Mice strains and genotyping.

To generate β-catenin-deficient mice, termed β-cat^S−/−, we first crossed FoxD1eGFPCre males (16) with mice containing loxP sites flanking exons 2–6 (β-cat^{Δ2–6/Δ2–6}) of the β-catenin allele (5). The Foxd1-Cre;β-cat^+/− males were then crossed with β-cat^{Δ2–6/Δ2–6} females to generate homozygous β-catenin loss-of-function mutants in the renal stroma. FoxD1eGFPCre mice were maintained on a CD1 genetic background, whereas β-cat^{Δ2–6/Δ2–6} mice were maintained on a C57BL/6J genetic background. Mice were genotyped using the following primers specific to the FoxD1eGFPCre allele, forward 5′-GCGGCATGGTGCAAGTTGAAT-3′ and reverse 5′-CGTTCACCGGCATCAACGTTT-3′; and forward 5′-AAGGTAGAGTGATGAAAGTTGTT-3′ and reverse 5′-CACCATGTCCTCTGTCTATTC-3′ for the floxed β-catenin allele.

Histology and immunofluorescence.

Whole kidney tissue was fixed in 4% paraformaldehyde for 24 h at 4°C. Kidneys were paraffin-embedded, sectioned to 5 μm, and mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA) and incubated overnight at 37°C. Sections were deparaffinized using xylene washes and rehydrated using graded ethanol washes (100%, 95%, 75%, 50%, H₂O) and stained with hematoxylin and eosin (Sigma, St. Louis, MO). For immunofluorescence, tissue was prepared as described above and antigen retrieval was performed for 5 min in 10 mM sodium citrate solution, pH 6.0, in a pressure cooker, followed by blocking with serum-free protein block (Dako, Carpinteria, CA). Sections were incubated with primary antibodies to Pbx1 (Cell Signaling; 1:250 dilution), Foxd1 (Santa Cruz Biotechnology; 1:200), p57Kip2 (Santa Cruz, CA; 1:200), Casp3 (Cell Signaling; 1:250), Aqp3 (Novus Biologicals; 1:250), Tenascin-C (AbCam, Cambridge, MA; 1:250), and GFP (AbCam; 1:200) overnight at 4°C. Tissue sections were washed in PBS, pH 7.4, incubated with secondary antibodies Alexa Fluor 488 or 568 (Invitrogen, Carlsbad, CA; 1:1,000 dilution) for 1 h at room temperature, and stained with DAPI (Sigma; 1:1,000 dilution) for 5 min. Tissue sections were mounted with glass coverslips and Fluoromount mounting media (Sigma) and photographed on a Nikon 90i-eclipse upright microscope.

In situ hybridization.

In situ hybridization for Bmp4, Pod1, Wnt11, and Wnt4 was performed using the Affymetrix QuantiGene ViewRNA assay. Briefly, paraffin-embedded WT and β-cat^S−/− kidneys were sectioned at a thickness of 5 μm, deparaffinized, boiled in pretreatment solution (Affymetrix, Santa Clara, CA), and digested with proteinase K. Sections were incubated with a custom-designed QuantiGene ViewRNA probe for 2 h at 40°C. Signal was amplified with Pre-Amp and Amp solutions, and the reaction was visualized with Fast-Red or Fast-blue Substrate. Slides were counterstained with DAPI, then mounted with glass coverslips using Fluoromount mounting media (Sigma) and photographed on a Nikon 90i-eclipse upright microscope.

Real-time reverse transcriptase-PCR.

Real-time PCR was performed using the Applied Biosystems 7900HT fast RT-PCR system (Applied Biosystems, Burlington, ON, Canada). cDNA was generated using first-strand cDNA synthesis (Invitrogen, Carlsbad, CA) from total RNA. Real-time PCR reaction mix contained 2.5 ng of each cDNA sample, SYBR green PCR Master Mix (Applied Biosystems, Burlington, ON, Canada), and 300 nM of each primer to a total volume of 25 μl. Primers for FoxD1, Pod1, Wnt4, Wnt11, Bmp4, and Tn-c were designed using the Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/) and verified using the UCSC genome bioinformatics website (genome.ucsc.edu). Relative levels of mRNA expression were determined using the 2^(−ΔΔCt) method. Individual expression values were normalized by comparison to β-2-microglobulin.

Analysis of cell proliferation and apoptosis.

