β-Catenin Causes Renal Dysplasia via Upregulation of Tgfβ2 and Dkk1

Abstract

Renal dysplasia, defined by defective ureteric branching morphogenesis and nephrogenesis, is the major cause of renal failure in infants and children. Here, we define a pathogenic role for a β-catenin–activated genetic pathway in murine renal dysplasia. Stabilization of β-catenin in the ureteric cell lineage before the onset of kidney development increased β-catenin levels and caused renal aplasia or severe hypodysplasia. Analysis of gene expression in the dysplastic tissue identified downregulation of genes required for ureteric branching and upregulation of Tgfβ2 and Dkk1. Treatment of wild-type kidney explants with TGFβ2 or DKK1 generated morphogenetic phenotypes strikingly similar to those observed in mutant kidney tissue. Stabilization of β-catenin after the onset of kidney development also caused dysplasia and upregulation of Tgfβ2 and Dkk1 in the epithelium. Together, these results demonstrate that elevation of β-catenin levels during kidney development causes dysplasia.

Congenital renal malformation is the major cause of childhood renal failure. Yet underlying pathogenic mechanisms are poorly defined and no specific treatments exist.¹ Formation of the mammalian kidney is dependent on reciprocal inductive tissue interactions between the ureteric bud and the metanephric blastema. These tissue interactions result in growth and branching of ureteric-derived tubules that differentiate into collecting ducts and formation of nephrons from mesenchyme-derived structures.² Failure of these processes results in kidney dysplasia, which is defined by a paucity of nephrons, decreased branching morphogenesis, abnormal differentiation of mesenchymal and epithelial-derived tissue elements, and abnormal cortico-medullary patterning.³

Kidney formation is regulated by secreted growth factors including members of the WNT family of secreted glycoproteins. Numerous WNT family members are required during kidney development.^4–6 WNT proteins elicit their biologic responses by activating canonical and noncanonical signaling pathways.⁷ Canonical WNT signaling is mediated by β-catenin, which controls cell adhesion and gene transcription. Canonical WNT signaling inhibits phosphorylation of β-catenin by glycogen synthase kinase-3 (GSK-3). Consequently, β-catenin degradation is inhibited, resulting in cytoplasmic accumulation, nuclear translocation of β-catenin, and regulation of target gene expression.⁸

Both the ureteric cell lineage and the mesenchyme cell population of committed nephron progenitor cells require canonical WNT/β-catenin signaling for their respective morphogenetic activities. Homozygous β-catenin deficiency targeted to metanephric mesenchyme cells inhibits nephron formation and decreases expression of genes including Wnt4, Lim1, and Fgf8, which are required for nephrogenesis.⁹ Homozygous β-catenin deficiency in ureteric cells causes a near arrest of ureteric branching, decreased expression of genes required for ureteric tip cell function (C-ret and Wnt11), and bilateral renal aplasia or severe hypodysplasia.^10,11

Abnormal canonical WNT/β-catenin signaling has been implicated in the pathogenesis of human kidney diseases with a developmental origin. In human renal dysplasia β-catenin is upregulated and misexpressed in ureteric cell–derived cysts.¹² Perturbation of ureteric branching in transgenic mice induces β-catenin expression and causes dysplasia, suggesting a secondary pathogenic mechanism whereby elevated β-catenin expression contributes to the genesis of renal dysplasia.^12–14 Wilms' tumor, a polymorphic childhood tumor derived from mesenchyme blastemal tissue elements, is characterized by β-catenin overexpression in blastemal and mesenchymal tumor tissue.¹⁵

Here, we report a functional contribution of elevated levels of β-catenin to kidney development using genetic murine models of β-catenin stabilization. Stabilization of β-catenin in the ureteric cell lineage, before the onset of kidney development, caused renal aplasia or dysplasia caused by a severe disruption of ureteric branching. Analysis of global gene expression in kidneys isolated from mutant mice revealed misexpression of Tgfβ2 and Dkk1, which inhibited ureteric branching and nephrogenesis, respectively, in embryonic kidneys cultured ex vivo. Stabilization of β-catenin at stages after nephrogenesis and ureteric branching are established also disrupted nephrogenesis and caused misexpression of Tgfβ2 and Dkk1. These results demonstrate a pathologic role for β-catenin in the genesis of renal dysplasia.

