Transcription Factor FoxP Controls Tetrapyrrole-based Body Pigmentation In Planarian

Aim:Pigmentation processes occur within almost every cell from invertebrates to mammals.Due to the complexity of the pigmentary system,in vivo animal models for pigmentation study are limited.Planarian is an emerging model organism for regeneration and adult stem cell study in vivo.However,the body pigment and pigmentation of planarian is not clear.Hence,we planned to study the pigmentation of planarian in vivo to reveal the mechanisms of pigment biosynthesis and maintainance as well as provide new prospects and models for pigmentation study.Method:Owing to the convenience of RNA interfere(RNAi)in planarian,this project started from a screening of transcription factors to find target genes that regulate planarian body color pigment biosynthesis and maintainance.Through long time observation and transmission electron microscopy,we further confirmed the phenotype.Together with spatio-temporal expression pattern examination,we further validated the function of objective genes.We also designed planarian specific micro-array to detect down-stream targets.Combining RNAi screen and in situ hybridization,we further revealed the signaling pathway that regulate planarian body pigmentation at cell biology and molecular biology levels.Results:We first selected 166 candidate transcription factors by bioinformatics analysis.RNAi screening revealed 3 genes required for planarian body color pigment biosynthesis and maintainance and only one of them,FoxP,is specifically required for pigmentation without affecting overall survival or regeneration.We focused on FoxP and found that FoxP mRNA expression displayed a close relationship with pigmentation process during planarian regeneration and homeostasis.Through customized micro-array and literature mining,FoxP target gene,PBGD,ALAD,ALAS and Kmo2 were identified.We further analyzed the function and expression of the downstream targets and our results demonstrated that PBGD RNAi phenocopy the phenotype resulted from FoxP RNAi and PBGD mRNA specifically expressed in planarian body pigment cell under the epidermis.We also examined the function and expression of kmo2,the key enzyme in ommochrome pathway.In the end we explored the expression of FoxP and PBGD during the differentiation from planarian adult stem cells to pigment cells.Conclusion:We identified a forkhead domain containing transcription factor,FoxP,that specifically required for the planarian body color pigment biosynthesis and maintainance through regulating tetrapyrrole biogenesis.Our results also showed that FoxP controls ommochrome pathway simultaneously.The downstream target PBGD,a key enzyme in tetrapyrrole biosynthesis is required for pigmentation and specifically labels planarian pigment cell severing as a perfect planarian pigment cell marker.Our results revealed the underlying mechanism of planarian body color pigmentation and set up a promising in vivo system for pigmentation study.At the mean time,our results shed lights on the study of porphyria pathogenesis.