Pregnant mice received an intraperitoneal (ip) injection of BrdU (100 mg/g body wt) 2 h before euthanasia. Cell proliferation was assayed in paraffin-embedded kidney tissue by incorporation of 5-bromo-2-deoxyuridine (Roche Molecular Biochemicals, Mannheim, Germany), as previously described (7). BrdU-positive cells in the stroma were identified by colabeling with the anti-BrdU antibody (Abcam, ab8152, 1:250) and Pbx1 antibody (Cell Signaling, Beverly, MA; 1:250). Apoptosis was assessed in paraffin-embedded kidney tissue using the cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) and visualized using 3,3′-diaminobenzidine (DAB) substrate solution (Vector).

Statistical analysis.

The RT-qPCR, stromal cell count, apoptosis, and cell proliferation were analyzed using a two-tailed Student’s t-test using GraphPad Prism software (version 5.0c). P < 0.05 indicates statistical significance.

Ethics statement.

All studies were performed in accordance with animal care and guidelines put forth by the Canadian Council for Animal Care, and McMaster’s Animal Research Ethics Board (AREB) (Animal Utilization Protocol no. 100855) approved the project described in this study.

Deletion of stromal β-catenin results in reduced medullary stroma.

Our previous studies demonstrate sparse loosely packed capsular, cortical, and medullary stroma in mice with a genetic ablation of β-catenin specifically in renal stroma cells (termed β-cat^S−/−) (4). Therefore, we wanted to investigate whether β-catenin plays a role in controlling the progenitor stromal population or the stroma subpopulations that are located in the cortical or medullary compartments. To begin this investigation, we performed a spatial and temporal analysis of the stromal cell population in developing kidneys. Our initial analysis of the stroma focused on the histology at E13.5 since no overt developmental abnormalities are observed in β-cat^S−/− kidneys at this time point (4) (Fig. 1, A and B). The organization of stromal cells throughout the developing kidneys was similar in WT and β-cat^S−/− kidneys. At the cellular level, the stromal cells exhibited a stellate cell morphology in both WT and β-cat^S−/− kidneys (Fig. 1, A′ and B′, insets). Next, we performed immunofluorescence (IF) using Pbx1, which is a marker of all stromal cells (4). This analysis demonstrated the spatial organization of the stromal cells were similar in both WT and β-cat^S−/−, confirming the histological findings. In addition, quantitative analysis of Pbx1 nuclei, representing all stromal cells, demonstrated no alterations in the total number of stromal cells (4.32 cells/mm² in WT vs. 4.33 cells/mm² in β-cat^S−/−, P < 0.05) (Fig. 1E). This demonstrates that the entire stromal cell population is not affected at E13.5.

We next performed a histological analysis of β-cat^S−/− kidneys at E15.5. This represents a developmental time point when the cortex and medulla are formed and all stroma subtypes, including capsular, cortical, and medullary stroma populations, are represented. In WT, the histological analysis of the stroma populations revealed distinct populations of capsular, cortical, and medullary stroma cells located between maturing and developing nephrons and collecting tubules (Fig. 1F). The population of cortical and medullary stromal cells were primarily composed of stellate-shaped cells (Fig. 1F′, inset). In contrast to WT, the β-cat^S−/− kidneys appeared to have fewer stromal cells surrounding the nephrogenic tubules and cortical and medullary collecting ducts (Fig. 1G). This reduction in stromal cells appeared to be more prominent in the medullary regions. Further, the stromal cells did not exhibit the stellate shape morphology that is typically observed in the WT kidneys (Fig. 1G′, inset). IF for Pbx1 demonstrated a 16.67% reduction in the number of Pbx1+ cells in β-cat^S−/− compared with WT littermate kidneys (4.20 cells/mm² in WT vs. 3.50 cells/mm² in β-cat^S−/−, P < 0.05) (Fig. 1J). We note that the reductions in the Pbx1 positive cells were primarily located deeper in the kidney (those below the dotted lines) in β-cat^S−/− (Fig. 1I) compared with WT (Fig. 1H). By PN0, the number of Pbx+ cells in β-cat^S−/− was reduced to 44.62% compared with WT (4.55 cells/mm² in WT vs. 2.52 cells/mm² in β-cat^S−/−, P < 0.05) (Fig. 1K). Together, these results demonstrate a loss of stromal β-catenin results in abnormal stroma cell morphology and progressive reductions in stromal cell number.