RESULTS

A Genetic Murine Model of β-Catenin Overexpression in Embryonic Mouse Kidney In Vivo

Because elevated β-catenin expression is associated with abnormal ureteric cell branching, we investigated the direct effects of increased β-catenin expression on ureteric cell function. Phosphorylation of serine/threonine residues in exon 3 of the β-catenin allele by GSK-3β targets β-catenin for intracellular degradation.⁸ We used CRE-mediated recombination targeted specifically to ureteric cells using Hoxb.7-Cre;GFP (GFP, green fluorescent protein) mice^16,17 and a β-catenin allele in which exon 3 is flanked by loxP sites (β-cat^Δ3 mice)¹⁸ to generate mice (termed β-cat^GOF-UB [Gain of Function-Ureteric Bud]) in which excision of exon 3 from the β-catenin allele is targeted to the ureteric cell lineage. To localize the spatial domain of CRE activity, we generated wild type (WT) and β-cat^GOF-UB mice containing a ROSA-26 reporter allele,¹⁹ which expresses β-galactosidase under the control of CRE recombinase. LacZ staining in WT and β-cat^GOF-UB in E12.5 urogential tissues demonstrated Hoxb.7-Cre mediated excision in the Wolffian duct, mesonephric tubules, and ureteric cells (Figure 1, A and B). β-Catenin protein expression, analyzed by immunofluorescence imaging of ureteric cells, was greatly increased throughout the cytoplasm and nucleus in β-cat^GOF-UB mutant mice when compared with WT (Figure 1, C and D). Next, we determined the effect of increased β-catenin levels on transcriptional activity in ureteric cells. We generated β-cat^GOF-UB mice containing TCF sites linked to a LacZ reporter gene.²⁰ Analysis of β-galactosidase activity in urogential tissue revealed an increase in the caudal portion of the Wolffian duct and ureteric cells of β-cat^GOF-UB mutants compared with WT littermates analyzed simultaneously (Figure 1, E and F). Increased levels of β-catenin in β-cat^GOF-UB mutant kidneys were confirmed using immunoblotting and protein lysates generated from four E18.5 β-cat^GOF-UB mutant kidneys. Quantitation of β-catenin controlled for by the expression of GAPDH demonstrated a 1.3-fold increase in β-catenin in β-cat^GOF-UB mutant kidneys compared with WT (note decreased loading of β-cat^GOF-UB kidney protein compared with WT) (Figure 1, G and H). The relatively modest increase in β-catenin expression is likely to underestimate the increase in ureteric cells (Figure 1, C and D) since ureteric cells constitute 10% or less of all cells in the intact kidney. Together, these data demonstrate that genetic stabilization of β-catenin in vivo increases β-catenin protein expression and β-catenin–dependent transcriptional activity in ureteric cells.

Figure 1.

Conditional overexpression of β-catenin in ureteric cells results in severe renal defects. (A, B) Whole mount X-gal staining in E12.5 urogenital ridges resected from WT (A) and β-cat^GOF-UB mice (B) containing a ROSA-26 reporter allele. LacZ staining in Wt and β-cat^GOF-UB in E12.5 urogential ridges demonstrated Hoxb.7-Cre mediated excision in Wolffian duct (WD), mesonephric tubules (MT), and ureteric bud (UB) tissue. (C, D) β-catenin immunofluoresence in E12.5 WT and β-cat^GOF-UB kidney tissue. (C) In WT kidney β-catenin is localized in the UB plasma membrane consistent with a role in adherens junctions (white arrow). β-Catenin expression was not observed in the cytoplasm or nucleus in WT UBs. (D) In β-cat^GOF-UB mutant mice β-catenin localized to the ureteric cell plasma membrane as well as the cytoplasm and nucleus (white arrow). (E, F) Whole mount X-gal staining in E11.5 urogenital ridges isolated from WT TCF reporter mice and β-cat^GOF-UB;TCF mice demonstrates activity in the WD and UB. The X-gal staining in β-cat^GOF-UB;TCF mice is noticeably more intense when compared with WT littermates, thus indicating increased transcriptional activity. (G, H) Western analysis and corresponding densitometry for β-catenin and GAPDH in WT and β-cat^GOF-UB mutant kidneys.

Overexpression of β-Catenin in the Ureteric Cell Lineage Causes Renal Agenesis and Hypodysplasia

