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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2022 Sep;33(9):1694–1707. doi: 10.1681/ASN.2021121559

Transcription Factors YAP/TAZ and SRF Cooperate To Specify Renal Myofibroblasts in the Developing Mouse Kidney

Keri A Drake 1, Christopher Chaney 2,3, Mohita Patel 1, Amrita Das 2,3, Julia Bittencourt 4, Martin Cohn 4, Thomas J Carroll 2,3,✉
PMCID: PMC9529188  PMID: 35918150

Significance Statement

Embryonic renal interstitial cells give rise to multiple cell types in the adult, including fibroblasts, myofibroblasts, mural cells, and smooth muscle. How the different cell types arise from a multipotent progenitor is unknown. In this study, the authors identified a subpopulation of stromal cells in mouse embryonic kidneys with enriched activity in the transcriptional regulators YAP and TAZ, and show that YAP/TAZ and the transcriptional regulator SRF have independent and codependent roles in the specification of unique subsets of interstitial cells. These findings offer insights into the role of the interstitium in kidney development and may inform efforts aimed at regenerating renal tissue and may aid efforts to understand the drivers of kidney fibrosis.

Keywords: Hippo/Warts, pericyte, fibroblast heterogeneity, stromal microenvironment, kidney development, myofibroblasts

Visual Abstract

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Abstract

Background

The embryonic renal stroma consists of multiple molecularly distinct cell subpopulations, the functional significance of which is largely unknown. Previous work has demonstrated that the transcription factors YAP and TAZ play roles in the development and morphogenesis of the nephrons, collecting ducts, and nephron progenitor cells.

Methods

In embryonic mouse kidneys, we identified a subpopulation of stromal cells with enriched activity in YAP and TAZ. To evaluate the function of these cell types, we genetically ablated both Yap and Taz from the stromal progenitor population and examined how gene activity and development of YAP/TAZ mutant kidneys are affected over a developmental time course.

Results

We found that YAP and TAZ are active in a subset of renal interstitium and that stromal-specific coablation of YAP/TAZ disrupts cortical fibroblast, pericyte, and myofibroblast development, with secondary effects on peritubular capillary differentiation. We also demonstrated that the transcription factor SRF cooperates with YAP/TAZ to drive expression of at least a subset of renal myofibroblast target genes and to specify myofibroblasts but not cortical fibroblasts or pericytes.

Conclusions

These findings reveal a critical role for YAP/TAZ in specific embryonic stromal cells and suggest that interaction with cofactors, such as SRF, influence the expression of cell type–specific target genes, thus driving stromal heterogeneity. Further, this work reveals functional roles for renal stroma heterogeneity in creating unique microenvironments that influence the differentiation and maintenance of the renal parenchyma.

The renal stroma (or interstitium) includes fibroblasts, vascular mural cells (vascular smooth muscle, pericytes, and mesangial cells), myofibroblasts, and smooth muscle.1–3 Stromal cells actively signal neighboring cell populations and regulate multiple aspects of renal development, including nephron progenitor cell maintenance/differentiation,4,5 ureteric bud branching,6–8 and arterial patterning5,9 and are also the source of renal hormones including erythropoietin10 and renin.11 Given the multitude of functions of these specialized cells, there is considerable interest in understanding mechanisms that drive cellular and molecular diversity of the interstitium.

We recently identified 17 transcriptionally unique interstitial cell clusters in E18.5 mouse kidneys, revealing previously unappreciated heterogeneity and regionalization of the stroma.12 How these different cell types arise is still unclear. Here, we report that transcription factors YAP and TAZ are active in a restricted domain of cells in the cortex of the kidney encompassing cortical pericytes, cortical fibroblasts, and corticomedullary myofibroblasts.

To evaluate the role of YAP/TAZ in these cell types, we coablated both factors using floxed alleles and a Cre line active within the stromal progenitor population (Foxd1cre;Yapc/c;Tazc/c). Whereas YAP/TAZ mutant kidneys are grossly normal in size and develop nephrons with expected segmentation, stromal patterning is severely perturbed, with loss of a subset of cortical pericytes, fibroblasts, and myofibroblasts and with nonautonomous effects on the cortical microvasculature.

Analysis of the transcriptome of YAP/TAZ mutants revealed the loss of several genes previously shown to be targets of serum response factor (SRF). Recent studies suggest that YAP/TAZ and SRF may cooperate in their regulation of transcriptional targets.13–16 Here, using loss and gain of function mutant analysis, we show that SRF cooperates with YAP/TAZ in differentiation of the myofibroblast population but not the cortical pericyte or fibroblast populations.

Together, our findings reveal how YAP/TAZ and SRF cooperate to pattern the renal stroma and reveal functional significance to a previously unappreciated stromal cell subtype. These findings will affect our understanding of renal development and disease and guide efforts to engineer renal replacement tissue that possesses the full complement of physiologically relevant cell types.

