The transcription factor Tcf21 is necessary for adoption of cell fates by Foxd1(+) stromal progenitors during kidney development.

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Published in final edited form as: Am J Physiol Renal Physiol. 2026 Feb 3;330(4):F347–F364. doi: 10.1152/ajprenal.00345.2025 Search in PMC Search in PubMed View in NLM Catalog Add to search The Transcription Factor TCF21 is Necessary for Adoption of Cell Fates by Foxd1+ Stromal Progenitors during Kidney Development Gal Finer Gal Finer 1 Division of Pediatric Nephrology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA 2 Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Find articles by Gal Finer 1, 2, * , George S Yacu George S Yacu 1 Division of Pediatric Nephrology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA Find articles by George S Yacu 1 , Mohammad D Khan Mohammad D Khan 3 Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Find articles by Mohammad D Khan 3 , Yalu Zhou Yalu Zhou 2 Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Find articles by Yalu Zhou 2 , Gaurav Gadhvi Gaurav Gadhvi 3 Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Find articles by Gaurav Gadhvi 3 , Sarah E Ward Sarah E Ward 1 Division of Pediatric Nephrology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA Find articles by Sarah E Ward 1 , Mohammed Sayed Mohammed Sayed 4 Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA Find articles by Mohammed Sayed 4 , R Ariel Gomez R Ariel Gomez 5 Department of Pediatrics, Child Health Research Center, University of Virginia, Charlottesville, VA, USA Find articles by R Ariel Gomez 5 , Maria Luisa S Sequeira-Lopez Maria Luisa S Sequeira-Lopez 5 Department of Pediatrics, Child Health Research Center, University of Virginia, Charlottesville, VA, USA Find articles by Maria Luisa S Sequeira-Lopez 5 , Joo-Seop Park Joo-Seop Park 2 Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Find articles by Joo-Seop Park 2 , Hee-Woong Lim Hee-Woong Lim 4 Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA 6 Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Find articles by Hee-Woong Lim 4, 6 , Susan E Quaggin Susan E Quaggin 2 Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 7 Division of Nephrology and Hypertension, Northwestern Memorial Hospital, Chicago IL, USA Find articles by Susan E Quaggin 2, 7 , Deborah R Winter Deborah R Winter 3 Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 8 Center for Human Immunobiology (CHI), Northwestern University Feinberg School of Medicine, Chicago, IL, USA Find articles by Deborah R Winter 3, 8 Author information Article notes Copyright and License information 1 Division of Pediatric Nephrology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA 2 Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 3 Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 4 Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA 5 Department of Pediatrics, Child Health Research Center, University of Virginia, Charlottesville, VA, USA 6 Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA 7 Division of Nephrology and Hypertension, Northwestern Memorial Hospital, Chicago IL, USA 8 Center for Human Immunobiology (CHI), Northwestern University Feinberg School of Medicine, Chicago, IL, USA * Correspondence: Gal Finer ( [email protected] ), Fax 312-227-9405: Deborah R. Winter ( [email protected] ) Issue date 2026 Apr 1. PMC Copyright notice PMCID: PMC13070429  NIHMSID: NIHMS2146041  PMID: 41632505 The publisher's version of this article is available at Am J Physiol Renal Physiol Previous version available: This article is based on a previously available preprint posted on bioRxiv on December 14, 2024: " The transcription factor TCF21 is necessary for adoption of cell fates by Foxd1+ stromal progenitors during kidney development ". Abstract The stromal compartment of the developing kidney arises from 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 day 14.5 (E14.5) Tcf21 conditional knockout ( Tcf21 -cKO) Foxd1 Cre /+ ;Rosa26 mTmG ;Tcf21 f/f 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. Additionally, we identified a novel cluster unique to Tcf21 -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 pro-fibrotic and perivascular transcriptional programs. Histological analyses at 2 months demonstrated lasting architectural disruption, interstitial fibrosis, and impaired renal function in Tcf21 -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. Keywords: Foxd1, kidney, single-cell-RNA-sequencing, stroma, Tcf21 Graphical Abstract INTRODUCTION Work that has spanned over seven decades increased our understanding of molecular mechanisms in kidney development and has been informing new regenerative based solutions for drug screening, disease modelling, and renal replacement 1 , 2 . The developing mammalian kidney is composed of four progenitor lineages: nephron (Six2+ cells), ureteric (Hoxb7+ cells), stromal/interstitial (Foxd1+ cells), and endothelial (c-kit+ cells) 3 , 4 . During kidney development, these progenitor populations signal to each other to ensure the ongoing expansion of the kidney to an organ of normal size and function 5 , 6 . The forkhead box D1 (Foxd1+) progenitors give rise to a diverse array of cell types including most of the renal stroma, VSMCs, pericytes, mesangial cells, renin-, and erythropoietin-producing cells 7 , 8 . Conventionally, renal stroma is divided into three anatomical regions: cortical, medullary, and papillary 9 . Recent research, however, has identified a dozen distinct anatomical positions within the stroma in mice, each characterized by unique cell types, highlighting its heterogeneity 10 . Foxd1+ cells play critical roles in branching morphogenesis, nephron development, and vascular patterning 11 , possibly by promoting maturation of neighboring epithelial and endothelial cells 12 – 15 . Despite these insights, understanding the molecular mechanisms through which the stroma influence non-stromal cells and guide the differentiation of Foxd1+ cells into distinct stromal populations is essential for normal kidney development, yet remains incomplete. TCF21 is a mesoderm-specific transcription factor of the bHLH family that forms homo/heterodimers upon binding to its target DNA sequence 16 . Originally named Pod1 17 , epicardin 18 , or capsulin 19 , 20 , Tcf21 is expressed in the developing urogenital, cardiovascular, respiratory, and gastrointestinal systems during embryonic development. Tcf21 has been recently implicated as a causal gene for coronary artery disease in humans 21 , 22 . Within the kidney, Tcf21 has been implicated in the formation of the renal stroma and perivascular cells, influencing the structural integrity and function of the organ 23 . To investigate the role of Tcf21 in kidney stroma development and uncover the developmental origins of cell diversity arising from Foxd1+ cells, we initially performed scRNA-seq on control and Tcf21-deficient Foxd1+ kidney cells from E14.5 mouse embryos, an early stage of nephrogenesis. To support and extend our initial findings, we subsequently incorporated scATAC-seq at E14.5, as well as both bulk RNA-seq and scRNA-seq at E18.5. Finally, to examine the long-term consequences of Tcf21 loss, we analyzed histological sections of adult kidneys at 2 months of age. MATERIALS AND METHODS Mouse strains and embryo staging Mice were maintained under pathogen-free conditions on a 12-hour light/dark cycle with ad libitum access to standard rodent chow and water. All mouse experiments were approved by the Animal Care Committee at the Center for Comparative Medicine of Northwestern University (Chicago, IL) and were