β-Catenin controls medullary stroma development.

We next performed a detailed analysis of the medullary stroma cell population since the reductions in the stromal population in β-cat^S−/− kidneys appeared more prominent in the medullary compartment. We analyzed the expression of Bmp4, a marker of the medullary stroma and regulator of medullary stromal expansion (24, 25). At E15.5, the expression of Bmp4 mRNA in WT was observed in medullary tubules (t) and the surrounding medullary stroma which is a pattern consistent with previous reports (Fig. 2, A and A′) (24, 25). While the tubular expression of Bmp4 mRNA was maintained, very little Bmp4 mRNA was observed in the stroma cell population surrounding the tubules in β-cat^S−/− kidneys (Fig. 2, B and B′). However, sporadic cells throughout the medullary stroma compartment did express some Bmp4. This reduction was confirmed by qQRT-PCR, which demonstrated a 44% reduction in Bmp4 expression in β-cat^S−/− kidneys compared with WT (1.01 vs. 0.57, P < 0.05) (Fig. 2C). We next analyzed the transcription factor Pod1 since it is essential for medullary stromal differentiation (9). Consistent with previous reports (9), Pod1 mRNA expression localized to the medullary stroma cells surrounding the tubules (t) and the ureter (u) in WT kidneys (Fig. 2, D and D′). Despite numerous DAPI-stained cell nuclei in the stromal compartment in the β-cat^S−/− kidneys, there is almost no Pod1 expression observed in the stromal compartment (ms). Whereas the Pod1 expression is maintained in the podocytes (Fig. 2, E and E′), the mRNA expression was reduced by 69% in β-cat^S−/− kidneys (1.11 vs. 0.42, P < 0.05) (Fig. 2F). These experiments establish that cells are occupying the medullary stromal compartment but are not expressing medullary stromal markers. This suggests the cells fated to become medullary stromal cells do not undergo the differentiation program.

Therefore, to further examine whether the cells fated to become the medullary stroma were differentiating, we analyzed the expression of additional medullary differentiation factors that are necessary for medulla stroma development (20). In WT kidneys at E15.5, Wnt4 (Fig. 3, A and A′) and Wnt11 (Fig. 3, D and D′) mRNA expression was observed in a punctatelike pattern throughout the medullary stroma cells. In the medulla Wnt4 expression was also observed in some of the tubular epithelia. In β-cat^S−/− kidneys, Wnt4 and Wnt11 mRNA expression was barely detectable in the majority of medullary stromal cells (Fig. 3, B, B′, E, and E′). Additionally, qRT-PCR analysis of Wnt4 revealed a 63% reduction in β-cat^S−/− kidneys (Fig. 3C) (1.05 vs. 0.42, P < 0.05), whereas Wnt11 levels were reduced by 48% (Fig. 3F) (1.18 vs. 0.89, P > 0.05). The lack of a statistically significant change for Wnt11 is likely due to the strong ureteric bud tip expression that is maintained in β-cat^S−/− kidneys (Fig. 3E) (4). We next analyzed the protein expression of p57Kip2, an essential protein for medullary development (37). Consistent with previous reports (28, 36), p57Kip2 demonstrated strong nuclear expression in the medullary stromal cells that are located around the tubules in WT kidneys at E15.5 (Fig. 3, G and G′). In β-cat^S−/− kidneys, the majority of medullary stroma cells did not express p57Kip2 (Fig. 3, H and H′). However, we do note that a few p57Kip2+ cells were observed surrounding the initial ureteric branch that is located deep in the kidney (Fig. 3, H and H′). The reduction of p57Kip2 mRNA demonstrated a 46% reduction in β-cat^S−/− kidneys (Fig. 3I) (1.00 vs. 0.54, P < 0.05). Combined, this detailed analysis of the cells occupying the medullary stroma compartment demonstrates a lack of medullary stromal differentiation markers suggesting defects in medullary stroma differentiation.

β-Catenin deficiency does not regulate the Foxd1 stromal progenitor pool.

