The Epigenetic Mechanisms Of Gonadal And Cardiac Plastic Regeneration In Zebrafish

Both stem cells and differentiated cells have potential to reversibly dedifferentiate or transdifferentiate into multi-lineages under certain conditions.This environment-guided cell plasticity is the basis for homeostasis,regeneration,and sex reversal.Epigenetic regulations such as DNA methylation,miRNA,histone modifications and chromatin remodeling,play an important role in cell plasticity.Zebrafish,a model animal with strong regenerative capacity,can completely regenerate the amputated tissues throughblastema formation and reformation during the regeneration of jaw,gonad,heart and caudal fin.In this study,zebrafish were used as examples to study the molecular mechanisms of miRNA and histone modification in gonad and cardiac plasticity regeneration.1.Regulatory mechanism of miRNA in zebrafish gonadal cell plasticityOur previous studies have screened many gender-related differentially expressed miRNAs and GPCR genes through miRNA-mRNA deep sequencing analyses of the gonads of five month-old zebrafishs.In this study,four specific miRNAs are selected by microinjection and quantitative PCR analysis: miR430 a,miR218a,miR734,miR141.To explore the interactions between specific miRNAs and Sox9 genes,we inject specific miRNAs into the sperm and ovary of zebrafish.As a result,injection of miR430 a can increase the expression of Sox9 a gene in testis while injection of miR218 a can increase the expression of Sox9 b in ovary.Over expression of Sox9 a and Sox9 b enhances the expression of testis miR430 a precursors and ovary miR218 a precursors,resepcvively.Moreoverknock down of Sox9 a and Sox9 b reduce the expression of miR430 a precursor in testis and the expression of miR218 a precursor in ovary,respectively.These results suggest that the miR430a-Sox9 a pathway biasly regulate the testis development,while the miR218a-Sox9 b pathway favors the development of the ovary.To testify this possibility,we inject a mixture of specific miRNA and Sox9 in the newly mature zebrafish gonads.It is found that the Sox9amiR430 a mixture can increase the ratio of chromosome-concentrated cells and undifferentiated spermatogonia;while the Sox9b-miR218 a mixture can induce follicular regeneration and reduce the proportion of chromosome-concentrated cells and sperm cells in the testis.When the gonads of two-year-old zebrafishs are injected,Sox9a-miR430 a can significantly increase the ratio of chromosomal-concentrated cells and undifferentiated spermatogonia,while Sox9b-miR218 a can induce follicular renewal.Meanwhile,we inject the miRNA-Sox9 mixture into the gonad of the vasaGFP transgenic medakas,and find that miR430a-Sox9 a can increase the fluorescent signal in the testis,while miR218a-Sox9 b can enhance the fluorescent signal in the ovary.These results confirm that miR430a-Sox9 a synergistically regulates the development of testis,while miR218a-Sox9 b promotes follicle renewal.On testing miR430 a and miR218 a targets of GPCRs: Gpr157,Grk5 l,Grk4,Lgr4,Pmir-GFP luciferase reporter assays show that miR430 a targets Lgr4,Grk5 l,Grk4,and miR218 a targets Gpr157,Grk5 l,Grk4.This suggests that the two miRNAs may regulate the activity of Sox9 a and Sox9 b via GPCR networks.Immunoprecipitation(IP)-mass spectrometry show that injection of miR430 a and miR218 a can affect the phosphorylation and acetylation of Sox9 a and Sox9 b.Several enzymes are identified to be involved in phosphorylation and acetylation of Sox9a/Sox9 b protein,including PI3 K,ROCK,PKC,etc.Immunoblot assays further validate the band size changes of Sox9 a and Sox9 b proteins before and after the mixed injection of miR430a/miR218 a.Furthermore,we find that injection of Sox9 a can increase the expression level of pPKC in testis,while co-injection of miR430a-Sox9 a mixture can simultaneously increase the activity of p-PKC and p-Akt.In the ovary,Sox9 b is able to increase the activity of p-PKC,but co-injection of miR218 a inhibits the activity of p-PKC and pAkt.Knockdown of lgr4,grk5 l and grk4 in the testis can significantly reduce the phosphorylation levels of p-PKC and p-Akt.However,knockdown of gpr157,grk5 l,and grk4 in the ovary promoted the expression of p-PKC and decrease the expression of P-Akt.At the same time,immunohistochemistry reaffirm that co-injection of Sox9 a and miR430 a could increase the expression of p-PKC and p-Akt in testis,while miR218 a and Sox9 b could inhibit the expression of p-PKC and p-Akt.2.Effect of histone modifications on cell plasticity during zebrafish heart regenerationOur previous experiments show that the cardiac regeneration process includes inflammatory hyperplasia,vascular remodeling and muscle regeneration,which are represented by ?-acta2,flk1 and pax3 a gene expression,respectively.In this experiment,high-throughput of chromosomal co-immunoprecipitation-sequencing analysis(ChIP-seq)combined with transcriptome sequencing analysis identify 57 genes with the high level of transcription and histone H3K9 ac modification during zebrafish heart regeneration.The genes related to cardiovascular production,extracellular matrix,cytoskeleton,and cell differentiation,such as Flk1,?-acta2,pax3 a and its neighboring genes,are significantly elevated.Their transcriptions are positively correlated with H3K9 Ac enrichment but negatively correlated with H3K9Methlation(H3K9Me3).Microinjection of H3K9 ac and H3K9me3 modification inhibitors reversely affect the blastema formation during zebrafish heart regeneration.In vitro inhibition of histone H3K9 ac H3K9me3 modifications in Pac2 cells also affects the cytoskeleton of fibroblasts,In conclusion,our study uncover that miR430a/mirR218 a can regulate the activity of p-PKC/p-Akt by regulating GPCR,thereby increasing the activity of Sox9 a and Sox9 b.Among them,miR430a-Sox9 a synergistically regulates the testis spermatogenesis,while miR218a-Sox9 b can promote follicle renewal.In addition,we find that histone H3K9 ac and H3K9me3 modifications reversely regulate the expression of ?-acta2,flk1 and pax3 a,and affect the blastema formation during zebrafish heart regeneration.Our study on regulatory netwroks via miRNAs and histone modifications can pave new ways to deeply understand the molecular mechanism of plasticity in zebrafish tissue regeneration,and provide new ideas for regenerative medicine.