DNA methylation regulates nephron progenitor cell renewal and differentiation

Nephron number is a major determinant of long-term renal function. We hypothesized a link between epigenetic regulation and nephron formation. In support of this hypothesis, expression analysis evidenced high levels of DNA methyltransferases Dnmt1 and Dnmt3a in the nephrogenic zone of the developing mouse kidney. Using targeted loss-of-function manipulations in mice, we show that deletion of Dnmt1 in nephron progenitor cells results in a marked hypoplasia and reduction of nephron number at birth. In contrast, deletion of Dnmt3a/3b in nephron progenitor cells or deletion of Dnmt1/3a/3b in differentiated renal cells did not lead to any overt kidney phenotype. Whole mount optical projection tomography and 3D-reconstructions uncovered a significant reduction of stem cell niches and progenitor cells in Dnmt1-deficient mice. Ultimately, RNA sequencing analysis revealed that Dnmt1 controls DNA transcription regulating progenitor renewal, identity and differentiation. In summary, this study establishes DNA methylation as key regulatory event of prenatal renal programming. RNA-seq of FACS-sorted cap mesenchyme of E18.5 Six2.Cre;Dnmt1 conditional heterozygous and homozygous knockout mice肾单位数量是决定肾脏长期功能的核心因素。我们提出假说：表观遗传调控与肾单位形成之间存在关联。为验证该假说，表达分析显示在发育中小鼠肾脏的生肾区中，DNA甲基转移酶Dnmt1与Dnmt3a呈现高表达水平。通过在小鼠中开展靶向功能缺失实验，我们发现：在肾单位祖细胞中敲除Dnmt1，会导致新生小鼠出现显著的肾脏发育不全以及肾单位数量减少。与之相反，在肾单位祖细胞中敲除Dnmt3a/3b，或在分化成熟的肾细胞中敲除Dnmt1/3a/3b，均未引发明显的肾脏表型异常。通过整体标本光学投影层析成像与三维重建技术，我们发现Dnmt1缺陷小鼠的干细胞龛与祖细胞数量显著减少。最终，RNA测序（RNA-seq）分析结果显示，Dnmt1通过调控DNA转录，进而影响祖细胞的自我更新、身份维持与分化过程。综上，本研究证实DNA甲基化是产前肾脏编程的关键调控事件。针对E18.5天Six2.Cre;Dnmt1条件性杂合及纯合敲除小鼠的荧光激活细胞分选（FACS）纯化帽状间充质样本进行RNA测序