β-cat^GOF-UB mice survived to term but died within hours of birth. Analysis of kidney tissue revealed bilateral renal aplasia in 45% and bilateral severe renal hypoplasia in 55% of mutant pups, respectively (Table 1 and Figure 2, A and B). Histologic analysis of kidney tissue from mutant mice at PN0 revealed disorganization of tissue elements, decreased number of nephrons, tubular dilation, and uninduced mesenchymal cells, the hallmark features of severe dysgenesis (Figure 2, C and D). Dilated tubules were bound by Dolichos biflorus agglutinin (DBA), a ureteric cell-specific marker (Supplemental Figure 1). Next, we examined embryologic mechanisms that underlie renal malformation in β-cat^GOF-UB mice. Although initial ureteric branches were comparable in WT and mutant mice at E11.5 (Figure 2, E and F), by E12.5, kidney tissue from β-cat^GOF-UB mutant mice was smaller than that in WT mice and appeared to consist of fewer ureteric branches (Figure 2, G and H). Ureteric branching was investigated directly using GFP fluorescence at E12.5. In contrast to WT mice, in which multiple generations of ureteric branches are formed, ureteric branching was greatly attenuated in mutant mice (Figure 2, I and J). Together, these data demonstrate that stabilization of β-catenin in the ureteric cell lineage results in arrested ureteric branching by E12.5.

Table 1.

Renal phenotype in β-cat^GOF-UB mice

	Mutants/WT	Aplasia	Dysplasia
Perinatal
β-catGOF-UB	11/36 (30.5%)	5/11 (45%)	6/11 (54.5%)
Embryonic
β-catGOF-UB	76/167 (40.2%)	13/76 (17.1%)	63/76 (82.8%)

Kidneys were isolated from newborn mice (P0) and from pregnant females at various gestational ages. In embryos in which kidney tissue could be identified, histology was analyzed and categorized as normal or dysplastic.

Figure 2.

Arrested branching morphogenesis in Hoxb.7;β-cat^Δ3/+ mutant mice. (A, B) Urinary systems from P0 WT and β-cat^GOF-UB mice, demonstrating severe renal malformations in β-cat^GOF-UB mice (white arrows). (C, D) H&E stained kidney sections from P0 WT and β-cat^GOF-UB mice, demonstrating a lack of cortical medullary patterning, cystic tubules (C), and a paucity of nephrogenic structures. (E through H) H&E stained cross sections of mouse embryos at E11.5 and E12.5 demonstrates a paucity of ureteric bud (UB) tissue and nephrogenic elements as early as E12.5 in β-cat^GOF-UB mice. White arrow, MM; black arrow, UB tissue. (I, J) GFP fluorescence demonstrating arrested branching morphogenesis in β-cat^GOF-UB mice. MM, metanephric mesenchyme.

Effect of β-Catenin Overexpression on Adherens Junctions, Apoptosis, and Cell Proliferation

Cellular processes, including cell-cell adhesion, cell proliferation, and apoptosis, which control branching morphogenesis,^21,22 are also controlled by canonical WNT/β-catenin signaling. We determined the effects of increased β-catenin expression on these processes using renal tissue isolated from β-cat^GOF-UB mice. To function in cell adhesion, cadherins must be associated with the actin cytoskeleton.²³ β-Catenin connects cadherins to the actin cytoskeleton.²⁴ Therefore, a disruption of adherens junctions could lead to a reduction in epithelial integrity and reduced branching morphogenesis. Adherens junctions, imaged in ureteric cells using electron microscopy, exhibited no morphologic difference in mutant mice compared with WT mice (Figure 3, A and B). Cell proliferation was quantitated using BrdU incorporation, which revealed a 1.24-fold increase (P = 0.03) in ureteric bud cell proliferation compared with WT (Figure 3, C through E). Examination of ureteric cell apoptosis by TUNEL analysis revealed no significant difference in the number of apoptotic ureteric cells between β-cat^GOF-UB and WT mice (P = 0.78) (Figure 3, F through H). However, apoptosis in the metanephric mesenchyme was increased 4.9-fold (P = 0.015) in β-cat^GOF-UB mice when compared with WT (Figure 3, I through K). The increase in mesenchyme cell apoptosis is consistent with the known requirement for ureteric tip cells to secrete signals required for metanephric mesenchyme cell survival.²⁵ Together, these results indicate a primary effect of increased β-catenin expression on ureteric cell proliferation. However, a modest increase in cell proliferation is unlikely to account for the severe disruption of branching morphogenesis observed in β-cat^GOF-UB mice.

Figure 3.

