Role Of Hdac9b During Glomerular Development

Objective:To explore the role of hdac9 b during glomerular development in zebrafish and the underlying regulating molecular mechanism.Methodolgy:(1)The pronephric glomeruli were isolated from transgenic reporter zebrafish Tg(pod:e GFP)at 3 dpf and subjected to RNA-sequencing,followed by results analysis;(2)The expression pattern of hdac9 b was determined by whole-mount in situ hybridization(WISH)in different developing stages;(3)CRISPR/Cas9 genome editing approach was utilized to generate hdac9 b gene knockout zebrafish lines,deformity ratio was calculated,the integrity of glomerular filtration barrier(GFB)was estimated through coomassie blue staining,the glomerular ultrastructure was examined by transmission electron microscopy(TEM)imaging,the expression level molecular markers of mature podocytes(podocin and nephrin)and renal progenitor cells(lhx1a/wt1b/mafba/pax2a)were detected by WISH;(4)hdac9b Crispants were generated based on a rapid method for directed gene knockout in F0 zebrafish using CRISPR/Cas9 system,to phenocopy hdac9b-deficient embryos,followed by capped hdac9 b mRNA injection into hdac9 b Crispants at one-cell stage to rescue the developmental abnormalities;(5)The expression level of ?-catenin and its downstream target genes in hdac9 b Crispants embryos were detected by WB and rt PCR,and resuced by capped ?-catenin and NLS-?-catenin mRNA injection,respectively,into hdac9 b Crispants at one-cell stage,the regulating effect on ?-catenin by hdac9 b was determined by WB.Results:(1)Through glomeruli RNA-sequncing,all the members of HDAC family were expressed in pronephric glomeruli of zebrafish except hdac2,among HDACs,hdac1 and hdac9 b were the most highly enriched;(2)hdac9b was continuously expressed in the glomerulus of zebrafish starting from 24 hpf and disappeared at 84 hpf using WISH;(3)Compared with wild type embryos,loss of hdac9 b lead to embryonic development defects and deformity,impaired GFB,leakage of macromolecular protein,podocyte foot process effacement,and decreased number of mature podocytes and renal progenitor cells;(4)hdac9b Crispant perfectly phenocopied hdac9b-deficient embryos and could be resuce by capped hdac9 b mRNA injection at one-cell stage;(5)Compared with wild type,the expression of ?-catenin and its downstream target genes myca and cyc D1 in the hdac9 b Crispants embryos were significantly suppressed,loss of hdac9 b resulted in cytosolic accumulation of ?-catenin and its failure translocation to the nucleus,injection of capped NLS-?-catenin mRNA,but not capped ?-catenin mRNA,could resuce the phenotype of hdac9 b Crispants..Conclusion:hdac9b is required for the transition from renal progenitor cells into normal podocytes during glomerular development by promoting nuclear translocation of ?-catenin.