p53 Enables Self Renewal of Nephron Progenitor Cells

p53 limits the self-renewing ability of a variety of stem cells. Here, contrary to its classical role in restraining cell proliferation, we demonstrate a divergent function of p53 in maintenance of self-renewal of the nephron progenitor population in the embryonic mouse kidney. p53-null nephron progenitor cells (NPC) exhibit progressive loss of the self-renewing progenitor niche in the cap mesenchyme, identified by Cited1 and Six2 expression, and loss of cap integrity. Nephron endowment is regulated by NPC availability and their differentiation to nephrons. Quantitatively, the Six2p53-/- cap has 30% fewer Six2GFP+ cells. While the apoptotic index is unchanged the proliferation index is significantly lower, in accordance with cell cycle analysis data showing less mutant Six2p53-/-;GFP+ cells in S and G2/M phases in comparison to Six2p53+/+;GFP+ cells. The mutant kidneys also show nephron deficit and decreased Fgf8 expression. To investigate the underlying changes in gene expression in the cap mesenchyme that contribute to the Six2p53-/- phenotype, we utilized RNA-Seq for transcriptome comparison. Top biological processes affected by p53 loss are development and morphogenesis, cell adhesion/migration, cell survival and metabolism. Cells from the mutant CM showed increased cellular ROS levels as well as deregulated expression of energy metabolism and mitochondrial genes suggesting metabolic dysfunction. Adhesion defects are visualized by decreased immunostaining of adhesion marker NCAM, and may possibly contribute to the differentiation defect as well. Altogether our data suggest a novel role for p53 in enabling self-renewal of the NPC and preservation of the progenitor niche, and thus regulating nephron endowment. mRNA profiles of wild-type (WT) and conditional p53 knockout (KO) of Six2+ mouse nephron progenitor cells (NPC) at embryonic day 15.5p53可抑制多种干细胞的自我更新能力。本研究中，与p53抑制细胞增殖的经典功能相悖，我们揭示了p53在维持胚胎小鼠肾脏肾单位祖细胞群自我更新中的全新功能。p53敲除（p53-null）的肾单位祖细胞（nephron progenitor cells, NPC）会在帽状间充质中出现自我更新祖细胞微环境的进行性丢失，该微环境可通过Cited1与Six2的表达特征进行鉴定，同时伴随帽状结构完整性的丧失。肾单位禀赋由NPC的数量及其向肾单位的分化进程共同调控。定量分析显示，Six2p53-/-小鼠的帽状结构中Six2GFP阳性细胞数量较对照组减少30%。尽管凋亡指数无明显变化，但突变体的增殖指数显著降低；细胞周期分析结果亦证实，与Six2p53+/+;GFP+细胞相比，Six2p53-/-;GFP+细胞处于S期及G2/M期的比例更低。突变体小鼠肾脏同时表现出肾单位发育缺陷以及Fgf8表达水平下调。为探究介导Six2p53-/-表型的帽状间充质基因表达调控机制，我们通过RNA测序（RNA-Seq）进行转录组比较分析。p53缺失所影响的核心生物学过程包括发育与形态发生、细胞黏附/迁移、细胞存活及代谢。突变体帽状间充质细胞的活性氧（reactive oxygen species, ROS）水平升高，同时能量代谢与线粒体相关基因表达失调，提示存在代谢功能异常。黏附标志物NCAM的免疫染色信号减弱，证实存在黏附功能缺陷，这或许同样参与了分化异常的发生。综上，本研究数据揭示了p53在维持NPC自我更新与祖细胞微环境稳态中的全新功能，进而调控肾单位禀赋。本数据集包含胚胎发育第15.5天的野生型（wild-type, WT）与Six2阳性细胞特异性条件性p53敲除（conditional p53 knockout, KO）小鼠肾单位祖细胞（NPC）的mRNA表达谱。