β-catenin stabilization modulates cellular events during ureteric branching. (A, B) Analysis of adherens junctions (white arrows) by transmission electron microscopy demonstrates the presence of junctional complexes in β-cat^GOF-UB tissue indistinguishable from WT. (C through E) Qualitative and quantitative analysis of cell proliferation in E12.5 WT and β-cat^GOF-UB kidney tissue using an in situ BrdU incorporation assay. Quantitative analysis of BrdU incorporation (red color) demonstrated a 1.24-fold increase (% BrdU-positive ureteric cells, WT [43.2%] versus β-cat^GOF-UB [53.7%], *P = 0.03) in β-cat^GOF-UB ureteric cell proliferation when compared with WT. (F through K) Qualitative and quantitative analysis of ureteric bud and mesenchymal apoptosis by TUNEL analysis. TUNEL-positive cells (brown color, arrowhead) are rarely detected in the ureteric bud (black arrow) in WT (F, G) and β-cat^GOF-UB (I, J) mutant mice (% TUNEL-positive ureteric cells, WT [0.33 ± 0.19%] versus β-cat^GOF-UB [0.40 ± 0.16%], P = 0.78). Apoptosis in the metanephric mesenchyme was increased 4.9-fold (number TUNEL-positive cells per mm², 0.1 ± 0.02 versus 0.55 ± 0.12, *P = 0.015) in β-cat^GOF-UB mice when compared with that in WT (J, K).

Overexpression of β-Catenin Causes Abnormal Regulation of Gene Expression in the Embryonic Kidney

During normal kidney development, β-catenin functions within ureteric cells to control the expression of a network of genes, each of which is required for branching morphogenesis.²⁶ We hypothesized that abnormally high levels of β-catenin may disrupt normal levels of gene transcription, as has been observed in nonrenal tissues.^27,28 We performed a global analysis of gene expression changes using WT and β-cat^GOF-UB kidney tissue isolated at E12.5, a stage at which decreased ureteric branching precedes histologic evidence of renal hypodysplasia. We reasoned that changes in gene expression detected at this stage would be more likely related to changes in β-catenin expression, itself, versus pathogenic changes associated more generally with hypodysplasia. In triplicate experiments, we compared mRNA species isolated from WT and β-cat^GOF-UB mutant kidneys (Figure 4, A and B) using the mouse genome 430 2.0 array (Affymetrix) which contains 45,000 probe sets representing over 20,000 genes. Investigation of microarray data by hierarchical cluster analysis revealed a low level of variability among biologic replicates (Figure 4C) and identified 1744 differentially expressed transcripts (993 upregulated and 751 downregulated) between WT and mutant kidneys using a statistical cutoff of P < 0.003 (Supplemental Tables 1 and 2).

Figure 4.

β-catenin stabilization in ureteric cells increases Tgfβ2 and DKK1 expression. (A, B) Ureteric bud–specific (UB-specific) green fluorescent protein fluorescence to identify the UB branching pattern in WT and β-cat^GOF-UB mice. Ureteric tissue is marked with a white arrow. WT and β-cat^GOF-UB mice were pooled to generate biologic triplicates. (C) Heat-map representation of differentially expressed genes between WT and β-cat^GOF-UB demonstrates a low level of variability of differentially expressed transcripts among sample replicates. Each lane represents individual biologic replicates normalized to each of three WT samples. (D through I) In situ hybridization for Gdnf, Ret, and Wnt11 in E12.5 WT and β-cat^GOF-UB kidneys. Gdnf was expressed in a mosaic pattern around the ureteric tips in β-cat^GOF-UB mice and Ret expression was limited to the tips of the UB tips in WT and β-cat^GOF-UB (black arrows). (H, I) In contrast to WT, Wnt11 was nearly absent in β-cat^GOF-UB ureteric tips (black arrows). (J) Validation of microarray data by quantitative real-time PCR confirmed a significant upregulation of Tgfβ2 (WT versus mutant: 0.013 ± 0.009 versus 0.067 ± 0.35, P = 0.03) and Dkk1 (0.9 ± 0.05 versus 1.41 ± 0.16. P = 0.038). (K through P) Spatial localization of the candidate genes. (K, L) Tgfβ2 mRNA is upregulated in E12.5 UB cells in β-cat^GOF-UB mutants. (M, N) Immunohistochemistry demonstrates a marked increase in Tgfβ2 protein expression in the metanephric mesenchyme (MM) in β-cat^GOF-UB mutants. (O,P) Dkk1 is markedly upregulated in E12.5 UB cells (UB) in β-cat^GOF-UB mutants. No changes in Tgfβ2 and Dkk1 mRNA were observed in MM cells.