As kidney development progresses, the stromal progenitor cells in the kidney cortex express FoxD1. The Foxd1+ stromal progenitors then differentiate into the capsular, cortical, and medullary stroma and express a unique set of genes in each population (18). Therefore we next determined whether changes in the FoxD1 stromal progenitor population were consequential to the alterations in the medullary stroma. We performed a FoxD1 IF analysis at E14.5, a time point just before the medullary stroma defects, and E15.5 when medullary stroma defects are observed. In both β-cat^S−/− and WT littermates at E14.5 and E15.5, the FoxD1 stromal progenitor population was limited to the cortex region surrounding the condensed mesenchyme in the nephrogenic zone (Fig. 4, A–E). Quantification of the mRNA levels revealed no changes in FoxD1 at E14.5 and E15.5 (Fig. 4, C and F). However, in the β-cat^S−/− kidneys, we observed that the FoxD1 stromal progenitors were also found outside of the nephrogenic zone and localized deeper in the kidney parenchyma (Fig. 4E′). Considering β-cat^S−/− kidneys exhibit defects in corticomedullary formation, we thought that these cells were part of the cortical region. However, using Aquaporin-3, a marker of the most distal part of the collecting ducts, we showed that the FoxD1 stromal progenitors were located directly adjacent to aquaporin-3 positive cells (Fig. 4, G and H). This was never observed in WT kidneys (Fig. 4G). This demonstrates that stromal progenitors, in the absence of β-catenin, maintain their Foxd1+ progenitor status and do not differentiate into medullary stromal cells. We next used the cortical marker Tenascin-C to determine if the stromal progenitor population leads to alterations in the cortical stroma (35). IF analysis of Tenascin-C in WT and β-cat^S−/− kidneys did not reveal any differences in the expression patterns surrounding the condensed mesenchyme and developing nephrons at E14.5 (Fig. 4, I and J) or E15.5 (Fig. 4, K and L). The levels of Tenascin-C mRNA expression were also unchanged in β-cat^S−/− kidneys compared with WT mice (Fig. 4M) (1.00 vs. 0.93, P > 0.05).

We next investigated if changes in cell proliferation contributed to the reduction in medullary stroma since the Wnt/β-catenin signaling pathway can regulate cell proliferation (22). We analyzed stromal cell proliferation by performing a BrdU incorporation assay. The colocalization experiment using an anti-BrdU antibody with the stromal marker Pbx1 (yellow cells represent the co-localization of BrdU and Pbx1) (Fig. 4, N and O) demonstrated no changes in stromal proliferation at E13.5, before the onset of medullary development (22.95 vs. 25.44, P = 0.38) (Fig. 4P). Similarly, no changes were observed at E15.5 (27.17 vs. 25.30, P = 0.1945) in β-cat^S−/− kidneys compared with WT littermates (data not shown). Combined our analysis demonstrates β-catenin is not required for the initial formation of the Foxd1 stromal progenitor population or in the formation of the cortical stroma. Additionally, β-catenin does not regulate stromal proliferation and therefore does not contribute to the reductions in medullary stroma.

β-Catenin deficiency increases apoptosis at the corticomedullary junction.

β-Catenin regulates cell survival during differentiation of the nephrogenic progenitors (30). Since no changes in proliferation were observed, we hypothesized that the reduction in medullary stroma could be caused by increased cell death. Thus we analyzed apoptosis using a terminal transferase-mediated dUTP nick end-labeling (TUNEL) experiment. In WT littermates at E13.5 and E15.5 a rare sporadic apoptotic cell was observed in the cortex region. Further, apoptotic cells were very rarely observed around the corticomedullary region in WT littermates at E13.5 and E15.5 (Fig. 5, A and F). In contrast, β-cat^S−/− kidneys exhibited a 2.2-fold increase in the number of apoptotic cells when compared with WT kidneys (0.54 vs. 1.2 cells/mm², P < 0.05) at E13.5 (Fig. 5C). At E13.5, the numerous apoptotic cells in β-cat^S−/− kidneys were always found in clusters, were in the compartment where the stromal cells reside, and were at the location where the corticomedullary junction is established (Fig. 5B). The apoptotic cells were rarely found in the nephrogenic zone in β-cat^S−/− kidneys (Fig. 5B). In β-cat^S−/−, at E15.5, an identical apoptotic pattern to E13.5 was also observed. At E15.5, a 3.3-fold increase in the number of apoptotic cells was observed and these cells were also located at the junction between the cortex and medulla (0.36 vs. 1.19 cells/mm², P < 0.05) (Fig. 5, G and H).