performed in accordance with institutional guidelines and the NIH Guide for the Care and Use of Laboratory Animals. The following strains were used: Tcf2 1 flox/flox 24 , Foxd1-eGFPCre (JAX Stock #012463), and Rosa26-mTmG (JAX Stock: #007576). Details about breeding schemes, genotyping. and embryo staging are in the Supplementary Methods . Histology, immunohistochemistry, and quantification Mouse kidneys were dissected and sectioned for histological analyses at E14.5, E18.5, and 2 months of age. At least three biological replicates including both female and male samples were used. Details about the staining procedures, antibodies, and quantifications are in the Supplementary Methods. Serum creatinine measurement Serum was collected from adult Tcf21 -cKO and control mice (female and male). Creatinine was measured using high-performance liquid chromatography-mass spectrometry at the Univeristy Alabama at Birmingham O’Brien Center Core Facility. Preparation of single cells from E14.5 and E18.5 mouse embryos Kidneys from control and Tcf21 -cKO embryos (female and male) were dissected under sterile conditions. Cell dissociation was performed using a previously-described cold-active protease dissociation protocol 25 that prevents artifactual changes and preserves transcriptomic profiles during enzymatic digestion. Following this, Foxd1-lineage stromal cells were purified by fluorescence-activated cell sorting. Cells that were GFP+ (either single-positive or double-positive for tdTomato) were collected for sequencing, ensuring the inclusion of both early progenitors and more differentiated stromal populations ( Suppl Fig. S1a ). Samples of the same genotype and sex were pooled and immediately submitted for sequencing. A detailed protocol and table with number of embryos/cells submitted for sequencing are reported in the Supplementary Methods . Processing and analysis of single cell RNA-seq at E14.5 Raw sequencing data was demultiplexed and processed using Cell Ranger v6.1.0 with a custom reference combining the mm10 transcriptome with the eGFP and tdTomato RNA sequences. After filtering, quality control, and unsupervised clustering, stromal clusters were annotated based on module scores for curated stromal markers, publicly available in situ hybridization atlases, and previously published clusters. Analyses of cell type composition, differential expression, GO enrichment, and pseudotime trajectory were performed between control and mutant stromal cells. The full workflow of the pre-processing and computational analyses is provided in the Supplementary Methods . Processing and analysis of single cell ATAC-seq at E14.5 Raw sequencing data was demultiplexed and processed using Cell Ranger ATAC with the mm10 genome. After filtering and quality control, we subset all stromal cells and transferred stromal subtype annotations from the E14.5 scRNA-seq dataset. Additionally, we pseudo-bulked signal tracks by genotype and visualized the signal in a genome browser. We chose to pseudo-bulk by genotype to account for the uneven representation of stromal clusters across genotypes. The full workflow is reported in the Supplementary Methods . Characterization of Emcn + stromal cells at E18.5 To characterize Emcn + stromal cells in late kidney development, we performed scRNA-seq on kidneys from control and Tcf21 -cKO embryos (female and male) at E18.5. After filtering, quality control, and unsupervised clustering, we used two complementary approaches to annotate clusters: label transfer from E14.5 scRNA-seq and de novo marker gene validation ( Suppl Fig. S8c-e; Suppl Table S5 ). For further clustering of Emcn + stromal cells at E18.5, we subset mutant cells with log 2 -transformed Emcn expression > 1.03 (corresponding to the median in mutant samples). Additionally, we evaluated the expression of matrisome-associated genes with a module score based on a published gene set 26 . The full workflow is reported in the Supplementary Methods . CellChat Analysis Intercellular communication networks were inferred among stromal, epithelial, and endothelial compartments in the E18.5 scRNA-seq dataset using CellChat (v2.1.2) 27 , as described in the Supplementary Methods . Bulk RNA-seq of E18.5 whole kidneys Whole kidneys from two E18.5 Foxd1 Cre /+ Tcf21 f/f embryos and two Foxd1 Cre /+ Tcf21 +/+ litermate controls (all males) were subjected to bulk RNA sequencing. Sample processing, sequencing, and bioinformatic analyses are described in the Supplementary Methods . Histological validation was performed on female and male samples. RESULTS Single-cell profiling of E14.5 Foxd1 GFP+ cells identifies diverse cell populations within the developing kidney To investigate the role of Tcf21 in the Foxd1+ stromal progenitors and their derivative cells at a single-cell level, we purified GFP+ and GFP+ tdTomato+ cells from female and male Foxd1 Cre /+ ;Rosa26 mTmG ;Tcf21 f/f ( Tcf21 -cKO) and littermate controls Foxd1 Cre /+ ;Rosa26 mTmG ;Tcf21 +/+ via flow cytometry ( Suppl Fig. S1a ). In this model, Tcf21 gene is permanently deleted, tdTomato expression is terminated, and GFP expression is maintained in all the progeny of Foxd1-expressing cells. We obtained 13,256 mutant and 19,205 control cells after QC ( Suppl Fig. S2a ). We defined 13 clusters using an unsupervised approach (see Methods) and assigned cell lineage annotations based on de novo markers and canonical gene expression ( Fig. 1a , b ; Suppl Fig. S2c,d; Suppl Table S1 ). This analysis identified a large population of stromal cells (Foxd1+ Meis1+ Pdgfra+) as well as representation of the nephron progenitor cell (NPC) lineage (Six2, Cited1, Crym), and significantly smaller populations of ureteric epithelium (Ret, Wnt11, Gata3), immune cells (Fcer1g, ​Lyz2), and neurons (Tubb3, Map2) ( Fig. 1b , Suppl Fig. S2e ). Cycling genes were expressed across cell lineages ( Suppl Fig. S2f ). We confirmed that GFP expression was robust in both stromal and non-stromal cells, though Foxd1 expression was largely limited to the stromal lineage ( Fig. 1c ). Low expression of tdTomato and depleted expression of Tcf21 in Tcf21 -cKO cells validate the activity of cre recombinase in these cells ( Fig. 1c , Suppl Fig. S2g ). Non-stromal GFP+ cells likely represent early transient expression of Foxd1 before the onset of lineage boundaries. All cell types were represented in both females and males of Tcf21 -cKO and control samples, however, the number and proportion of stromal cells was lower in sex-matched mutant samples ( Fig. 1d , Suppl Fig. S2h ). These differences in cell numbers are likely attributable to experimental variation rather than being biologically driven. Figure 1: scRNA-seq analysis of the Foxd1-GFP+ tdTomato+ sorted cells from E14.5 embryonic mouse kidneys of Tcf21-conditional knockout ( Tcf21 -cKO) and controls. Open in a new tab (a) UMAP plot of 32,461 individual cells combined from male and female Tcf21 -cKO and control kidneys colored by cell type annotation (E14.5 scRNA-seq: n =2 female control, n= 4 male control , n= 3 female mutant , n= 1 male mutant embryos). (b) Dot plot illustrating expression of canonical genes across cell types. Size of dot illustrates percent of cells expressing the gene and color scale indicates relative expression across cell types. (c) Feature plots of EGFP, Foxd1, tdTomato, and Tcf21 gene expression (log 2 transformed). (d) UMAP plots of samples by experimental group and sex. See Suppl Fig. S2h for number of cells per experimental group. E14.5 stromal progenitors express genes associated with different cell fates Next, we sought to explore the heterogeneity within the developing stromal cell populations by sub-clustering