We performed a gene ontology analysis to identify functionally related groups of differentially expressed genes. A complete list of the functional categories and their accompanying genes are shown in Supplemental Tables 3 and 4. Among the functional gene groups downregulated in mutant kidneys were those containing genes involved in ureteric bud development and nephrogenesis (Table 2). Among these categories were genes that make a functional contribution to kidney development. These genes include Gdnf, Lhx1, Pax8, Cited 1, and Sall1. Inspection of the complete list of downregulated transcripts for genes not categorized within the gene ontology analysis but known to be important for renal development also identified Wnt4 (Table 3). Next, we used in situ hybridization to examine the expression of genes that play a pivotal role in ureteric branching. During ureteric branching, glial cell line–derived neurotrophic factor (GDNF) acts within a GDNF-RET-WNT11 positive autoregulatory feedback loop to regulate branching morphogenesis.⁴ Although Ret expression was maintained in ureteric tip cells in β-cat^GOF-UB mice (Figure 4, F and G), Gdnf demonstrated a mosaic pattern of mRNA expression (Figure 4, D and E) and Wnt11 was markedly reduced in ureteric tip cells (Figure 4, H and I). Together, these findings would predict a more modest effect on renal development than that observed in β-cat^GOF-UB mice^4,29,30 and suggest that additional mechanisms contribute to this severe phenotype. Indeed, although treatment of WT embryonic kidney explants with exogenous GDNF induced a 1.5-fold increase in the number of ureteric branches, no difference in ureteric branching was observed in GDNF-treated β-cat^GOF-UB embryonic kidney explants (Supplemental Figure 2).

Table 2.

Gene ontology analysis of selected functional categories of genes decreased in kidney tissue of β-cat^GOF-UB mice

GO-ID	P	Description	Genes in test set
1822	1.25 × 10−8	Kidney development	GDNF SPRY1 BMP2 SALL1 PAX8 PBX1 BMP7 SLIT2 SHH
1656	2.08 × 10−7	Metanephros development	SPRY1 BMP2 SALL1 PAX8 PBX1 GDNF SHH
1657	4.22 × 10−7	Ureteric bud development	BMP2 TCF7 CHERP JARID2 PDGFA JAG1 SHH BRCA1 THY1 MYCN MINA IGF1R OSR2 BCL2 ID4 PBX1 LAMC1

Table 3.

Selected mRNA transcripts significantly decreased (P < 0.003) in kidney tissue of β-cat^GOF-UB mice

Probe set	Gene title	Gene symbol	Fold change
1450428	LIM homeobox protein 1	Lhx1	4.92
1450782	Wingless-related MMTV integration site 4	Wnt4	3.25
1418208	Paired box gene 8	Pax8	2.83
1426155	Odd-skipped related 2 (Drosophila)	Osr2	2.64
1440650	Slit homolog 2 (Drosophila)	Slit2	2.46
1448738	Calbindin-28K	Calb1	2.30
1456258	Empty spiracles homolog 2 (Drosophila)	Emx2	2.30
1421106	Jagged 1	Jag1	2.30
1419080	Glial cell line derived neurotrophic factor	Gdnf	2.14
1448886	GATA binding protein 3	Gata3	1.87
1431225	Bone morphogenetic protein 7	Bmp7	1.62
1431225	Sprouty homolog 1 (Drosophila)	Spry1	1.52
1431225	Cbp/p300-interacting transactivator	Cited2	1.52
1431225	Sal-like 1 (Drosophila)	Sall1	1.41
1431225	Glypican 3	Gpc3	1.32

Identification of upregulated genes in β-cat^GOF-UB mice using a gene ontology analysis revealed an enrichment of genes involved in cell morphogenesis, cell motility, muscle cell development, vasculogenesis, and negative regulation of signal transduction (Table 4). Inspection of upregulated genes for those that regulate branching morphogenesis and that were not categorized within the ontology analysis identified effectors in signaling pathways controlled by TGFβ (Tgfβ2 and Tgfβr2) and WNT proteins. Interestingly, those genes in the WNT pathway encoded WNT inhibitors including Dickkopf 1 (Dkk1), WNT inhibitory factor 1 (Wif1), and secreted frizzled-related protein (sFrp) (Table 5). Because previous studies demonstrated that Dkk1 and Tgfβ2 inhibit ureteric branching and nephrogenesis, respectively,^31,32 we proceeded with further analysis of these effectors. Upregulation of Tgfβ2 and Dkk1 was verified using quantitative real-time PCR on mRNA isolated from β-cat^GOF-UB and WT kidneys (P = 0.038) (Figure 4J). The spatial expression of Tgfβ2 and Dkk1 was next determined by in situ hybridization. In E12.5 WT mice, Tgfβ2 mRNA transcripts were detected at low levels in the metanephric mesenchyme and ureteric cells (Figure 4K). In contrast, Tgfβ2 mRNA transcripts were expressed at high levels in the ureteric cells of β-cat^GOF-UB mice (Figure 4L). We hypothesized Tgfβ2 protein is generated in the UB and then secreted to the mesenchyme. Consistent with our hypothesis, examination of Tgfβ2 protein expression by immunohistochemistry revealed marked up regulation of Tgfβ2 protein in the metanephric mesenchyme of β-cat^GOF-UB mice (Figure 4, M and N). In WT E12.5 tissue, Dkk1 mRNA transcripts were expressed at low levels in the mesenchyme and were virtually absent from the ureteric cells (Figure 4O). In contrast, in β-cat^GOF-UB mice, a significant increase in Dkk1 expression was observed in ureteric cells, whereas low levels were maintained in the metanephric mesenchyme (Figure 4P). Together, these results demonstrate increased Tgfβ2 and Dkk1 mRNA expression in ureteric cells in β-cat^GOF-UB mice.