Methods

Animal Models

All mice were bred on a mixed genetic background. Foxd1cre transgenic mice were bred with mice carrying loxP-flanked Yap (Yapflox/flox or Yapc/c), Taz/Wwtr1 (Tazflox/flox or Tazc/c), Srf (Srfflox/flox or Srfc/c), Lats1 (Lats1flox/flox or Lats1c/c), and Lats2 (Lats2flox/flox or Lats2c/c) alleles as described in the supplemental methods (Supplemental Table 1). Timed matings were generated, with day of plug counted as embryonic day (E) 0.5. Pregnant females were euthanized at various gestational time points. Lineage tracing experiments were performed by crossing Rosa26YFP reporter mice with the above mouse lines. Mice with the desired genotype were randomly selected regardless of sex with Cre-negative littermates used as controls. All animals were housed, maintained, and used according to National Institutes of Health and Institutional Animal Care and Use Committees approved protocols at the University of Texas Southwestern Medical Center (Office of Laboratory Animal Welfare Assurance Number D16-00296).

Histologic Analysis

Embryonic kidney tissue was fixed in 4% paraformaldehyde, washed with PBS, dehydrated to 50% alcohol, embedded in paraffin, sectioned into 5-μm slices, and subjected to hematoxylin and eosin staining.

Immunofluorescence on Paraffin and/or Optimal Cutting Temperature Sections

Embryonic tissue was fixed in 4% paraformaldehyde, either embedded in optimal cutting temperature (OCT) media and cryosectioned to 10-μm slices or embedded in paraffin and sectioned into 5-μm slices, and subjected to immunofluorescence (IF). Slides for IF were immersed and boiled with either 10 mM sodium citrate or Tris-EDTA antigen retrieval buffer and blocked with a solution of 5% FBS/PBS for 1 hour at room temperature followed by the application of primary antibodies (as provided in the supplemental methods, Supplemental Table 1) diluted in blocking solution.

In Situ Hybridization on OCT Sections

For in situ hybridization (ISH) assays, tissue was fixed with 4% paraformaldehyde, cryoprotected with 30% sucrose, embedded in OCT medium (TissueTek), and sectioned into 10-μm slices. ISH was performed as previously described.17 After color reaction, slides were fixed with 4% PFA and mounted using Permount.

Nano-Computed Tomography Imaging

Embryonic kidneys at E18.5 were isolated, fixed overnight in 4% PFA, and stored in PBS before preparation for nano-computed tomography (CT) using Lugol’s iodine solution (1.25% I2, 2.5% KI, with DEPC water) at 4°C for contrast enhancement before scanning, as previously described.18 Excess iodine solution was pipetted off the specimen, and the specimens were washed in double-deionized water and wiped off with a Kimwipes wipe. Embryos were placed in a small plastic capsule to prevent desiccation during scanning. Kidneys were scanned in a GE V|TOME|X M 240 Nano CT scanner (General Electric) at the University of Florida Nanoscale Research Facility. TIFF stacks and 3D reconstruction/volume processing were generated using Phoenix Datos|x 2 and sample segmentation/manipulation was done using VG Studio Max.

RNA Sequencing

Single-cell RNA sequencing (RNA-seq) of enriched Foxd1-derived interstitial cells from E18.5 kidneys was previously performed.12 This dataset was utilized to perform gene regulatory network reconstruction and measurement of regulon activity using SCENIC.19 Briefly, the regulon activity was binarized using the strategy to model the regulon’s activity as a mixture of two normal distributions using the mixtools20 R package. If such a model could be fit using expectation maximization, then those cells for which the regulon activity were assigned higher probability by the distribution with the greater mean were assigned a binarized regulon activity of one and zero otherwise. If a two-component mixture of normal distributions could not be fit, then a beta distribution was fit to the regulon’s activity and cells for which the regulon’s activity was greater than one mean absolute deviation above the mean were assigned a binarized regulon activity of one and zero otherwise. Additionally, RNA-seq was performed on mouse kidneys (E15.5 whole kidneys; three Cre-negative controls and three Foxd1cre;Yapc/c;Tazc/c mutants). RNA was isolated from dissected kidneys stored in RNAlater solution. RNA-seq was performed using paired-end 100 bp with a minimum of 20 million reads per sample. Transcript abundance was estimated without aligning reads using Salmon21 against an index of coding sequences from the Ensembl GRCm38 assembly. Transcript-level abundance was imported and count and offset matrices generated using the tximport R/Bioconductor package.22 Differential expression analysis was performed using the DESeq2 R/Bioconductor package.23 Gene sets for enrichment analysis to assign identities to clusters 6–9 were constructed from the union of GeneRIF Biologic Term Annotations24 and the TISSUES Curated Tissue Protein Expression Evidence Scores and TISSUES Text-Mining Tissue Protein Expression Evidence Scores25 gene sets curated at the Harmonizome database (http://amp.pharm.mssm.edu/Harmonizome/) that matched the search terms “fibroblast,” “myofibroblast,” and “pericyte.” The gene sets were filtered to include only those genes with a standardized value >1 for the text-mining datasets. A set representing genes lost on ablation of Yap and Taz was created by identifying all genes with a log fold change less than −0.2 at a false discovery rate <0.05. Gene ontology of downregulated genes (Supplemental Table 1) in Foxd1cre;Yapc/c;Tazc/c mutants versus control kidneys was analyzed using ToppGene (https://toppgene.cchmc.org; P value Bonferroni correction). Gene set enrichment analysis was performed using the fgsea26 Bioconductor package. Over-representation analysis of transcription factor binding sites was performed using the clusterProfiler27 Bioconductor package with the C3:TFT gene sets from MSigDB.28,29