cells annotated as stroma ( Suppl Fig. S3a,b ). We defined seven stromal subpopulations (numbered 0–6) in Tcf21 -cKO and controls with distinct gene expression profiles based on de novo marker expression ( Fig. 2a , b ). All clusters expressed similar levels of GFP and low levels of tdTomato while expression of Foxd1 and Tcf21 was variable ( Suppl Fig. S3c ). To annotate these clusters, we created modules associated with different subpopulations based on canonical markers from the literature ( Suppl Fig. S4 ) and visualized their expression along with canonical cell cycle genes ( Fig. 2c ). Based on module expression and de novo gene markers, we annotated Nephrogenic zone associated stroma, Proliferating stroma, Medullary/Perivascular associated stroma, Collecting duct associated stroma, Differentiating stroma, and Ureteric stroma. To confirm our annotations, we compared expression of key genes in our single-cell dataset vs. spatial expression from the GenePaint gene expression atlas ( Fig. 3 ). Our annotations largely agreed with previously published stromal clusters during kidney development (Combes et al 28 ), with subtle differences ( Suppl Fig. S3d,e ). Our annotated Differentiating stroma cluster (cluster 3) more closely resembles the published Ureteric cluster. However, the lower levels of Tbx18 , a gene known to play a critical role in early separation of the ureteric mesenchymal lineage from other mesenchymal lineages 11 29 , in these cells compared with those in cluster 6 did not support its classification as Ureteric. Moreover, de novo markers with unique expression in cluster 6 demonstrated high spatial specificity to the ureteric zone ( Fig. 3 ). Instead, cluster 3 was annotated as “Differentiating Stroma” due to more diffuse expression of associated markers, consistent with its lack of a distinct anatomical location ( Fig. 3b ). Additionally, we annotated cluster 2 as Emcn-expressing based on unique expression of the gene Emcn, further discussed in the following section ( Fig. 2 ; Suppl Fig. S3f-h ). Figure 2: Stromal lineage sub-clustering identifies populations of distinct cell fate within the developing mouse kidney. Open in a new tab (a) Sub-clustering of stromal cells ( n =22,297) from E14.5 Foxd1GFP+ kidney cells (stromal cluster in Fig. 1a ) identifies seven transcriptionally distinct populations and their suggested annotation. (b) Heatmap depicting the relative expression of five of the top de novo marker genes for each cluster across individual cells. Column color coding is consistent with Fig. 2a . (c) Feature plots of the module score distribution in UMAP space based on canonical gene signatures (listed in Suppl Fig. S4 ). Figure 3: Spatial annotation of stromal clusters of control and Tcf21 -cKO. Open in a new tab (a) Feature plots showing the log 2 -transformed gene expression of markers for Nephrogenic zone stroma (Foxd1, Crabp2, Gdnf, Aldh1a2), Medullary and Perivascular stroma (Pdgfrb, Ace2, Rgs5, Heyl), Collecting duct stroma (Penk, Pfn2, Tcf21, Thy1), Differentiating stroma (Smoc2, Igf1, Dcn, Fbln5), and Ureteric stroma (Tbx18, Syt1, Npy1r). (b) Expression of the same marker genes by in situ hybridization in control E14 mouse kidneys as per GenePaint gene expression atlas ( https://gp3.mpg.de/ , cropped images from whole mount), including a schematic illustration of the spatial location of each stromal population in the developing kidney. Cluster 4 (Nephrogenic zone-associated stroma), Cluster 0 (Medullary/Perivascular stroma), cluster 5 (Collecting duct stroma), cluster 3 (Differentiating stroma), and cluster 6 (Ureteric stroma). Tcf21 is required for emergence of stromal sub-populations during kidney development Next, we sought to determine how stromal progenitor cells in Tcf21 -cKO have deviated from controls using a downsampled dataset to control for sex and cell number. Strikingly, Medullary/Perivascular stroma, Collecting duct associated stroma, Proliferating stroma, and Nephrogenic zone associated stroma clusters were dramatically reduced in Tcf21 -cKO compared to control ( Fig. 4a – c ). Conversely, the cell numbers of Differentiating stroma and Ureteric stroma clusters were moderately increased in Tcf21 -cKO, though this may be due to reduced total stromal cell numbers. Notably, the most profound change was that the Emcn-expressing cluster was virtually exclusive to Tcf21 -cKO kidneys, with only few cells (7 cells) of shared transcriptional profile identified in stromal cells of controls ( Fig. 4a – c ). These results were conserved when male and female samples were compared independently ( Suppl Fig. S6c-e ). Figure 4: Tcf21 is critical for the development of specific stromal cell fates. Open in a new tab (a) UMAPs of Foxd1GFP+ 7,108 stromal cells (downsampled to equally reflect 4 samples) from male and female E14.5 control and Tcf21 -cKO mouse kidneys ( Foxd1 Cre ;Rosa26 mTmG ;Tcf21 +/+ and Foxd1 Cre ;Rosa26 mTmG ;Tcf21 f/f , respectively). (b) Stacked bar chart indicating subpopulation composition by sample. Percent of each subpopulation in a sample are given. (c) Relative differences in cell proportions for each cluster between the Tcf21 -cKOs versus controls. Dashed vertical lines mark absolute log 2 fold change (FC) > 1 compared with the control. All changes in proportion were significant after FDR was applied (scProportion permutation test; n =3,554/group). Bi-sex comparison in Suppl Fig. S6c-e . (d) Volcano plot of differentially expressed genes (DEGs) between all stromal cells in Tcf21 -cKOs versus controls defined by log 2 (fold change) >0.25 and adjusted p-value <0.05. Red and blue dots denote significantly up- and down-regulated differential genes, respectively. (e) Dot plots of GO Terms significantly enriched among up-regulated (top panel) and down-regulated (bottom panel) DEGs. X axis denotes the overlap ratio (# genes in overlap/# genes in GO term), size of dot denotes the #genes in overlap, and color scale denotes the −log 10 adjusted p-value (p-adj). (f) Feature plot of stromal cells with color-coding based on pseudotime bins. (g) Box and whiskers plot depicting the range of pseudotime by bin in each stromal cluster. In parallel, we performed differential gene expression analysis of the whole stromal compartment between Tcf21 -cKO and control ( Suppl Table S3a-c ). We identified 214 genes up-regulated and 64 genes down-regulated in Tcf21 -cKO compared with control cells ( Fig. 4d ). Tcf21 is among the most down-regulated genes, as expected, while genes associated with connective tissue development, epithelial cell proliferation, extracellular matrix organization, and WNT pathway were up-regulated ( Fig. 4e ). In order to determine how the Emcn-expressing cells fit into healthy stromal cell differentiation, we ran Monocle on control stromal cells to estimate the underlying pseudotime trajectory, assuming Nephrogenic zone-associated cells represented the most progenitor-like subpopulation. By transferring control-based pseudotime to Tcf21 -cKO cells, we found that the Emcn-expressing cluster comprised cells spanning across all pseudotime ( Fig. 4f , g ). Moreover, when we defined modules of co-regulated genes along the control trajectory, we found that specific cell subsets within the Emcn-expressing cluster expressed genes associated with a variety of other subpopulations including Nephrogenic (module 18), Proliferating (module 20), and Medullary/Perivascular (module 14) ( Suppl Fig. S5c,d, Suppl Table S4a,b ). Taken together, these results suggest that the Tcf21 -cKO is associated with the emergence of cells displaying altered differentiation outcomes, including a potential aberrant differentiation. To