Tet2- andTet3-Mediated Cytosine Hydroxymethylation inSix2Progenitor Cells in Mice Is Critical for Nephron Progenitor Differentiation and Nephron Endowment

BackgroundNephron endowment is a key determinant of hypertension and kidney disease in later life. Epigenetic changes have been proposed to mediate fetal programming and nephron number. DNA cytosine methylation, which plays a critical role in gene regulation, is affected by proteins encoded by the 10-11 translocation (TET) DNA demethylase gene family (Tet1,Tet2, andTet3), but the roles of TET proteins in kidney development and nephron endowment have not been characterized.MethodsTo study whether epigenetic changes—specifically, active DNA hydroxymethylation mediated byTet1,Tet2, andTet3—are necessary for nephron progenitor differentiation and nephron endowment, we generated mice with deletion ofTet1,Tet2, orTet3in Six2-positive nephron progenitors cells (NPCs). We then performed unbiased omics profiling, including whole-genome bisulfite sequencing on isolated Six2-positive NPCs and single-cell RNA sequencing on kidneys from newborn mice.ResultsWe did not observe changes in kidney development or function in mice with NPCspecific deletion ofTet1,Tet2,Tet3orTet1/Tet2orTet1/Tet3. On the other hand, mice with combinedTet2andTet3loss inSix2-positive NPCs failed to form nephrons, leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2-positive NPCs resulted in defective mesenchymal to epithelial transition and renal vesicle differentiation. Whole-genome bisulfite sequencing, single-cell RNA sequencing, and gene and protein expression analysis identified a defect in expression in multiple genes, including the WNT-β-catenin signaling pathway, due to a failure in demethylation of these loci in the absence ofTet2andTet3.ConclusionsThese findings suggest thatTet2- andTet3-mediated active cytosine hydroxymethylation in NPCs play a key role in kidney development and nephron endowment.