Statistical Analyses

Data presented in figures are representative examples from one of at least three different experiments on at least three different embryos/organs. No significant variability was noted in tissues of the same genotype; all animals with correct genotypes were included in the analysis. Bioinformatic statistics on single-cell RNA-seq data were carried out as described above. Morphometric analyses were carried out using ImageJ software. Transverse nano-CT whole kidney scans were used to identify the image showing the maximal transverse length of the kidney and included a scale bar used for calibration of each individual image. The total kidney length (from tip of the papilla to outer cortex) and the papilla length (from tip of the papilla to start of the corticomedullary zone) were measured. The percentage of interstitial space was quantified in ImageJ using color thresholding of nano-CT images to identify “black/low intensity” areas reported as a percentage of total kidney area. A similar technique was used to quantify the stromal marker expression by IF using color thresholding to label antibody signal in ImageJ, with this measured area divided by kidney section area on the image (subtracting out any glomerular staining), with three images analyzed per reported measure. One-way ANOVA with Tukey’s test was used for both the nano-CT and IF quantification. Two-tailed t test was used to quantify apoptosis by determining the number of cleaved caspase 3–positive cells divided by the total number of stromal cells per high power field. In all analyses, P<0.05 was considered statistically significant.

Results

YAP and TAZ Are Active in a Subset of Renal Interstitium

To identify transcription factors active within distinct stromal cell types, we evaluated previously generated single-cell RNA-seq data from isolated E18.5 stromal cells.12 Gene set enrichment and regulon analysis comparing transcription factor activity among the different cell clusters identified a subset of stromal clusters (6–9) with highly enriched expression of YAP/TAZ target genes (Figure 1). Genes within these clusters show increased expression in the cortical stroma adjacent to proximal tubules (clusters 6–8), and in the corticomedullary stroma (cluster 9) just medullary to the proximal tubule region.12 To clarify the cellular identity of the cells in clusters 6–9, we used a computational comparison of genes differentially expressed in the distinct clusters with genes expressed in various stromal gene sets including fibroblasts, mesangial cells, myofibroblasts, smooth muscle, pericytes, and vascular smooth muscle. Based on normalized enrichment scores, cluster 6 is annotated as pericyte, cluster 7 as fibroblast, and clusters 8 and 9 as myofibroblasts (Supplemental Figure 1). It is important to note that clusters 7 and 8 are not as well distinguished as 6 and 9, suggesting they may still be in the process of differentiating.

Figure 1.

Figure 1.

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YAP/TAZ are transcriptionally active in the cortical and corticomedullary stroma of the developing kidney. (A–C) Regulon analysis of single cell RNA-seq of isolated stromal cells (A and B), with YAP/TAZ activity represented by black circles (A) or lines (C). (C) Upregulated YAP/TAZ activity corresponds to clusters 6–9 localized to the cortical/corticomedullary stroma based on expression of validated anchor genes as previously published.12 (D) Phosphorylated-YAP (green) localizes to the nephrogenic zone (NZ) with minimal expression in the cortical/medullary stroma. (E) Total YAP (green) shows strong nuclear staining in the stroma of the cortical and medullary zones but not the NZ, with arrows showing stromal cells labeled with MEIS1/2/3 (red) and tubules labeled with ECAD (E-cadherin; purple). Scale bar = 100 μm.

To determine whether YAP was active in these cells, we performed IF staining with antibodies to YAP and other renal cell types. Although YAP protein appears broadly present in the renal stroma, in the nephrogenic zone it is predominantly cytoplasmic (Figure 1D). However, YAP localization abruptly shifts to nuclear in stroma adjacent to the proximal tubules and more medullary populations (Figure 1E) encompassing the presumptive cortical fibroblast, pericyte, and myofibroblast populations.

Stromal-Specific Inactivation of YAP/TAZ Disrupts Normal Renal Development

Yap and Taz were ablated individually and in tandem from the renal stroma using Foxd1cre, which targets a progenitor population giving rise to the majority of the renal interstitium.1 Foxd1cre;Yapc/c;Tazc/c mutants die within 24 hours of birth. Although grossly similar in size, mutant kidneys are distinguishable from littermate controls at birth by the appearance of large transparent regions when illuminated with transmitted light. Large empty spaces in the renal cortex adjacent to the proximal tubules were confirmed by both histology and nano-CT on intact kidneys (Figure 2, A and B). Nano-CT was used to quantify kidney morphology and interstitial spaces (Figure 2C). Further characterization showed that loss of Yap or Taz alone (Foxd1cre;Yapc/c or Foxd1cre;Tazc/c) or loss of both alleles of Taz and a single copy of Yap (Foxd1cre;Yapc/+;Tazc/c) had no detectable effect. However, deletion of both alleles of Yap and a single allele of Taz (Foxd1cre;Yapc/c;Tazc/+) led to histologic defects that are similar, but less severe, than double mutants (Supplemental Figure 2). These findings suggest that Yap and Taz have redundant, dosage-sensitive roles within the renal interstitium. All subsequent analyses have been performed on double mutants.