To confirm the cells undergoing apoptosis in β-cat^S−/− kidneys were of the stromal origin, we performed co-IF at E13.5 for stromal marker Pbx1 and activated Caspase-3, which is required for DNA fragmentation and chromatin condensation during apoptosis (27). Caspase-3 and Pbx1 colocalized in β-cat^S−/− kidneys, confirming that the TUNEL+ apoptotic cells are stromal cells (Fig. 5, D and E). Clusters of Casp-3+ cells were found below the nephrogenic zone in β-cat^S−/− kidneys in a pattern identical to the TUNEL+ cells (Fig. 5E′). This analysis confirms that as stromal cells become located deeper in the kidney they undergo apoptosis and therefore do not integrate into the medullary compartment.

In WT developing kidneys the Foxd1+ progenitor cell population reaches the deeper cortical zone, loses Foxd1 expression, and medullary stromal genes are upregulated (18). In β-cat^S−/− kidneys we observed numerous Foxd1+ cells deep to the nephrogenic zone. This was never observed in the WT littermates (Fig. 4, G and H). We suspected the apoptotic cells observed at the corticomedullary junction in β-cat^S−/− kidneys are FoxD1+ stromal progenitors that do not differentiate to medullary stromal cells. Therefore we performed a co-IF analysis to determine whether these apoptotic cells in β-cat^S−/− kidneys were the FoxD1 + cells. In WT kidneys, no FoxD1+ cells were undergoing apoptosis (Fig. 5I, arrowheads). In contrast, the majority of the FoxD1+ stromal progenitors deep to the nephrogenic zone were undergoing apoptosis in β-cat^S−/− kidneys, as evidenced by Caspase3 and FoxD1 colocalization (Fig. 5J, arrowheads). This demonstrates that the apoptotic cells undergoing apoptosis are indeed the mislocalized Foxd1 stromal progenitors. These findings demonstrate that a loss of stromal β-catenin results in increased apoptosis in the stromal cells located around the region where the corticomedullary junction would be established.

β-Catenin does not play a functional role in cortical stroma or stromal progenitor formation.

The factors that control the differentiation of stromal progenitors into mature stromal cells that integrate the capsular, cortical, and medullary compartments are not established (18). In pursuing the role that β-catenin plays in this process, our analysis, combined with our previous data (4), has provided insights into the roles of β-catenin in the differentiation and formation of cortical stromal cells. Here we demonstrate that β-catenin is not required for the formation of the Foxd1 progenitors since we observed no changes in the numbers of Foxd1 progenitors population or in the levels of Foxd1 expression. In addition we showed no differences in the differentiation state of the cortical stroma between WT and mutants. Further our data showed no alterations in apoptosis in the cortical stroma in the absence of β-catenin. Therefore the data in this manuscript show no evidence to support a developmental role for β-catenin in the initial formation of the Foxd1 progenitor pool or in maintaining the survival of this population. However, our previous data demonstrated a role for stromal β-catenin in the modulation of ureteric epithelial Wnt9b expression and proliferation of nephrogenic progenitors (4). This suggests β-catenin plays a functional role in the cortical stroma to regulate gene expression in adjacent cell populations, such as the nephrogenic progenitors and ureteric epithelium. Some potential candidates that could be regulated by β-catenin are Fat4 (1, 10, 23) and Raldh2 (29). Further analysis of the gene expression patterns in the cortical stroma population is required to identify β-catenin-dependent factors that control communication with adjacent cell populations.

β-Catenin is required for medullary stroma formation.

