The transcription factor Tcf21 is necessary for adoption of cell fates by <i>Foxd1</i> ⁺ stromal progenitors during kidney development

The stromal compartment of the developing kidney arises from forkhead box D1 (Foxd1)-expressing progenitors and gives rise to diverse cell types essential for nephrogenesis, including the renal stroma, capsule, mesangial cells, renin cells, pericytes, and vascular smooth muscle cells (VSMCs). However, the molecular mechanisms guiding their fate specification remain incompletely defined. Here, we identify the basic helix-loop-helix transcription factor Tcf21 as a critical determinant of stromal cell identity during kidney development. We performed single-cell RNA sequencing (scRNA-seq) on Foxd1-lineage cells isolated from embryonic <i>day 14.5</i> (E14.5) Tcf21 conditional knockout (<i>Tcf21</i>-cKO) <i>Foxd1^Cre/+</i>;<i>Rosa26^mTmG</i>;<i>Tcf21^f/f</i> and control kidneys, revealing seven transcriptionally distinct stromal subpopulations. Loss of Tcf21 resulted in marked depletion of medullary/perivascular stroma, collecting duct-associated stroma, proliferating stroma, and nephrogenic zone-associated subpopulations, confirmed by immunostaining, which revealed severe constriction of medullary and collecting duct stromal spaces. In addition, we identified a novel cluster unique to <i>Tcf21-</i>cKO kidneys, characterized by high expression of endomucin (Emcn). These cells spanned pseudotime trajectories and were distributed broadly across the mutant kidney. These findings were corroborated by E14.5 single-cell ATAC sequencing (scATAC-seq), which confirmed altered chromatin accessibility in Tcf21-deficient stroma. To assess the persistence and downstream impact of these defects, we performed bulk and scRNA-seq at E18.5, revealing sustained expansion of Emcn⁺ cells with profibrotic and perivascular transcriptional programs. Histological analyses at 2 mo demonstrated lasting architectural disruption, interstitial fibrosis, and impaired renal function in <i>Tcf21-</i>cKO mice. Our results identify Tcf21 as a key regulator of stromal progenitor fate and establish a developmental origin for fibrotic remodeling and kidney dysfunction.<b>NEW & NOTEWORTHY</b> This study identifies Tcf21 as a key regulator of kidney stromal fate. Loss of Tcf21 disrupts the emergence of key stromal cell types and leads to the expansion of a dysregulated, Emcn-expressing stromal population. Integrating single-cell transcriptomics, chromatin accessibility, and histology, we show that this misdifferentiation contributes to fibrosis in adulthood. These findings suggest that TCF21-dependent stromal differentiation restrains maladaptive remodeling and links developmental fate decisions to later fibrotic disease.