Figure 2.

Figure 2.

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Stromal-specific inactivation of YAP/TAZ disrupts normal kidney development. (A) Hematoxylin and eosin–stained sections of E18.5 control and Foxd1cre;Yapc/c;Tazc/c mutants. Asterisks indicate interstitial spaces. Arrows indicate expanded cortical interstitial space. Scale bar = 100 μm. (B) Nano-CT of wild-type and mutant E18.5 whole kidneys showing single optical slices through the papilla in gray and 3D reconstructions from stacked images with low-density regions colored in blue, medium-density in red, and high-density in green. Scale bar = 0.4 mm. (C) Morphometric quantification of traverse sections of nano-CT images shows increased interstitial space (quantified as percentage of measured area of interstitial space divided by total kidney area) in Foxd1cre;Yapc/c;Tazc/c mutant kidneys (P=0.001). SRF mutant kidneys showed an increase in interstitial space from control (P=0.003) but were less affected than YAP/TAZ mutants (P=0.003). No differences in kidney size were found (measured as the total length from the papilla to outer cortex) with SRF mutants trending smaller than control and YAP/TAZ kidneys (P=0.12 and 0.14, respectively). Papillary length of both YAP/TAZ and SRF mutants showed a trend toward shortened papilla, but was not significantly different from controls (P=0.13 and P=0.07, respectively).

To determine the etiology of the defects, we analyzed YAP/TAZ mutants over a developmental time course. Before E15.5, there were no obvious histologic defects in mutants. At E15.5, coinciding with the initiation of renal filtration, an expansion of interstitial cell space was observed in the cortex of mutant kidneys, although no interstitial cavities were evident. By E16.5, interstitial cavities were clearly evident (Supplemental Figure 2).

To determine whether the interstitial spaces represented epithelial or vascular cysts, we performed IF on mutant kidneys. As shown in Supplemental Figure 3, the spaces were not lined by markers of the nephron epithelia (proximal tubule, loop of Henle, distal tubule, or collecting duct), vasculature (general endothelia, arteries/arterioles), or lymphatics. These immunoassays suggest normal formation of the above-mentioned cell types.

To determine whether the interstitial cavities resulted from stromal cell death, we stained mutants with antibodies to cleaved caspase 3 at E15.5, a time point just before the first appearance of the cavities at E16.5 (Supplemental Figure 4). There was no significant difference in the rate of apoptosis between mutants and controls. To confirm the presence of stromal cells in this region, we crossed the Rosa26YFP lineage tracer into the Foxd1cre;Yapc/c;Tazc/c mutant background. Analysis of YFP expression showed Foxd1cre derivatives interspersed with endothelial cells surrounding the interstitial spaces (Figure 3A).

Figure 3.

Figure 3.

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Stromal-specific YAP/TAZ mutant kidneys develop interstitial defects and lack differentiated myofibroblast cells normally present in the corticomedullary stroma. (A) The interstitium normally comprises stromal and endothelial cells in close contact with epithelial tubules. YAP/TAZ mutant kidneys develop interstitial spaces, not lined by a contiguous layer of any one cell type but instead by interspersed stromal lineage positive cells (identified by lineage tracing of Foxd1cre; green; arrowhead) and endothelial cells (labeled with endomucin; red; arrow). (B) Foxd1cre;Yapc/c;Tazc/c mutant kidneys show decreased expression of alpha–smooth muscle actin (aSMA), transgelin (SM22a), and myosin light chain 9 (MYL9) in the cortical/corticomedullary stroma, with intact expression in the vascular smooth muscle surrounding large vessels (arrow; inserts). Scale bar = 100 μm.

We next sought to determine whether the spaces were lined with Foxd1cre-derived myofibroblast cells similar to the phenotype described in Angpt1/2 mutants.30 Mutant kidneys were stained with antibodies to aSMA, SM22a, and MYL9. Analysis revealed that not only were the spaces in YAP/TAZ mutants not lined by cells expressing these markers but, in fact, there was a significant reduction in the number of cells expressing these proteins in the cortical interstitium of mutants (Figure 3B). Importantly, vascular smooth muscle cells showed normal expression of these markers (Figure 3B, insets, and Supplemental Figure 3).

