组蛋白去乙酰化酶9对肾小球发育的调控

Histone deacetylase 9 regulates glomerular development in zebrafish

目的:研究组蛋白去乙酰化酶9(histone deacetylase,hdac9)缺失对肾小球发育的影响.方法:利用全胚胎原位杂交检测斑马鱼hdac9在受精后24h、36h、48h、60h、72h的表达分布;应用基因编辑技术CRISPR/Cas9构建hdac9基因敲除模型;透射电镜观察hdac9基因敲除对肾脏超微结构的影响;利用全胚胎原位杂交检测hdac9敲除后对肾脏祖细胞和足细胞标志物表达的影响;在hdac9基因敲除斑马鱼中,回输hdac9 mRNA以恢复hdac9缺失的表型.结果:全胚胎原位杂交检测发现hdac9在斑马鱼前肾发育阶段高峰度表达,即受精后24h开始表达,直至肾小球发育完成hdac9表达消失;透射电镜观察发现hdac9基因敲除导致肾小球发育障碍,肾脏祖细胞标志物(lhx1a,pax2a,mafba和wt1b)和足细胞标志物(podocin和nephrin)表达均显著抑制;在hdac9基因敲除斑马鱼中回输hdac9 mRNA可明显恢复hdac9基因敲除的表型.结论:hdac9在肾小球发育阶段表达,为肾小球发育所必需的,可能具有调控肾脏祖细胞增殖以及祖细胞向足细胞分化的功能.

Objective:To investigate the loss of histone deacetylase(hdac)9 function during glomerular development. Methodology:Whole mount in situ hybridization(WISH) was utilized to examine the expression pattern of hdac9 in zebrafish larvae at 24,36,48 and 72 hours post-fertilization(hpf) stages;hdac9 mutant zebrafish was generated through CRISPR/Cas9 editing method,ultrastructural changes were observed with transmission electron microscopy(TEM),expression of renal progenitor cells and mature podocytes were examined by WISH.Capped hdac 9 messenger RNA(mRNA) was injected into hdac9 mutant embryos at one-cell stage for rescuing the phenotype. Results:Zebrafish hdac9 started expression in renal progenitor cells and developing glomerulus until its maturation;loss of hdac9 led to glomerular developmental defects,including reduced number of renal progenitor cells and podocytes;Injection of hdac9 mRNA reversed glomerular filtration barrier and podocyte maturation in hdac9 knock-out(KO) embryos. Conclusion:hdac9 is required for normal glomerular development and maturation,probably regulate the transition from renal progenitor cells to podocytes.