spatially define the defect in the stromal lineage of the Tcf21 -cKO, we performed lineage-tracing of Foxd1 (GFP+) in Foxd1 Cre /+ ;Rosa26 mTmG ;Tcf21 f/f and control mice from E14.5 to E18.5. Tcf21 -cKOs demonstrated a marked constriction of the medullary stroma and the stromal space adjacent to the collecting ducts compared with controls ( Fig. 5a ). Additionally, immunostaining for Emcn, a marker of the stromal population exclusively expressed in Tcf21 -cKO, confirmed robust expression in Tcf21 -cKO kidneys at E14.5 and E16.5 ( Fig. 5b – e ). Furthermore, staining with alpha smooth muscle actin (Acta2), which marks medullary stromal cells 10 , 23 , demonstrated a loss of Acta2-expressing stroma and the emergence of Emcn + stromal cells in Tcf21 -cKO kidneys ( Fig. 5e , Suppl Fig. S7 ), supporting a population shift. Taken together, these data support the scRNA-seq analysis above and indicate a requirement for Tcf21 in the emergence of specific derivatives of the stromal lineage during kidney development. Figure 5: Histological characterization of stromal changes in Tcf21 -cKO kidneys. Open in a new tab Immunofluorescence analysis of Foxd1 Cre ;Rosa26 mTmG ;Tcf21 f/f and control kidneys reveals structural and molecular alterations in stromal identity following loss of Tcf21. (a) Anti-GFP staining (marking the Foxd1+ lineage) shows a significant reduction in GFP+ stromal area in Tcf21 -cKO kidneys at E14.5. This reduction persists at E15.5, E16.5, and E18.5, especially in the medullary stroma and regions adjacent to the collecting ducts (dotted line), consistent with scRNA-seq data. n =4 control and n =4 mutant embryos, sex unknown. (b,c) Emcn staining at E14.5 reveals the presence of novel Emcn + stromal cells (arrowheads) occupying the majority of the stromal compartment in Tcf21 -cKO kidneys. Quantification of Emcn + stromal cells is shown on the right ( p = 0.003, two-tailed unpaired t-test). n= 2 control (1 male and 1 female) and n =2 mutant (both male) embryos. (d) At E16.5, Emcn + stromal cells are again evident in Tcf21 -cKOs. In contrast, in control kidneys, Emcn expression remains restricted to the endothelium, where it co-localizes with CD31 and is absent from stromal cells. n =2 control (both male) and n =2 mutant (1 male and 1 female) embryos. (e) Staining for α-smooth muscle actin (Acta2) shows a marked loss of Acta2 + stromal cells in Tcf21 -cKO kidneys at E16.5, coinciding with the expansion of Emcn + stromal cells. n =3 control (2 males, 1 sex unknown) and n =3 mutant (1 male, 1 female, 1 sex unknown) embryos. (f) Co-staining for Emcn (magenta) and Meis1/2/3 (green) at E18.5 demonstrates that Emcn + cells in Tcf21 -cKOs co-express Meis1/2/3 (white signal), indicating that these cells retain a stromal lineage identity despite their aberrant marker expression. n =1 control and n =1 mutant, both male. (g) Dual staining for Emcn (magenta) and GFP (green) at E18.5 further confirms co-localization (white signal) of Emcn + and GFP + stromal lineage cells in cortical, perivascular, and medullary compartments, revealing integration of Emcn + cells into the vasculature and stroma of Tcf21 -cKOs. (h) Whole kidney images highlight the widespread distribution of Emcn + GFP + double-positive cells in Tcf21 -cKO kidneys, consistent with transcriptional evidence of Emcn up-regulation across multiple stromal subtypes including Medullary, Perivascular, and Collecting duct-associated stroma shown in Fig. 6a , b . n =2 control and n =2 mutant, both male. Scale bars: (a) 500 μm, (b) 5 μm, (c-h) 100 μm Emcn + stromal cells persist at E18.5 and exhibit vascular and ECM-associated features To investigate the persistence and molecular characteristics of the Emcn + stromal population identified at E14.5, we performed immunofluorescence analysis at E18.5. Emcn + cells remained abundant and broadly distributed in Tcf21 -cKO kidneys, continuing to co-express GFP and canonical stromal markers such as Meis1/2/3 and Pdgfrb ( Fig. 5f – h ; Fig. 8g ), consistent with a retained stromal identity despite signs of dysregulation. At this stage, Emcn + cells occupied a large portion of the stromal compartment and were prominently expanded around the medullary vasculature, where they appeared to integrate with the vascular network ( Fig. 5g , middle panel ). Additionally, Tcf21 -cKO kidneys exhibited an expansion of Emcn + vascular structures ( Fig. 8g ), suggestive of vascular remodeling. Consistent with these histological observations, scRNA-seq analysis of E18.5 stromal cells (see clustering strategy in Suppl Fig. S8 ) revealed a marked expansion of Emcn-expressing cells in Tcf21-deficient kidneys, broadly distributed across multiple stromal clusters ( Fig. 6a , b , d ). Notably, Tcf21 -cKO kidneys continued to show enrichment for Postn, Ecm1, Sparcl1, and Rgs5 ( Fig. 6c ), all previously up-regulated at E14.5, indicating a sustained shift in stromal cell identity following Tcf21 deletion. Differential expression analysis comparing control stromal cells to mutant Emcn + cells demonstrated up-regulation of ECM-associated genes (matrisome gene module in Fig. 6e ; Suppl Table S6 ). Further re-clustering of Tcf21 -cKO Emcn + cells revealed their distribution across multiple stromal subtypes ( Fig. 6f ), with high overall expression of matrisome-associated genes ( Fig. 6g ), supporting the persistence and expansion of ECM-producing Emcn + cells in a dysregulated transcriptional state at E18.5. Figure 8: Functional impact of stromal changes on kidney development. Open in a new tab (a) Heatmap depicting the relative expression of selected differentially expressed genes (DEGs) from bulk RNA-seq of whole kidney lysates from E18.5 control ( Foxd1 Cre /+ ;Tcf21 +/+ ) and Tcf21 -cKO ( Foxd1 Cre /+ ;Tcf21 f/f ) kidneys. Significant genes were defined by absolute log 2 fold change > 1 and q-value < 0.05 (E18.5 bulk RNA-seq: n =2 control and n =2 mutant embryos, all male). Tcf21 -cKO kidneys exhibit upregulation of genes associated with fibrosis (Fap, Cyp1b1, Inhba) and muscle markers (Myh8, Myh3, Atp2a1), and downregulation of genes related to renal tubular cell identity and function (Slc14a2, Corin, Aqp4) involved in urine concentration, as well as medullary stroma differentiation (Alx1) and anti-fibrotic activity (Runx2, Bmp6). (b) Bar plots showing FPKM (fragments per kilobase of transcript per million mapped reads) values for representative genes differentially expressed. (Bars represent mean ± standard error of mean). (c) Gene Ontology enrichment analysis shows the top enriched biological processes, ranked by log adjusted p -value. Tcf21 -cKO kidneys exhibit enrichment of terms associated with muscle contraction, consistent with a shift in cell identity following the loss of Tcf21, while downregulated genes were enriched for processes related to water transport (urine concentration). (d,e) Analysis of intercompartmental signaling in E18.5 control and Tcf21 -cKO kidneys using CellChat. Bubble plots depict significant differential ligand–receptor interactions between stromal, epithelial, and endothelial compartments, stratified by genotype. In control kidneys, stromal signals to endothelium and epithelium are enriched for developmental and morphogenic pathways (Bmp4-Bmpr1a, Sema3a-Nrp1+Plxna2, Wnt5a-Fzd4). In contrast, Tcf21 -cKO kidneys exhibit emergence of maladaptive or fibrotic cues (Wnt4-Fzd4+Lrp6, Sema3c-Nrp2+Plxna2, Col4a5-Itga2+Itgb1), alongside diminished epithelial-to-stromal feedback. Dot size represents p value, and color indicates communication probability. (f) IF for E-cadherin (yellow) marks tubular epithelial cells. In control kidneys, it outlines well-organized