Ablation of YAP/TAZ Leads to Defects in Cortical Stroma Formation

To gain insight into the cause of the YAP/TAZ defect, we performed whole kidney RNA-seq at E15.5, a time point just before the appearance of the interstitial edema (Supplemental Figure 2). We identified 643 genes that were differentially expressed at a log2 fold change >0.2 and an adjusted P≥0.05 (Supplemental Table 1), with the top 25 upregulated and top 25 downregulated shown by heatmap (Figure 4A). Comparison of the YAP/TAZ mutant transcriptome to our single-cell analysis confirmed that the vast majority of genes with significantly decreased expression in YAP/TAZ mutants are normally enriched in the myofibroblast (8 and 9), cortical pericyte (6), and fibroblast (7) populations (Supplemental Figure 1A).

Figure 4.

Figure 4.

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Ablation of YAP/TAZ results in loss of cortical stromal cell types and expansion of the medullary stroma. (A) Whole kidney RNA-seq identified over 600 differentially expressed genes at a log2 fold change >0.2 and an adjusted P≥0.05, with the top 25 upregulated and top 25 downregulated shown by heatmap. (B) In comparison to controls, stromal-specific YAP/TAZ mutants show altered patterning of the cortical/corticomedullary stroma. Pleiotrophin (Ptn) expression appears unaffected in the nephrogenic zone. However, other regionally specific stromal markers, including calcium-activated chloride channel isoform (Clca3a1), lysyl oxidase (Lox), SPARC related modular calcium binding 2 (Smoc2), and claudin 11 (Cldn11) are lost, whereas markers of the medullary stroma, including α chain of type XV collagen (Col15a1) and APC downregulated 1 (Apcdd1), appear to be expanded into the cortex. Scale bar = 100 μm. (C) Gene ontology analysis performed using ToppGene (https://toppgene.cchmc.org) shows decreased expression of genes involved in extracellular matrix organization/structure and circulatory/vascular development.

To validate this observation, we performed ISH with several genes whose expression was significantly decreased in mutants according to RNA-seq. Genes that are differentially expressed in cluster 7 (Clca3a1), clusters 6–8 (Lox), cluster 9 (Smoc2), and clusters 8 and 9 (Cldn11) all showed significantly reduced/absent expression in YAP/TAZ mutants (Figure 4B). In contrast, but in agreement with RNA-seq data, genes normally expressed in the stroma that lies more cortical (clusters 4 and 5) or medullary (clusters 10–12) to clusters 6–9 showed either no effect (e.g., Ptn) or expanded expression (e.g., Apcdd1 and Col15a1). These findings strongly suggest that YAP and TAZ are necessary for the specification of a subset of the renal stroma represented by single-cell clusters 6–9.

YAP/TAZ Mutants Develop Defects in Differentiation of the Cortical Pericytes and Peritubular Capillaries

In addition to the observed defects in myofibroblast formation, gene ontology analysis of our RNA-seq data (Figure 4C) identified a significant effect on vascular/circulatory system development in YAP/TAZ mutants (P<0.001). Expression of multiple angiogenic factors, including Vegfd and Angpt2, along with other established pericyte markers, including Ren1, Ace2, Thbs2, Hey2, Rgs5, Anxa2, Nrp1, and Pdgfrb, were significantly reduced in mutants (Supplemental File 1). We used antibody staining to validate the expression of several of these targets in YAP/TAZ mutants, which show significantly reduced stromal expression of PDGFRB, NRP1, ANXA2, and THBS231–34 surrounding peritubular capillaries of mutants (Figure 5 and Supplemental Figure 5). Importantly, expression of these factors was not significantly affected in other mural cell populations, including mesangial cells and vascular smooth muscle cells (Figure 5, asterisks) or medullary pericytes (Supplemental Figure 6).

Figure 5.

Figure 5.

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YAP/TAZ stromal mutant kidneys show defects in pericyte and peritubular capillary development. (A–D) Several known pericyte markers, including platelet-derived growth factor receptor beta (PDGFRB), neuropilin 1 (NRP1, expressed in both the cortical stroma and developing vasculature), annexin A2 (ANXA2), and thrombospondin 2 (THBS2) are decreased in the cortical/corticomedullary stroma of YAP/TAZ mutants, with preserved expression in mesangial cells/glomeruli (asterisks). (E and F) Endothelial cells of the cortical microvasculature (i.e., peritubular capillaries) labeled by endomucin (ENDO, red) and VE cadherin (VECAD, red) show decreased expression of VEGFR2 (E) and PLVAP (F) in YAP/TAZ mutant kidneys. Scale bar = 100 μm.

Although pericytes are mainly studied in regard to vascular physiology, they have been shown to have profound effects on the development/differentiation of endothelia.35 Given our observation of interstitial edema in YAP/TAZ mutants, we next assessed whether deletion of YAP/TAZ nonautonomously affected the adjacent vasculature. In agreement with our observations that there were no defects in vascular smooth muscle, we did not detect morphologic, histologic, or molecular changes in large blood vessels or lymphatics (Supplemental Figure 3). In contrast, the cortical microvasculature/peritubular capillaries of YAP/TAZ mutant kidneys showed a significant decrease in VEGFR2 and PLVAP despite normal endomucin staining (Figure 5E, Supplemental Figure 3, M and N, and Supplemental Figure 5). Loss of PLVAP staining was of particular interest as this protein marks fenestrated membranes and peritubular capillaries are a known fenestrated endothelia. These findings suggest that deletion of YAP/TAZ from the interstitium affects formation of the cortical pericytes, which nonautonomously affects the differentiation of the fenestrated peritubular capillaries. In addition, it suggests that pathways controlling vascular permeability are affected, potentially explaining the edema observed in mutant kidneys.

