Tet2 and Tet3 mediated active cytosine hydroxymethylation in Six2 progenitor cells is critical for nephron progenitor differentiation and nephron endowment

Nephron endowment is a key determinant of later life hypertension and kidney disease. Here we studied whether epigenetic changes, specifically the ten-eleven translocation (Tet) DNA demethylase family, Tet1, Tet2, and Tet3-mediated active DNA hydroxymethylation is necessary for gene expression regulation and kidney differentiation. We generated mice with deletion of Tet1, Tet2 or Tet3 in Six2 positive nephron progenitors (NP). We did not observe changes in development or kidney function in mice with nephron progenitor-specific deletion of Tet1, Tet2, Tet3 or Tet1/Tet2 or Tet1/Tet3. On the other hand, mice with combined Tet2 and Tet3 loss in Six2-positive NPCs failed to form nephrons leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2-positive NPs resulted in defect in mesenchymal to epithelial transition and renal vesicle differentiation. Whole genome bisulfite sequencing, single cell RNA sequencing, and gene and protein expression assay identified a defect in expression in genes in the WNT-β-catenin signaling pathway in absence of Tet2 and Tet3 due to a failure in demethylation of these loci. Our results indicate the key role of Tet2 and Tet3-mediated active cytosine hydroxymethylation in NPs in kidney development and nephron endowment. scRNA-seq of kidneys from newborn Six2Cre and Six2CreTet2/3f/f mice. WGBS of Six2+ NP isolated from kidneys of newborn Six2Cre and Six2CreTet2/3f/f mice.肾单位储备（nephron endowment）是晚年高血压与肾脏疾病的关键决定因素。本研究旨在探究表观遗传调控变化——尤其是十-十一易位（ten-eleven translocation, Tet）DNA去甲基化酶家族成员Tet1、Tet2与Tet3介导的主动DNA羟甲基化——是否对基因表达调控与肾脏分化至关重要。我们构建了在Six2阳性肾单位祖细胞（nephron progenitors, NP）中分别敲除Tet1、Tet2或Tet3的小鼠模型。在肾单位祖细胞特异性敲除Tet1、Tet2、Tet3，或共敲除Tet1/Tet2、Tet1/Tet3的小鼠中，我们未观察到发育或肾脏功能的异常变化。而在Six2阳性肾单位祖细胞中同时敲除Tet2与Tet3的小鼠，则无法形成肾单位，进而引发肾衰竭并导致围产期死亡。Six2阳性肾单位祖细胞中Tet2与Tet3的缺失，会导致间充质-上皮转化（mesenchymal to epithelial transition, MET）缺陷与肾小泡分化异常。通过全基因组亚硫酸氢盐测序（WGBS）、单细胞RNA测序（scRNA-seq）以及基因与蛋白质表达检测，我们发现：在Tet2与Tet3缺失的情况下，由于这些基因位点无法完成去甲基化，WNT-β-连环蛋白信号通路相关基因的表达存在缺陷。本研究结果证实，Tet2与Tet3介导的主动胞嘧啶羟甲基化在肾单位祖细胞的肾脏发育与肾单位储备过程中发挥关键作用。本研究数据集包含：新生Six2Cre与Six2CreTet2/3f/f小鼠肾脏的单细胞RNA测序数据；以及从新生Six2Cre与Six2CreTet2/3f/f小鼠肾脏中分离的Six2阳性肾单位祖细胞的全基因组亚硫酸氢盐测序数据。