Table 4.

Gene ontology analysis of selected functional categories of genes increased in kidney tissue of β-cat^GOF-UB mice

GO-ID	P	Description	Genes in test set
9653	2.83 × 10−10	Morphogenesis	MYO7A IGFBP6 TBX20 HOXD13 TGFB2 OSR1 GATA6 SOX17 SLIT3 CNTN4 TBX18 GAP43 SOX2 COL8A2 COL8A2 TGFBR2 IGF1 DKK1 NFIB
7148	5.74 × 10−5	Cell morphogenesis	MYO7A IGFBP6 TGFB2 ACTN2 SLIT3 NTRK2 CNTN4 GAP43
1570	8.22 × 10−4	Vasculogenesis	PTPRJ NTRK2 ZFPM2 WARS2 SOX17
3528	2.87 × 10−3	Segmentation	SFRP1 ZFHX1B MAFB PCDH8 TBX18
30111	3.16 × 10−3	Regulation of Wnt receptor signaling pathway	DKK2 DKK1 MDFIC WIF1

Table 5.

Selected mRNA transcripts significantly increased (P < 0.003) in kidney tissue of β-cat^GOF-UB mice

Probe set	Gene title	Gene symbol	Fold change
1425425	Wnt inhibitory factor 1	Wif1	6.96
1449350	Odd-skipped related 1 (Drosophila)	Osr1	1.74
1458232	Dickkopf homolog 1 (Xenopus laevis)	Dkk1	1.74
1423250	Transforming growth factor, beta 2	Tgfb2	1.52
1455851	Bone morphogenetic protein 5	Bmp5	1.52
1426397	Transforming growth factor, beta receptor II	Tgfbr2	1.41
1418876	Secreted frizzled-related sequence protein 1	Sfrp1	1.41
1418876	Forkhead box D1	Foxd1	1.32
1443221	Wilms tumor homolog	Wt1	1.23

TGFβ2 Inhibits Ureteric Branching and Expands the Population of Committed Nephrogenic Precursor Cells

We investigated the functional contribution of increased Tgfβ2 expression to renal dysplasia using an in vitro culture model of murine renal development. Embryonic kidney explants isolated at E12.5 and cultured for 48 hours are characterized by multiple ureteric branches, each of which is adjacent to a two- to three-cell-thick layer of condensed cap mesenchyme. This condensed mesenchyme is distinct from uninduced mesenchyme, which exists at a greater distance from ureteric branch tips and is loosely organized (Figure 5A). Treatment with recombinant TGFβ2 (50 ng/ml) for 48 hours resulted in decreased ureteric branching (Figure 5, B versus A), and condensation of metanephric mesenchyme cells throughout almost the entire explant, even at sites distant from ureteric branch tips (Figure 5B). Next, we determined if the condensed appearance of the mesenchyme indeed reflected induction of these cells by investigating the expression of PAX2, neural cell adhesion molecule (NCAM), and CITED1, markers of induced mesenchyme cells. In untreated kidneys, PAX2 was expressed in ureteric cells, the condensing mesenchyme around the tips of the ureteric bud, and in developing nephrogenic structures (Figure 5E). Remarkably, in TGFβ2-treated kidneys, nearly the entire mass of the metanephric mesenchyme was PAX2-positive (Figure 5F). In untreated explants, NCAM expression was restricted to mesenchymal cells in close proximity to ureteric tips (Figure 5E). In contrast, in TGFβ2-treated explants, NCAM was coexpressed with PAX2 in cells distant from ureteric branch tips (Figure 5F). During renal embryogenesis, CITED1 expression is normally limited to mesenchyme cell aggregates, approximately two- to three-cells-thick around the ureteric tips (Figure 5I). In contrast, TGFβ2-treated kidneys demonstrated CITED1 expression throughout the entire mass of the metanephric mesenchyme (Figure 5J). Next, we determined the relevance of these findings to the cellular changes observed in β-cat^GOF-UB mutant mice. Analysis of marker expression in renal tissue from mutant mice revealed striking similarities to TGFβ2-treated kidney explants (Figure 5, C through L). Kidney tissue isolated from β-cat^GOF-UB mice was characterized by aggregation of virtually the entire mass of the metanephric mesenchyme (Figure 5, D and C). Moreover, expression of PAX2, NCAM, and CITED1 was detected throughout the entire mesenchyme cell mass (Figure 5, G through L). To determine if alterations in the stroma were also observed in β-cat^GOF-UB mutant kidneys, we analyzed the expression of Foxd1, a cortical stromal marker at E12.5. Foxd1 was expressed in a similar pattern in the cortical stroma in β-cat^GOF-UB versus WT kidneys (Supplemental Figure 3, A and B). Similarly, analysis of α-smooth muscle actin protein demonstrated no evidence of ectopic expression in E13.5 mutant kidney tissue (Supplemental Figure 3, C and D). Together, these studies indicate that TGFβ2 inhibits ureteric branching and induces metanephric mesenchyme cells to initiate a nephrogenic program even in the absence of signaling by adjacent ureteric cells as observed in β-cat^GOF-UB mice.