SRF Target Genes Are Repressed in YAP/TAZ Mutants

Our observations suggest that YAP/TAZ activity is necessary for the development of a subset of the cortical stroma including pericyte, fibroblast, and myofibroblast subpopulations, whereas it is dispensable for the formation of other stromal cell types including mesangial cells, vascular smooth muscle, and medullary fibroblasts/pericytes. We next asked how these two paralogous transcription factors could regulate the differentiation of multiple different cell types. To test the hypothesis that transcriptional coregulators cooperated with YAP and TAZ in different cell types, we assessed the presence of binding sites for other transcription factors in the enhancers of stromal YAP/TAZ target genes.

Of the top 15 transcription factor motifs enriched among YAP/TAZ stromal target genes (log2 fold change less than −0.2 and false discovery rate <0.05), three were annotated as SRF binding sites (Supplemental Figure 7). A similar analysis of genes whose expression is enriched in clusters 6–9 versus all other stromal cells demonstrated increased representation of SRF binding motifs, especially for the putative myofibroblast population represented by cluster 9 (Supplemental Figure 1, C–F). SRF is a transcription factor known to play multiple critical roles in tissue development and maintenance and previous in vitro studies suggest YAP/TAZ and SRF may cooperate in the activation of target genes.14,16,36–38 SRF is broadly expressed in the renal interstitium (Supplemental Figure 8) and regulon analysis indicates activity in multiple interstitial cell types (Figure 6A).

Figure 6.

Figure 6.

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Loss of SRF partially phenocopies YAP/TAZ stromal-specific mutants. (A) Regulon analysis of SRF shows broad activity throughout stromal subpopulations. (B) Compared with control kidneys, SRF stromal mutants show increased interstitial space. (C) Mutant kidneys also show decreased expression of alpha–smooth muscle actin (aSMA), transgelin (SM22a), and myosin light chain 9 (MYL9), similar to YAP/TAZ mutants. (D and E) However, SRF mutants show maintained expression of Clca3a1, Lox, Smoc2, Cldn11, PDGFRB, NRP1, ANXA2, and THBS2, with peritubular capillaries expressing VEGFR2 and PLVAP, unlike YAP/TAZ mutants in which these stromal/microvasculature markers are lost. Scale bar = 100 μm.

To determine whether SRF was necessary for the expression of YAP/TAZ myofibroblast targets, we generated Foxd1cre;Srfc/c mutants. Similar to YAP/TAZ mutants, Foxd1cre;Srfc/c mutants die shortly after birth. Histologic examination of E18.5 mutants revealed a similar expansion of interstitial spaces, as was observed in YAP/TAZ mutants; however, they did not exhibit the large interstitial cavities that were observed in YAP/TAZ mutants (Figure 6, B and C).

Antibody staining revealed that YAP/TAZ myofibroblast targets aSMA, Tagln/SM22a, and MYL9 were also lost in SRF mutant interstitial cells (Figure 6C and Supplemental Figure 9). However, Smoc2 and Cldn11, YAP/TAZ targets that are expressed in the myofibroblast cell type, were not affected by SRF deletion (Figure 6D). In addition, expression of cortical fibroblast- and pericyte-specific YAP/TAZ targets Clca3a1, Lox, PDGFRB, NRP1, ANXA2, and THBS2 were unaffected (Figure 6, D and E and Supplemental Figure 5, D–G). Expression of endothelial VEGFR2 and PLVAP were also normal, suggesting that SRF deletion did not affect peritubular capillary formation (Figure 6E and Supplemental Figure 5, B and C). Finally, expression of medullary stromal markers, including Apcdd1, was unaffected in SRF mutants (Supplemental Figure 8).

SRF mutants showed additional defects not observed in YAP/TAZ mutants, including abnormal glomeruli, containing aneurysm-like structures (Supplemental Figure 8). This phenotype is consistent with defects in formation of the mesangial cells, a Foxd1cre-derived glomerular stromal cell type that is unaffected by deletion of YAP/TAZ.39–41 Loss of SRF also caused abnormal lymphatic development, with LYVE1 positive vessels traversing the corticomedullary region of the kidney (Supplemental Figure 8).

Our findings suggest that YAP/TAZ and SRF mutants share overlapping defects in myofibroblast population, but have unique/nonoverlapping roles in other stromal populations.

