

Aim: Planarian is an emerging model organism which process powerful regenerative ability.Transcription factor regulates the expressions of a single gene or a set of genes and plays vital roles in development.We applied genome-wide RNAi screening and identified critical transcription factors during regeneration and homeostasis of planarian.Meanwhile,we further explored the underlying mechanisms of the transcription factors that required for regeneration or homeostasis of planarian.Contents: We first focus on the general information of existing transcription factors in planarian and how many of them have effects on regeneration or homeostasis of planarian.We next categorized the transcription factors by phenotypes and analyzed the phenotypes preliminary.We also examined whether there was specific m RNA expression pattern with genes with similar phenotypes.We paid special attentions to genes with unique phenotype.We selected several genes and further studied the expression and the relationship between their functions and regeneration.Importantly,we performed m RNA array to further reveal the mechanisms that how these genes affects regeneration and homeostasis.Methods: We applied RNAi screening in order to study the function of each transcription factor in regeneration and homeostasis.Whole mount fluorescence in situ hybridization and immunofluorescence are the main techniques used to dissect the function of single gene.We also performed m RNA array to further study the detail molecular mechanisms.Results: We started from existing transcription factors in higher organisms and identified 686 transcription factors in planarian genome by protein and RNA sequence alignments.Since 88 of the 686 transcription factors have already been reported,we performed RNAi screen against the remaining 598 genes and revealed that 60 of them show various phenotypes upon RNAi.Among them,RNAi of 34 genes resulted in defects in both regeneration and homeostasis and 13 genes RNAi caused regeneration defects alone while another 13 genes RNAi caused homeostasis failure.We further analyzed the phenotypes and found that loss of three genes resulted in polarity formation failure;4 genes RNAi led to eye spot regeneration failure;3 genes control the speed of regeneration and loss of another 3 genes caused normal regeneration failure.Among the 13 genes that affects homeostasis alone,3 genes were critical for planarian pigmentation;another 3 genes were required for normal locomotion;and 7 genes lysed soon after RNAi.We analyzed the m RNA expression pattern in 10 time point post amputation and found that most genes that required for regeneration and homeostasis have an expression increase at 3 days post amputation.Among the genes with phenotypes,we identified a gene zbtb6,whose RNAi led to rapid lysis phenotype of planarian.Interestingly,zbtb6 does not express in planarian adult stem cell but showed an adjacent expression pattern to adult stem cells.Meanwhile,zbtb6 RNAi lead to a significant increase of proliferation.In the screen,we identified a gene,pogz that expresses within adult stem cells and is required for planarian regeneration.We further revealed that pogz is required for the regional proliferation peak.By m RNA array analysis,we found that dis3l2 is required for the pogz RNAi phenotype.Meanwhile,we also demonstrated that pogz is required for the stem cell recognition and stem cell differentiation upon tissue lost in planarian.m RNA array also revealed that pogz functions by regulating the expression of zfp-1 and inx-11 to promote adult stem cell differentiation.Conclusion:We totally screened 598 genes and identified 60 genes with various phenotypes.zbtb6 indirectly controls the proliferation of planarian adult stem cell.During regeneration,pogz promote the expression of dis3l2 and initiate the regeneration specific local stem cell proliferation.During homeostasis,pogz regulates the expression of zfp-1 and inx-11 to promote adult stem cell differentiation.