Figure 5.

Expanded and ectopic metanephric mesenchyme (MM) cell induction in β-cat^GOF-UB mutants. (A, B, E, F, I, J) Analysis of TGFβ2-treated E12.5 kidney explants and (C, D, G, H, K, L) kidney tissue isolated from E12.5 WT and β-cat^GOF-UB mice. (A through D) H&E stained kidney tissue in untreated and TGFβ2-treated kidney explants (A, B) and WT and β-cat^GOF-UB mutants (C, D) (black arrows). TGFβ2-treated (B) and β-cat^GOF-UB kidneys (D) demonstrate an expansion of mesenchymal aggregates around the tips of the UB (black arrows). (E through H) Dual-label immunofluorescence imaging of PAX2 and NCAM demonstrating expanded and ectopic NCAM and PAX2 expression in TGFβ2-treated kidney explants (E, F) and β-cat^GOF-UB mice (white arrow) (G, H). (I through L) Expression of CITED1, a nephrogenic lineage–specific marker, confirms an expansion of induced mesenchyme in TGFβ2-treated kidney explants (white arrow) (I, J). (K, L) CITED1 and NCAM co-immunofluorescence in WT and β-cat^GOF-UB mice confirms an expanded domain of cells expressing CITED1 adjacent to the UB (white arrows).

WNT-Dependent Nephrogenesis Is Disrupted in β-cat^GOF-UB Mouse Kidney

In contrast to our results demonstrating that Tgfβ2 promotes nephrogenesis, β-cat^GOF-UB kidney tissue is characterized by nephron deficiency. We investigated this apparent paradox by examining the expression of the WNT-dependent genetic pathway that is required for nephrogenesis using in situ hybridization in WT and mutant kidney tissue. Wnt9b, Wnt4, and Lim1 act sequentially to control nephrogenesis.^5,6,33 Wnt9b is the primary inductive signal required for the formation of the renal vesicle and acts upstream of Wnt4.⁵ Wnt9b was expressed in a normal pattern in both WT and β-cat^GOF-UB kidney tissue (Figure 6, A and B). In WT mice, Wnt4 is expressed in pretubular aggregates (Figure 6C). In contrast, in β-cat^GOF-UB mutant kidneys, Wnt4 expression was misexpressed in mesenchyme cells lining ureteric branches (Figure 6D). Lim1 is expressed in ureteric cells, pretubular aggregates, and renal vesicles in WT mice and is activated by Wnt4^6,9,33,34(Figure 6E). In β-cat^GOF-UB mutants, Lim1 was observed in the ureteric tissue but was absent from the metanephric mesenchyme (Figure 6F).

Figure 6.

Disruption in nephrogenesis is Dkk1-dependent. (A through F) Analysis of genes necessary for nephrogenesis. Wnt9b localizes to the ureteric bud (UB) in E12.5 β-cat^GOF-UB mice in a pattern similar to WT (A, B). In contrast to WT, Wnt4 is ectopically expressed in the induced mesenchymal aggregates surrounding the ureteric tips in β-cat^GOF-UB mice (black arrow) (C, D). Lim1 mRNA is expressed in UB cells in both WT and β-cat^GOF-UB mutants. In contrast to WT, Lim1 mRNA expression is practically absent from the metanephric mesenchyme. (E, F). Resected E11.5 kidney explants were treated with DKK1 for 96 hours. CYTOKERATIN (red) demonstrates a similar ureteric branch pattern in untreated and DKK1-treated samples. In contrast, a noticeable decrease in brush boarder–positive structures (proximal tubules, green, white arrows) is observed in DKK1-treated samples (G, H). (I, J) Analysis of activated caspase-3 expression (apoptotic marker) in DKK1-treated explants. Activated caspase-3 expression is increased within metanephric mesenchyme in DKK1-treated kidney explants. n, nephrogenic structure.