tubular structures. In Tcf21 cKO kidneys, E-cadherin+ cells exhibit loss of tubular organization, suggesting architectural disarray. However, E-cadherin staining alone does not allow definitive conclusions about epithelial cell identity. ( n =2 control, 1 male and 1 sex not determined, and n =2 mutant, 1 male and 1 sex not determined, embryos). (g) IF of EMCN (magenta) and PDGFRB (green) illustrates vascular (EMCN+ endothelial cells) and perivascular (EMCN+ PDGFRB+) organization, indicating an expanded perivascular compartment and altered organization of endothelial cells in Tcf21 -cKO kidneys. Scale bar, 100 μm. Figure 6: Fate of Emcn + cells at E18.5. Open in a new tab (a) Feature plots and (b) violin plot showing log 2 -transformed gene expression of Emcn across stromal sub-populations in control (blue) and Tcf21 -cKO (red) at E18.5. (E18.5 scRNA-seq: n =2 female control, n =2 male control , n= 2 female mutant , n= 1 male mutant embryos). (c) Violin plots depicting log 2 -transformed gene expression for significant upregulated genes (Postn, Ecm1, Sparcl1, and Rgs5) across stromal populations in Tcf21 -cKO kidneys (red) compared with controls (blue), consistent with expression pattern in E14.5 Tcf21 -cKO kidneys (see Suppl Fig. S5 ). (d) UMAP highlighting the distribution of Emcn + mutant cells (magenta; defined by log 2 -transformed Emcn expression > 1.03) and control stromal cells (blue). Emcn + cells are broadly distributed across the stroma, consistent with expansion of a dysregulated stromal subset identified at E14.5. (e) Violin plot illustrating the expression of canonical matrisome gene module in Emcn + mutant cells versus control stromal cells ( p = 2.2e-16 by Wilcoxon rank-sum test, ***p < 0.001). See Suppl Table 6 for genes. Emcn + mutant cells exhibit elevated expression, suggesting enhanced ECM production and a shift toward a fibrotic phenotype with Tcf21 loss. (f) UMAP of reclustered Emcn + stromal cells from E18.5 Tcf21 -cKO kidneys. Eight distinct clusters were identified, broadly distributed across multiple stromal subtypes, suggesting a loss of fate restriction in the absence of Tcf21. (g) Feature plot of Emcn + cells with color-coding based on matrisome gene module quartile, demonstrating enrichment of Emcn + cells with highest expression across the Differentiating stroma, Medullary, Collecting duct-associated, and Proliferating stromal clusters. Tcf21 deletion alters chromatin accessibility and reveals elevated Emcn gene activity in E14.5 Tcf21 -cKO stroma To assess the impact of Tcf21 deletion on stromal chromatin accessibility, we performed scATAC-seq on E14.5 kidneys from control and Tcf21 -cKO embryos. Clustering identified a predominant stromal population, along with smaller populations of non-stromal cells ( Suppl Fig. S9a,b ). Label transfer from E14.5 scRNA-seq identified seven major stromal sub-populations ( Fig. 7a ). Mutant kidneys showed significant depletion of Medullary/Perivascular, Proliferating, Nephrogenic zone-associated, and Collecting duct-associated stroma, alongside enrichment of Emcn-expressing stroma, consistent with E14.5 scRNA-seq findings ( Fig. 7b ). Tcf21 motif activity was robust in multiple stromal populations in controls, particularly Medullary/Perivascular, Proliferating, and Collecting duct-associated stroma, but markedly reduced in mutants, confirming effective gene excision ( Fig. 7c , d ). Notably, Emcn emerged as the top marker gene for the Emcn-expressing population in our analysis of de novo cluster markers, based on estimated gene activity from chromatin accessibility at the promoter and gene body ( Suppl Fig. 9c, Suppl Table S7 ). Pseudobulked ATAC-seq tracks revealed increased chromatin accessibility at the Emcn promoter in mutants and identified a Tcf21 binding motif 80 bp downstream, supporting the possibility that Tcf21 may normally repress Emcn expression ( Fig. 7e ). Figure 7: scATAC-seq analysis of chromatin accessibility and Emcn locus activity at E14.5. Open in a new tab (a) UMAP visualizations of control and Tcf21 -cKO samples following label transfer from the E14.5 scRNA-seq dataset. UMAPs were subsampled to include an equal number of cells per condition. (E14.5 scATAC-seq: n =4 control and n =4 mutant embryos, all male.) (b) Relative differences in cell composition between control and Tcf21 -cKO kidneys. Proliferating, Collecting duct-associated, Medullary/Perivascular, and Nephrogenic zone-associated stroma were significantly depleted in mutants, whereas Emcn-expressing stroma was enriched. Dashed vertical lines mark absolute log 2 fold change > 1. (c) Feature plots and (d) violin plots showing Tcf21 motif activity across cell types, with prominent motif activity in Collecting Duct, Medullary/Perivascular, and Proliferating stroma in controls, and markedly reduced activity in Tcf21 -cKO. (e) Pseudobulked ATAC-seq tracks showing increased chromatin accessibility at the Emcn promoter in Tcf21 -cKO stromal cells. A predicted Tcf21 binding motif is located 80 bp downstream of the promoter, supporting the possibility that Tcf21 may normally repress Emcn expression. Stromal Tcf21 deficiency drives non-cell-autonomous disruption of kidney differentiation, maturation, and vascular integrity To evaluate the downstream, non–cell-autonomous consequences of aberrant stromal differentiation beyond E14.5, we performed bulk RNA sequencing on whole kidney lysates from E18.5 Foxd1 Cre /+ ;Tcf21 f/f and control embryos. Differential gene expression analysis revealed significant upregulation of transcripts associated with extracellular matrix remodeling and fibrosis (Fap, Cyp1b1, Inhba) and markers of ectopic muscle lineage identity (Myh8, Myh3, Atp2a1). Conversely, genes critical for renal tubular differentiation and function, including Aqp4, Slc14a2, and Corin were markedly downregulated ( Fig. 8a , b , Suppl Table S8a ). Gene Ontology enrichment analysis further supported these findings, identifying over-represented biological processes among up-regulated genes related to muscle contraction, while down-regulated genes were enriched for water transport ( Fig. 8c , Suppl Table S8b,c ). To further probe intercompartmental communication, we applied, to infer ligand–receptor interactions between stromal, epithelial, and endothelial compartments at E18.5. In control kidneys, stromal-derived cues to the epithelium and endothelium were enriched for developmental and morphogenic pathways such as Bmp4 - Bmpr1a, Wnt5a - Fzd4, and Sema3a - Nrp1+Plxna2. In contrast, Tcf21-deficient kidneys showed emergence of maladaptive and fibrotic signals, including Wnt4 - Fzd4+Lrp6, Sema3c - Nrp2+Plxna2, Col4a5 - Itga2+Itgb1 ( Fig. 8d , e ), accompanied by a reduction in epithelial-to-stromal feedback ( Fig. 8d , Suppl Table S9 ). These shifts suggest a breakdown in stromal signaling and increased ECM remodeling, likely contributing to aberrant epithelial and vascular patterning. Histological and immunofluorescence analyses corroborated the transcriptomic results: Tcf21 -cKO kidneys exhibited reduced and disorganized E-cadherin expression, consistent with impaired maturation of the renal tubular epithelium ( Fig. 8f ). Immunostaining also revealed an expanded perivascular compartment and disorganized Emcn + endothelial structures ( Fig. 8g ), indicative of vascular remodeling and disruption of endothelial-stromal interactions in the absence of Tcf21. Together, these findings suggest that loss of Tcf21 in stromal progenitors induces widespread paracrine dysregulation, impairing epithelial and vascular differentiation during late fetal kidney development. Loss of Tcf21 results in long-term fibrotic remodeling and functional decline