The overall reduction in medullary stroma, combined with very little expression of medullary stroma genes, suggested a functional role for β-catenin in medullary stroma formation. Specifically, our data demonstrate an absence or significant reduction of key medullary stroma markers (Pod1, Bmp4, Wnt4, p57Kip2, and Wnt11), in the absence of stromal β-catenin. Studies have demonstrated that these genes are essential for medullary formation and expressed in fully differentiated medullary stromal cells (9, 24, 25, 32, 37). Based on the lack of medullary stroma gene expression in the absence of β-catenin, it is possible that β-catenin regulates the transcription of some of these genes. In support of this, studies in other cells of the kidney where the levels of β-catenin are specifically deleted or overexpressed have identified changes of genes Bmp4, Wnt4, and Wnt11. Specifically, in the ureteric epithelium, a deletion of β-catenin results in a complete absence of Wnt11 expression (6). In the condensed mesenchyme, deletion of β-catenin prevents Wnt4 expression, whereas its overexpression results in increased Wnt4 levels (26). Similarly, we have previously shown that the overexpression of β-catenin in stromal progenitors results in increased levels of Wnt4 and Bmp4. Combined, these studies suggest that Wnt4, Wnt11, and Bmp4 are candidate downstream transcriptional targets of β-catenin. The direct binding of β-catenin/Tcf transcriptional complexes to the promoter regions of other medullary stroma genes (i.e., Pod1 and p57Kip2) is not understood. Previous reports have demonstrated a downregulation of p57Kip2 in β-catenin deficient stromal cells (36), which is consistent with our data. However, this interaction was not demonstrated in any other cell types. Similarly, no reports have demonstrated a potential regulation of Pod1 by β-catenin in stromal cells or any other cell types. Therefore, taken together, our findings suggest the potential for novel transcriptional β-catenin targets involved in medullary stroma differentiation. However, during the course of this work we performed an analysis of the promoter region of these five putative target genes (Bmp4, Pod1, Wnt4, Wnt11, p57Kip2) demonstrated no conserved TCF/LEF transcription factor binding sites within the initial 5kb of the proximal promoter region (22). This suggests that β-catenin may activate factors upstream of these candidates to initiate their expression, or β-catenin partners with other transcription factors and coactivators or the consensus sites lie outside the proximal promoter region. Further work is required to establish whether the initial activation of this differentiation program leads to a sequential activation of medullary stromal genes, whereby markers such as Wnt4, Bmp4, and Wnt11 are first activated and in turn promote the activation of additional medullary markers, such as Pod1 and p57Kip2.

Mechanisms contributing to reduced medullary stroma.

We suspected apoptosis accounts for the reduced medullary stromal cell population. This is supported by our data demonstrating the numerous apoptotic stroma cells located specifically at the junction between the cortex and medulla, a location where the stroma cells would begin adopting medullary stromal specific genes. Therefore, in the absence of β-catenin the cortical stromal cells fail to differentiate into medullary stromal cells and are eliminated by apoptosis. This concept is supported by other studies in the kidney. In the absence of β-catenin in nephron progenitors, the progenitor population fails to differentiate into nephrons. The maldifferentiated nephron progenitors are eliminated through apoptosis (2, 30). This mechanism is also observed in nonrenal tissues. For example, reduced levels of β-catenin in mammary gland epithelial cells results in failed differentiation and elimination through apoptosis (14). Similarly, the lack of β-catenin in T cells prevents their proper maturation and results in apoptosis (17). Together, these studies support a role for β-catenin in cell differentiation and survival, and demonstrate that apoptosis is triggered as a mechanism to eliminate improperly differentiating stromal cells.

β-Catenin also modulates apoptosis in the kidney (33, 38) and other organ systems (17, 34) via direct transcription of antiapoptotic genes, such as Bcl2l1, and repression of proapoptotic factors like p53 and Bax (38). Our data demonstrate that cells undergoing apoptosis in β-cat^S−/− kidneys are the stromal progenitors that fail to differentiate. Therefore it is possible that rather than apoptosis being secondary to improper differentiation, β-catenin may directly activate a survival program in differentiating cells. This is supported by studies performed in differentiating embryonic stem cells. Just before differentiating the stem cells upregulate antiapoptotic genes (11). Therefore, it is possible that the renal stromal progenitors that are about to differentiate into medullary stromal cells could upregulate antiapoptotic and prosurvival genes and therefore protect them from apoptosis in a β-catenin-dependent manner. This suggests β-catenin could contribute to the regulation of medullary stroma differentiation through the activation of a survival program, but future studies will be required to define this pathway.

In summary, we demonstrate here that β-catenin is essential for the differentiation and survival of medullary stromal cells. Our findings are consistent with the work of others with respect to the medullary defects observed in this model (36) and provide further insight into the role of β-catenin in stroma differentiation. Our work clarifies β-catenin’s role in the stroma progenitors by demonstrating that β-catenin is not required for the induction of the initial stromal progenitors or in the differentiation of the cortical stroma. Our findings support a functional role for β-catenin in medullary stroma differentiation and demonstrate that the reduction of medullary stroma in β-cat^S−/− mutant mice is caused by apoptosis of maldifferentiated stromal cells.