This is in agreement with previous work showing that SRF/MRTF and YAP/TAZ binding sites sit in close proximity in the enhancers of a subset of common target genes. However, it is not clear whether the factors cooperate, are epistatic, or work in parallel to drive gene expression. To determine the relationship of YAP/TAZ and SRF in the renal stroma, we generated kidneys in which LATS1 and 2 had been ablated by Foxd1Cre. LATS1 and 2 phosphorylate YAP and TAZ preventing their nuclear accumulation. Ablation of these kinases is predicted to lead to loss of YAP/TAZ phosphorylation, resulting in their accumulation in the nucleus and a gain-of-function phenotype. Foxd1cre;Lats1c/c;Lats2c/c were not viable beyond E15.5 because of extrarenal defects (not shown). Examination of E15.5 mutant kidneys revealed a severe defect in kidney histology with a near complete loss of nephron structures and the ectopic presence of myofibroblast-like cells in the cortex (Figure 7A). As expected, mutants showed a loss of phosphorylated YAP and ectopic localization of total YAP protein within the nuclei of cortical stroma (Figure 7B). Multiple YAP/TAZ myofibroblast target genes (including aSMA, SM22a, and MYL9) were ectopically and precociously expressed throughout the mutant stroma (Figure 7B).

Figure 7.

Figure 7.

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Activation of YAP in stromal progenitor cells drives ectopic stromal development. (A) Compared with control kidneys, activation of YAP through the deletion of LATS1 and 2 (Foxd1cre;Lats1/2c/c) severely disturbs the nephrogenic zone with the development of ectopic stroma (arrow) that is maintained despite the loss of SRF (Foxd1cre;Lats1/2c/c;Srfc/c). (B) Increased nuclear YAP is confirmed in the developing ectopic stroma (arrows) labeled with a Rosa26YFP reporter (red). Ectopic stroma in mutant kidneys express aSMA, SM22a, and MYL9, with these markers lost after the additional ablation of SRF (Foxd1cre;Lats1/2c/c;Srfc/c). Scale bar = 100 μm.

To determine whether SRF was necessary for the ectopic expression of YAP/TAZ targets in LATS mutants, we analyzed Foxd1cre;Lats1c/c;Lats2c/c;Srfc/c mutant mice. Although triple mutant kidneys showed similar histology to LATS1/2 mutants, they failed to express aSMA, SM22a, and MYL9 (Figure 7B). These data suggest that SRF cooperates with YAP/TAZ, either directly at gene enhancers or epistatically, to drive expression of at least a subset of renal myofibroblast target genes and to specify this cell type.

Discussion

We recently showed that the renal interstitium consists of numerous molecularly distinct cell populations that map to specific anatomic domains along the corticomedullary axis.12 How this heterogeneity arises and what its functional significance is still remains largely unknown. Here, we show that a region of the renal interstitium adjacent and just medullary to the proximal tubules (referred to as cortical and corticomedullary interstitium) is significantly enriched for YAP transcriptional activity and that ablation of YAP and its paralog TAZ from these cells disrupts the formation of regional fibroblast, pericyte, and myofibroblast populations.

YAP and TAZ have been shown to be essential for the development of numerous tissues. Within the kidney, previous work has revealed distinct roles in the development and morphogenesis of the nephrons and collecting ducts42–44 and the nephron progenitor cells.45 At least a part of the pleiotropic function of YAP and TAZ is determined by their interaction with additional transcriptional coregulators. Here we report that SRF is one of these coregulators. SRF is a transcription factor known to play multiple critical roles in tissue development and maintenance. SRF and YAP/TAZ share several transcriptional targets, and previous in vitro studies suggested that they interact to regulate transcriptional activation.14,15,36,37 Our analysis of the targets of YAP/TAZ in the renal stroma revealed that a subset is predicted SRF targets. Indeed, stromal-specific deletion of SRF partially phenocopies the YAP/TAZ deletion in that the myofibroblast population does not differentiate. Further, SRF is necessary for ectopic expression of myofibroblast gene products upon ectopic YAP activation (through ablation of the upstream kinases LATS1/2). In contrast, SRF deletion does not appear to be necessary for cortical fibroblast or pericyte differentiation, whereas it is necessary for differentiation of additional stromal cell types that do not require YAP (including the mesangial cells). These findings suggest that YAP/TAZ and SRF not only cooperate to promote myofibroblast differentiation through coregulation of a subset of targets but also have nonshared targets. Other regionally restricted cofactors may cooperate with these proteins in nonmyofibroblast cell types.

Interestingly, peritubular capillary differentiation was affected in the YAP/TAZ mutants whereas other capillary beds and larger endothelial subtypes appeared to be unaffected. This is consistent with our observation that YAP/TAZ are active in the cortical pericytes but not the medullary pericytes or other mural cell types. Although stromal cells/pericytes have previously been shown to regulate vascular development and maturation,46–48 the data described here are of interest in that they suggest that (1) there are molecularly distinct pericyte populations (YAP/TAZ+ and YAP/TAZ−) associated with different capillary beds in the kidney; (2) these different populations contribute to endothelial heterogeneity; and (3) the pericytes play important roles in blood vessel regionalization. Although it has previously been postulated that local environmental cues control the terminally differentiated phenotype of blood vessels resulting in the vascular heterogeneity within the segment of the renal vascular bed,49–51 the findings from this study provide some of the first direct evidence of unique pericyte populations contributing to this phenomenon.