in adult kidneys Histological analysis of adult Tcf21 -cKO kidneys revealed lasting structural consequences stemming from embryonic Tcf21 deletion. Masson’s Trichrome and Picrosirius Red staining demonstrated prominent interstitial fibrosis, glomerulosclerosis, and tubular atrophy in mutant kidneys compared to controls ( Fig. 9a , b ). Quantitative image analysis confirmed a significant increase in interstitial collagen deposition in Tcf21 -cKO samples ( Fig. 9c ). Immunofluorescence for fibronectin and CD31 further revealed extensive extracellular matrix accumulation and disrupted vascular architecture in Tcf21 -cKO kidneys ( Fig. 9d , e ). Additionally, mutant samples exhibited reduced glomerular number ( Fig. 9f ), and total kidney surface area was lower ( Fig. 9g ), reflecting a global defect in kidney growth and morphogenesis consistent with a hypodysplastic phenotype. Correspondingly, serum creatinine levels were significantly elevated in Tcf21 -cKO mice ( Fig. 9h ), indicating impaired renal function. Importantly, immunostaining of adult kidneys confirmed the persistence of the aberrant Emcn + stromal population originally identified at E14.5 and E18.5. These Emcn + cells continued to co-express stromal markers such as Meis1/2/3 ( Fig. 9i ) and were spatially associated with fibrotic and vascular lesions, suggesting that they contribute to long-term ECM remodeling and vascular dysfunction. Together, these findings establish Tcf21 as a critical regulator of kidney development and homeostasis. Its loss in stromal progenitors leads to persistent mis-specification of stromal fate, resulting in progressive fibrosis, impaired vascular organization, and decline in renal function into adulthood. Figure 9. Long-term structural and functional impact of Tcf21 loss. Open in a new tab (a,b) Representative histological images of adult kidneys from control and Foxd1 Cre /+ ;Tcf21 f/f mice stained with Masson’s Trichrome (a) and Picrosirius Red (b). Control kidneys show preserved cortical and medullary architecture, well-defined glomeruli, and minimal interstitial collagen deposition. In contrast, Tcf21 -cKO kidneys exhibit tubular dilation, glomerulosclerosis, and marked interstitial fibrosis, with extensive collagen accumulation particularly around glomeruli and vessels. High-power images highlight fibrotic bands separating tubules and encasing glomeruli. Scale bars, 500 μm (low magnification), 100 μm (high magnification). (c) Quantification of interstitial collagen-positive area in Picrosirius Red-stained sections reveals a significant increase in Tcf21 -cKO kidneys ( p = 0.0172). (d) Representative IF images of adult kidneys stained for CD31 (green, endothelium) and Fibronectin (red, ECM) show minimal fibronectin deposition in controls and marked interstitial ECM expansion in Tcf21 -cKO kidneys. (e) Quantification of fibronectin-positive area demonstrates a significant increase in Tcf21 -cKO kidneys ( p = 0.0047). (f) Quantification of glomerular number in representative kidney mid-sections reveals a significant reduction in Tcf21 -cKO mice ( p = 0.0412). (g) Quantification of kidney surface area shows a trend toward reduction in Tcf21 -cKO mice, though not statistically significant ( p = 0.0850). (h) Serum creatinine concentrations measured by LC-MS/MS are significantly elevated in Tcf21 -cKO mice ( p = 0.0060). (i) IF for Meis1/2/3 (stroma, green) and Emcn (magenta) in adult kidneys demonstrates intact vascular networks in controls and abnormal Emcn expression in interstitial compartments of Tcf21 -cKO kidneys. Scale bars, 100 μm. For histology: n = 3 control kidneys (2 male and 1 female) and n =3 mutant mice (2 male and 1 female). For serum creatinine: n =2 control (1 male and 1 female) and n =3 mutant mice (2 male and 1 female). Bars represent mean ± standard error of mean. Statistical significance determined by unpaired two-tailed t test. DISCUSSION Our findings underscore the critical role of the transcription factor Tcf21 in orchestrating the differentiation trajectories of Foxd1+ stromal progenitors during kidney development. Using scRNA-seq of Foxd1+ cells at E14.5, we identified seven distinct stromal subpopulations with unique gene expression signatures and anatomical affiliations. Deletion of Tcf21 resulted in a profound loss of key stromal derivatives, including medullary/perivascular stroma, collecting duct–associated stroma, nephrogenic zone–associated stroma, and proliferating stroma, demonstrating that Tcf21 is essential for the emergence of these lineages. This was accompanied by the appearance of a novel stromal population defined by expression of Emcn, which is typically restricted to endothelial cells. scATAC-seq analysis at E14.5 revealed that Emcn emerged as the top marker of the mutant-enriched population, with increased chromatin accessibility at the Emcn promoter and a predicted Tcf21 binding motif located 80 bp downstream. While these data do not conclusively demonstrate direct repression, they support a model in which Tcf21 helps maintain chromatin boundaries and prevents premature or ectopic activation of stromal gene programs. The Emcn + stromal population exhibited transcriptional features of multiple stromal lineages, as revealed by pseudotime and gene module analysis, suggesting aberrant or incomplete differentiation. Spatial mapping confirmed the widespread presence of these cells across Tcf21-deficient kidneys, particularly in perivascular regions, and their persistence from E14.5 through adulthood suggests a stable alteration in fate rather than a transient state. Emcn + stromal cells emerged in anatomical regions where Acta2 + medullary stromal cells were localized, supporting the hypothesis that Emcn + cells may arise at the expense of normal stromal derivatives. The stromal compartment is known for its heterogeneity, arising from Foxd1+ progenitors and giving rise to mesangial cells, renin cells, pericytes, and fibroblasts involved in supporting nephron and vascular development 7 , 10 , 30 . Our work adds mechanistic insight to how these diverse fates are specified. Previous studies 23 demonstrated Tcf21’s broad role in medullary stroma formation and associated urinary concentration defect but lacked mechanistic insight into stromal heterogeneity or its structural and functional consequences in the mature kidney. At E18.5, Emcn + cells continued to occupy the majority of the stroma in mutants and co-expressed canonical stromal markers including Meis1/2/3 and Pdgfrb, suggesting retention of stromal identity despite their transcriptional deviation. These cells exhibited enrichment for ECM-related and vascular-associated transcripts (Postn, Ecm1, Sparcl1, Rgs5), consistent with features of fibrotic and perivascular cells. Notably, these genes were also upregulated at E14.5, suggesting that Tcf21 loss programs stromal cells toward a stable maladaptive identity. Transcriptomic analyses of whole E18.5 kidneys confirmed the broader, non–cell-autonomous consequences of stromal dysregulation. Genes related to fibrosis, myogenesis, and ECM remodeling were upregulated, while renal epithelial markers involved in tubular transport and differentiation were downregulated. Inference of ligand-receptor interactions using CellChat revealed that stromal-epithelial and stromal-endothelial signaling was significantly altered, with increased expression of fibrotic ligands (Col4a5, Sema3c) and reduced morphogenic signals (Bmp4, Wnt5a). These changes were corroborated by histological findings, including diminished E-cadherin expression, disrupted endothelial network, and expanded perivascular zones in mutant kidneys. Importantly, the consequences