Mechanism Investigation Of Wdr5 Coordinated Regulation On Digestive Organogenesis In Zebrafish

Liver,pancreas and intestine are the three main digestive organs in vertebrates,all of which originate from the endoderm.Different digestive organs require different signals and transcription factors to regulate their differentiation.However,the digestion and absorption of food requires the synergistic action of three organs.Is there a mechanism that regulates the maturation and differentiation of these three organs during organogenesis?In 2019,our laboratory found that WDR5-mediated H3K4me3 modification was involved in genetic compensation response.Unexpectedly,we found that the expression of liver-specific differentiation gene:fabp10a could not be detected.in zebrafish wdr5^-/-mutant Because Wdr5^+/-mouse embryos die before early organogenesis,little is known about the regulatory role of wdr5 in organogenesis.The maternal effect of wdr5 gene in zebrafish may be responsible for the relatively normal development of wdr5^-/-homozygous mutants at day 2,compared to the early embryo death caused by half dose of Wdr5 knockout mice.This provides a good platform for us to use this mutant to study the role of Wdr5-mediated histone modifications in organ development.This paper will take advantage of the zebrafish wdr5^-/-mutant to study the role of Wdr5 in the development of digestive organs.Wdr5,a highly conserved WD40-containing domain repeat protein,has two main functions:catalyzed histone H3K4 di/trimethylation as a core member of COMPASS complex;involved in NSL complex mediated H4K16 acetylation.Previous experiments showed that in zebrafish wdr5^-/-mutant had relatively normal specialization and development in liver,pancreas and intestine at the early stage,but could not undergo mature differentiation.In the wdr5^-/-mutant,the cells of the digestive organs are at the progenitor like stage where they want to differentiate but cannot.Western blot and immunofluorescence results showed that H3K4me3 modification decreased significantly in wdr5^-/-mutants,but H4K16ac modification did not change significantly.Further analysis of H3K4me3-ChIP-seq showed that some of the digestive differentiation genes with reduced expression in wdr5^-/-mutants accompanied by reduced H3K4me3 modification in their transcription start sites（TSS）,while H3K4me3modification in the TSS region of digestive organs development related regulator was not reduced.Transgenic zebrafish experiments showed that only wild-type Wdr5 could rescue the phenotype of wdr5^-/-mutant,while the Wdr5 without H3K4me3 modification function could not rescue the phenotype of wdr5^-/-mutant.These results indicate that H3K4me3 modification mediated by Wdr5 regulates the development of digestive organs.During wild-type zebrafish embryos development,the proliferation of digestive organs gradually disappeared with cell differentiation.However,the proliferation of wdr5^-/-mutant’s three digestive organs,liver,pancreas and intestine,maintained a high level.To explore why the cell cycle could not be terminated in the wdr5^-/-mutant,we found that the level ofβ-Catenin protein was significantly increased in the wdr5^-/-mutant,and that Wnt/β-Catenin inhibitors can reduce the accumulation ofβ-Catenin protein and the level of proliferation in the wdr5^-/-mutant’s digestive organs.Further studies showed that the expression of apc gene,an important component ofβ-Catenin degradation complex,was down-regulated in wdr5^-/-mutants accompanied by decreased H3K4me3 modification in the TSS region.Increasedβ-Catenin protein levels and proliferation of digestive organs were also observed in apc^+/-heterozygous.Our results suggest that Wdr5-mediated H3K4me3 modification inhibits the Wnt/β-Catenin signaling pathway by increasing apc expression,thereby terminating the proliferation of differentiated cells.We found that the p53 signaling pathway was activated in the digestive organs of the wdr5^-/-mutant,a large amount of apoptosis caused by P53 protein accumulation was observed.This is due to the down-regulated expression of some anti-apoptotic genes（e.g.,XIAP-like gene）in wdr5^-/-mutants accompanied by reduced H3K4me3 modification in the TSS region.P53 protein accumulation was increased in XIAP-like^-/-mutants,and apoptosis was increased in digestive organs.It was proved that Wdr5-mediated H3K4me3 modification ensured the survival of differentiated cells by promoting the expression of anti-apoptotic gene and inhibiting p53 signaling pathway.To sum up,this paper draws the following conclusions:1)Wdr5 synergistically regulates the differentiation and development of digestive organs by mediating H3K4me3 modification;2)Wdr5-mediated H3K4me3 modification regulates the differentiation and development of digestive organs by promoting the expression of differentiation genes in digestive organs;3)Wdr5-mediated H3K4me3 modification inhibits the Wnt/β-Catenin signaling pathway by increasing apc expression,thus terminating the proliferation of differentiated cells;4)Wdr5-mediated H3K4me3 modification inhibits the p53 signaling pathway by increasing the expression of anti-apoptotic genes（e.g.XIAP-like）,thereby allowing the survival of differentiated cells.This study not only revealed a new mechanism of synergistic regulation of digestive organ differentiation and development,but also provided new implications for the study of pathogenesis of human digestive organ related diseases.