The interstitial spaces in YAP/TAZ mutant kidneys are reminiscent of phenotypes described upon loss of stromal integrin or angiopoietin.30,52,53 Given that YAP/TAZ mutant kidney RNA-seq shows decreased expression of genes involved in both these pathways, it will be of interest to determine if either or both contribute to YAP/TAZ mutant deficiencies. Further study of these mutants will facilitate future endeavors focused on elucidating the pericyte produced signals that regulate peritubular capillary differentiation, and will help identify the factors that promote endothelial heterogeneity and differentiation, advancing efforts to generate functional, vascularized organoids/bioengineered kidney tissue. Further investigation is warranted in this area.

Although we did not identify defects in the adjacent proximal or distal tubules upon stromal deletion of YAP/TAZ, it is important to note that our RNA-seq analysis was performed at a stage before nephron differentiation. Further analysis will be required to determine whether the cortical fibroblast cells have a role in the maturation or function of the adjacent epithelia.

Finally, we propose that our findings further support a role for the renal stroma in directing cellular heterogeneity within the developing kidney. Multiple distinct phenotypes have now been ascribed to primary defects in the stroma, including mesenchymal to epithelial transition of the nephron progenitors,4,5 defects in vascular patterning,5,9,54 ureteric bud branching,6–8 and extension of the loop of Henle and papillary collecting ducts.55 Based on this study and our previous work,12,56 we hypothesize that unique stromal subtypes underlie the diverse functional roles of the stroma in regulating the multifaceted aspects of kidney development. Given that we identified 17 molecularly distinct clusters in our previous single-cell analysis, it will be of great interest to further define their functions in the developing kidney, as we predict additional roles are likely to be revealed.

Disclosures

A. Das reports employment with Amgen and Oric Pharmaceuticals; reports ownership interest with Amgen and Oric Pharmaceuticals; reports preclinical research funding from Amgen and Oric Pharmaceuticals; and reports patents for Amgen. All remaining authors have nothing to disclose.

Funding

Work in the Carroll laboratory was supported by National Institutes of Health grants R01DK095057, R01DK080004, R01DK106743, and R24DK090127 to T. Carroll. K. Drake was funded by Children’s Clinical Research Advisory Committee early career research award UTSW-21002111. This work was also supported by University of Texas Southwestern Medical Center George O’Brien Kidney Research core grant DK079328.

Supplementary Material

Supplemental Data
ASN.2021121559SupplementaryData.pdf (11.5MB, pdf)

Acknowledgments

We would like to acknowledge Drs. Ondine Cleaver, Eric Olson, and Denise Marciano and members of the Carroll, Cleaver, and Marciano laboratories for their helpful discussions.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

Present address: Dr. Amrita Das, Amgen, Inc., San Francisco, California.

Author Contributions

T. Carroll, C. Chaney, A. Das, and K. Drake conceptualized the study and were responsible for investigation, and supervision; J. Bittencourt and C. Chaney were responsible for methodology; J. Bittencourt, A. Das, and M. Patel were responsible for validation and visualization; M. Patel was responsible for investigation; C. Chaney was responsible for data curation; T. Carroll and M. Cohn were responsible for funding acquisition; A. Das and K. Drake were responsible for formal analysis; T. Carroll was responsible for project administration; T. Carroll and K. Drake wrote the original draft; and T. Carroll, C. Chaney, and K. Drake reviewed and edited the manuscript.

Data Sharing Statement

RNA-seq data presented in this manuscript have been deposited in the Gene Expression Omnibus (GEO) under the series accession number GSE181400. Single-cell RNA-seq data from the original analysis12 are available in the GEO under accession number GSE155794.

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2021121559/-/DCSupplemental.

Supplemental Table 1. Supplemental methods.

Supplemental Figure 1. Analysis of stromal cell clusters altered by ablation of YAP and TAZ.

Supplemental Figure 2. Stromal-specific YAP/TAZ allelic combinations and time course.

Supplemental Figure 3. Evaluation of YAP/TAZ stromal mutant kidneys.

Supplemental Figure 4. Stromal-specific YAP/TAZ mutants do not show increased apoptosis.

Supplemental Figure 5. Quantification of stromal and vascular IF in YAP/TAZ and SRF mutant kidneys.

Supplemental Figure 6. Medullary expression of NRP1 and PDGFRB in YAP/TAZ mutants.

Supplemental Figure 7. Transcription factor motifs enriched among those genes downregulated in YAP/TAZ mutants.

Supplemental Figure 8. Additional characterization of stromal-specific SRF mutant kidneys.

Supplemental Figure 9. Quantification of SM22a, aSMA, and MYL9 in YAP/TAZ and SRF stromal mutant kidneys.

Supplemental File 1. Differential gene expression from RNA-seq control versus YAP/TAZ mutant kidneys.

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Supplementary Materials

Supplemental Data
ASN.2021121559SupplementaryData.pdf (11.5MB, pdf)

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