of stromal Tcf21 loss persisted into adulthood. Tcf21 -cKO mice developed progressive interstitial fibrosis, glomerulosclerosis, tubular atrophy, and impaired renal function. Emcn + stromal cells remained abundant and were spatially associated with fibrotic and vascular lesions, suggesting they contribute to chronic kidney damage. These data establish a developmental origin for fibrotic remodeling, highlighting the importance of stromal fate control in long-term kidney health and offering broader insight into mesenchymal cell specification and regenerative therapy. The increase in Ureteric stroma and Differentiating stroma in Tcf21 -cKO kidneys may reflect reduced overall stromal cell numbers or compensatory expansion of populations such as Tbx18 + stroma, which may originate outside the Foxd1 + lineage and are likely not regulated by Tcf21. A similar mechanism may underlie changes in the Differentiating stroma cluster, though its identity and anatomical location remain unclear. Lineage tracing studies could determine whether these populations expand in response to the loss of other stromal cells. This study has several limitations. The use of scRNA-seq to define the role of Tcf21 in kidney development is complicated by the potential inclusion of GFP + non-stromal cells, which may arise from transient Foxd1 expression or autofluorescence overlapping with the GFP channel. To address this, we reclustered stromal cells based on canonical stromal markers (Foxd1, Meis1, Pdgfra), allowing for more accurate identification of bona fide stromal populations. Expression of Foxd1 in NPC has been reported by others 8 , 31 – 34 , likely reflecting a shared transcriptional signature during early nephrogenesis 28 . Although potential disruption of normal Foxd1 expression by the Cre model remains a consideration, pooling mice and including Foxd1-Cre controls mitigates this issue. The limited cell numbers obtained from single embryos necessitated pooling, and uneven sex representation among biological replicates, due to Mendelian distribution, posed additional challenges. We sequenced male and female embryos separately and then sub-sampled to match the smallest genotype/sex group before comparing genotypes, in order to minimize sex-related bias. The variation in the number of replicates per sex and genotype reflects biological and logistical constraints: due to Mendelian ratios, it is uncommon to obtain multiple embryos of the same sex and genotype from a single litter. Additionally, our requirement to process all samples on the same day further limited the feasibility of increasing biological replicates. However, both female and male samples were included in all scRNA-seq, sq-ATAC-seq, RNA-seq, and in immunostaining validation. Consistent findings across sexes helped address this limitation. Supplementary Material Please refer to the following link for all Supplementary Methods, Supplementary Figures, and Supplementary Tables: 10.18131/8sh31-6cz40 TRANSLATIONAL STATEMENT. This study identifies Tcf21 as a pivotal regulator of kidney stromal cell fate, revealing that its loss disrupts the emergence of key stromal cell types and leads to the expansion of a dysregulated, Emcn-expressing stromal population. Using a multimodal approach combining single-cell transcriptomics, chromatin accessibility profiling, and long-term histological analysis, we demonstrate that this misdifferentiation contributes to progressive fibrosis, vascular remodeling, and impaired kidney function in adulthood. These findings uncover a developmental origin for chronic kidney disease and suggest that targeting stromal fate specification pathways may hold therapeutic potential for congenital and fibrotic kidney disorders. Our work also informs strategies to support ex vivo kidney cell growth and lineage differentiation by highlighting the importance of recapitulating stromal complexity to more accurately model developmental and disease processes. ACKNOWLEDGMENTS This work was supported by the Northwestern University Robert H Lurie Comprehensive Cancer Center Core Facilities, including Flow Cytometry Core, Mouse Histology and Phenotyping Laboratory (MHPL) and NUSeq Core. Microscopy was provided by the Center for Advanced Microscopy of Northwetsern University and by the Microscopy and Histology Group at Stanley Manne Children’s Research Institute affiliated with Ann and Robert H. Lurie Children’s Hospital of Chicago. We thank Dr. Matthew Schipma and Ms. Wai at NUSeq, Mr. Mehl at flow cytometry, Ms. Acar at MHPL for technical supports. We also thank Dr. Benjamin Thomson and other members of the Quaggin lab for helpful discussions. This research was supported in part through the computational resources and staff contributions provided by the Genomics Compute Cluster which is jointly supported by the Feinberg School of Medicine, the Center for Genetic Medicine, and Feinberg’s Department of Biochemistry and Molecular Genetics, the Office of the Provost, the Office for Research, and Northwestern Information Technology. The Genomics Compute Cluster is part of Quest, Northwestern University’s high performance computing facility, with the purpose to advance research in genomics. With gratitude to the Zell Family Foundation. GRANTS This work was supported by NIH/NIDDK (K08 DK118180–01A1, G.F.) and NIH/NIDDK U54DK137516, S.E.Q.), an NIH/NEI funded program (R01 EY025799–05, S.E.Q.) The Winter Lab is supported by the American Heart Association (AHA: 18CDA34110224), the American Thoracic Society (ATS), and the NIH (R01 AI163742; R01AR080513; R21AR080351; R01AR075423). Footnotes DISCLOSURES Susan E. Quaggin holds patents related to therapeutic targeting of the ANGPT-TEK pathway in ocular hypertension and glaucoma and owns stock in and is a director of Mannin Research. S. Quaggin also receives consulting fees from AstraZeneca, Janssen, the Lowy Medical Research Foundation, Roche/Genentech, Novartis, and Pfizer and is a scientific advisor or member of AstraZeneca, Genentech/Roche, JCI, the Karolinska CVRM Institute, the Lowy Medical Research Institute, Mannin, Novartis and Goldilocks. Deborah R. Winter received consulting fees from Pfizer during the duration of this project. Maria Luisa S. Sequeira-Lopez is an associate editor of AJP Renal Physiology and was not involved and did not have access to information regarding the peer-review process or final disposition of this article. An alternate editor oversaw the peer-review and decision-making process for this article. All other authors have nothing to disclose. DATA SHARING STATEMENT Data will be uploaded to the Gene Expression Omnibus upon acceptance of the manuscript. REFERENCES 1. Lawlor KT, Vanslambrouck JM, Higgins JW, Chambon A, Bishard K, Arndt D, et al. Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation. Nat Mater. 20(2):260–71,2021. 10.1038/s41563-020-00853-9. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. Dorison A, Forbes TA, Little MH. What can we learn from kidney organoids? Kidney Int. 102(5):1013–29,2022. 10.1016/j.kint.2022.06.032. [ DOI ] [ PubMed ] [ Google Scholar ] 3. Walker KA, Bertram JF. Kidney development: core curriculum 2011. Am J Kidney Dis. 57(6):948–58,2011. 10.1053/j.ajkd.2011.03.009. [ DOI ] [ PubMed ] [ Google Scholar ] 4. Merlet-Benichou C Influence of fetal environment on kidney development. Int J Dev Biol. 43(5):453–6,1999. [ PubMed ] [ Google Scholar ] 5. Little MH, McMahon AP. Mammalian kidney development: principles, progress, and projections. Cold Spring Harb Perspect Biol. 4(5)2012. 10.1101/cshperspect.a008300. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 6. McMahon AP. Development of the Mammalian Kidney. Curr Top Dev Biol. 117:31–64,2016. 10.1016/bs.ctdb.2015.10.010. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 7. Sequeira-Lopez ML, Lin EE, Li M, Hu Y, Sigmund CD, Gomez RA. The earliest metanephric arteriolar progenitors and their role in kidney vascular development. Am J Physiol Regul Integr Comp Physiol. 308(2):R138–49,2015. 10.1152/ajpregu.00428.2014. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 8. Kobayashi A, Mugford JW, Krautzberger AM, Naiman N, Liao J, McMahon AP. Identification of a multipotent self-renewing stromal progenitor population during mammalian kidney organogenesis. Stem Cell Reports. 3(4):650–62,2014. 10.1016/j.stemcr.2014.08.008. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 9. Little MH, Brennan J, Georgas K, Davies JA, Davidson DR, Baldock RA, et al. A high-resolution anatomical ontology of the developing murine genitourinary tract. Gene Expr Patterns. 7(6):680–99,2007. 10.1016/j.modgep.2007.03.002. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 10. England AR, Chaney CP, Das A, Patel M, Malewska A, Armendariz D, et al. Identification and characterization of cellular heterogeneity within the developing renal interstitium. Development. 147(15)2020. 10.1242/dev.190108. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 11. Airik R, Bussen M, Singh MK, Petry M, Kispert A. Tbx18 regulates the development of the ureteral mesenchyme. J Clin Invest. 116(3):663–74,2006. 10.1172/JCI26027. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 12. Drake KA, Chaney CP, Das A, Roy P, Kwartler CS, Rakheja D, et al. Stromal beta-catenin activation impacts nephron progenitor differentiation in the developing kidney and may contribute to Wilms tumor. Development. 147(21)2020. 10.1242/dev.189597. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 13. Fetting JL, Guay JA, Karolak MJ, Iozzo RV, Adams DC, Maridas DE, et al. FOXD1 promotes nephron progenitor differentiation by repressing decorin in the embryonic kidney. Development. 141(1):17–27,2014. 10.1242/dev.089078. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 14. Levinson RS, Batourina E, Choi C, Vorontchikhina M, Kitajewski J, Mendelsohn CL. Foxd1-dependent signals control cellularity in the renal capsule, a structure required for normal renal development. Development. 132(3):529–39,2005. 10.1242/dev.01604. [ DOI ] [ PubMed ] [ Google Scholar ] 15. Yallowitz AR, Hrycaj SM, Short KM, Smyth IM, Wellik DM. Hox10 genes function in kidney development in the differentiation and integration of the cortical stroma. PLoS One. 6(8):e23410,2011. 10.1371/journal.pone.0023410. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 16. Lotfi CFP, Passaia BS, Kremer JL. Role of the bHLH transcription factor TCF21 in development and tumorigenesis. Braz J Med Biol Res. 54(5):e10637,2021. 10.1590/1414-431X202010637. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 17. Quaggin SE, Vanden Heuvel GB, Igarashi P. Pod-1, a mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mech Dev. 71(1–2):37–48,1998. 10.1016/s0925-4773(97)00201-3. [ DOI ] [ PubMed ] [ Google Scholar ] 18. Robb L, Mifsud L, Hartley L, Biben C, Copeland NG, Gilbert DJ, et al. epicardin: A novel basic helix-loop-helix transcription factor gene expressed in epicardium, branchial arch myoblasts, and mesenchyme of developing lung, gut, kidney, and gonads. Dev Dyn. 213(1):105–13,1998. 10.1002/(SICI)1097-0177(199809)213:1<105::AID-AJA10>3.0.CO;2-1. [ DOI ] [ PubMed ] [ Google Scholar ] 19. Lu J, Richardson JA, Olson EN. Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs. Mech Dev. 73(1):23–32,1998. 10.1016/s0925-4773(98)00030-6. [ DOI ] [ PubMed ] [ Google Scholar ] 20. Hidai H, Bardales R, Goodwin R, Quertermous T, Quertermous EE. Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries. Mech Dev. 73(1):33–43,1998. 10.1016/s0925-4773(98)00031-8. [ DOI ] [ PubMed ] [ Google Scholar ] 21. Nagao M, Lyu Q, Zhao Q, Wirka RC, Bagga J, Nguyen T, et al. Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway. Circ Res. 126(4):517–29,2020. 10.1161/CIRCRESAHA.119.315968. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 22. Wirka RC, Wagh D, Paik DT, Pjanic M, Nguyen T, Miller CL, et al. Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis. Nat Med. 25(8):1280–9,2019. 10.1038/s41591-019-0512-5. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 23. Finer G, Maezawa Y, Ide S, Onay T, Souma T, Scott R, et al. Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/beta-Catenin Signaling. Kidney360. 3(7):1228–41,2022. 10.34067/KID.0005572021. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 24. Quaggin SE, Schwartz L, Cui S, Igarashi P, Deimling J, Post M, et al. The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development. 126(24):5771–83,1999. [ DOI ] [ PubMed ] [ Google Scholar ] 25. Adam M, Potter AS, Potter SS. Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development. Development. 144(19):3625–32,2017. 10.1242/dev.151142. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 26. Naba A, Clauser KR, Ding H, Whittaker CA, Carr SA, Hynes RO. The extracellular matrix: Tools and insights for the “omics” era. Matrix Biol. 49:10–24,2016. 10.1016/j.matbio.2015.06.003. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 27. Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan CH, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 12(1):1088,2021. 10.1038/s41467-021-21246-9. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 28. Combes AN, Phipson B, Lawlor KT, Dorison A, Patrick R, Zappia L, et al. Single cell analysis of the developing mouse kidney provides deeper insight into marker gene expression and ligand-receptor crosstalk. Development. 146(12)2019. 10.1242/dev.178673. [ DOI ] [ PubMed ] [ Google Scholar ] 29. Bohnenpoll T, Bettenhausen E, Weiss AC, Foik AB, Trowe MO, Blank P, et al. Tbx18 expression demarcates multipotent precursor populations in the developing urogenital system but is exclusively required within the ureteric mesenchymal lineage to suppress a renal stromal fate. Dev Biol. 380(1):25–36,2013. 10.1016/j.ydbio.2013.04.036. [ DOI ] [ PubMed ] [ Google Scholar ] 30. Lin EE, Sequeira-Lopez ML, Gomez RA. RBP-J in FOXD1+ renal stromal progenitors is crucial for the proper development and assembly of the kidney vasculature and glomerular mesangial cells. Am J Physiol Renal Physiol. 306(2):F249–58,2014. 10.1152/ajprenal.00313.2013. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 31. Mugford JW, Sipila P, McMahon JA, McMahon AP. Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney. Dev Biol. 324(1):88–98,2008. 10.1016/j.ydbio.2008.09.010. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 32. Naiman N, Fujioka K, Fujino M, Valerius MT, Potter SS, McMahon AP, et al. Repression of Interstitial Identity in Nephron Progenitor Cells by Pax2 Establishes the Nephron-Interstitium Boundary during Kidney Development. Dev Cell. 41(4):349–65 e3,2017. 10.1016/j.devcel.2017.04.022. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 33. Brunskill EW, Park JS, Chung E, Chen F, Magella B, Potter SS. Single cell dissection of early kidney development: multilineage priming. Development. 141(15):3093–101,2014. 10.1242/dev.110601. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 34. Magella B, Adam M, Potter AS, Venkatasubramanian M, Chetal K, Hay SB, et al. Cross-platform single cell analysis of kidney development shows stromal cells express Gdnf. Dev Biol. 434(1):36–47,2018. 10.1016/j.ydbio.2017.11.006. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement Data will be uploaded to the Gene Expression Omnibus upon acceptance of the manuscript. 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