Our finding that Dkk1 is upregulated in mutant kidney tissue was particularly interesting given the disruption in WNT signaling. Consistent with the disruption of WNT-dependent nephrogenesis, our global gene expression analysis indicated upregulation of Dkk1. DKK1 inhibits canonical WNT signaling by binding to the frizzled coreceptor, LRP5/6, thereby blocking ligand-receptor interactions.³⁵ We determined the effects of DKK1 on nephrogenesis using recombinant DKK1 in embryonic kidney explant cultures. Kidney explants isolated from E11.5 mice were incubated in the presence or absence of 1 μg/ml DKK1. In contrast to untreated kidneys, which were characterized by multiple nephron structures identified by brush boarder positive structures (Figure 6G), DKK1-treated explants demonstrated a marked reduction in these nephron structures (Figure 6H). Remarkably, exogenous DKK1 resulted in an increase in the metanephric mesenchyme cell apoptosis (Figure 6, I and J). Together, these data demonstrate an interruption of WNT-dependent signaling in β-cat^GOF-UB mice and suggest a requirement for WNT signaling in cell survival.

Temporal Stabilization of β-Catenin after the Onset of Renal Development Causes Renal Dysplasia

Overexpression of β-catenin is observed in human and murine renal dysplasia.^12–14 Analyses of β-catenin expression in some genetic mouse models indicate that upregulation begins after the onset of tissue malformation.¹⁴ These findings suggest that β-catenin may participate in a secondary pathway of tissue injury in renal dysplasia. To investigate this possibility, we generated mice in which β-catenin overexpression was timed to occur after the onset of kidney development. We determined whether these result in similar cellular and molecular consequences to those observed in mice with β-catenin overexpression starting at the onset of kidney development. Stabilization of β-catenin after the initiation of renal morphogenesis was achieved by breeding transgenic mice containing a ubiquitous Cre recombinase fused to the ligand-binding domain of the estrogen receptor (CreER^T)³⁶ to β-cat^Δ3 mice. Pregnant mice, 14.5-days postcoitus, were treated with a single intraperitoneal injection of tamoxifen and pups were isolated after 48 hours. Histologic examination of E16.5 CreER^T;β-cat^Δ3/+ embryonic kidney tissue revealed reduced kidney size (Figure 7, A and C). Because nephrons are formed in an axis from the interior to the periphery of the kidney, nephrons formed during the early stages of kidney development are located in the kidney interior that will become the medulla. Indeed, glomeruli were observed in the deep cortex, which likely formed before the stage at which tamoxifen was administered to CreER^T;β-cat^Δ3/+ mice (Figure 7, C and D). In contrast, in mutant mice the peripheral nephrogenic zone was devoid of condensing mesenchyme and developing nephrogenic intermediate structures and contained an increased mass of loosely organized stromal cells (Figure 7, C and D). To further investigate the differentiated state of the cells in the nephrogenic zone, we defined the expression pattern of PAX2 and CITED1, which are misexpressed in TGFβ2-treated WT kidney explants. In CreER^T;β-cat^Δ3/+ renal tissue, CITED1 was misexpressed in nonaggregated cortical mesenchyme and in epithelial tubules also located in the cortex. CITED1-expressing tubules did not coexpress cytokeratin, a marker of ureteric cells, suggesting that they are derived from mesenchyme. This expression pattern was in sharp contrast to the restricted expression of CITED1 in cap mesenchyme cells in WT mice (Figure 7, E and F). In contrast to WT mice, PAX2 was virtually absent from the cortical mesenchyme in CreER^T;β-cat^Δ3/+ mice (Figure 7, G and H). Taken together, these results indicate that upregulation of β-catenin abrogates nephron formation in the presumptive nephrogenic zone and causes misexpression of genes that control early stages of the mesenchymal to epithelial transformation. These deleterious effects are more severe than those observed in mice in which stabilization of β-catenin is targeted to the Six-2–positive subpopulation of metanephric mesenchyme cells, which are committed to forming nephrons at the time of β-catenin stabilization.⁹

Figure 7.

Tamoxifen-induced overexpression of β-catenin in E14.5 embryos results in renal dysplasia. (A, D) H&E stained sections from resected E16.5 kidneys from WT and CreER^T;β-cat^Δ3/+ mice. CreER^T;β-cat^Δ3/+ mice demonstrate slightly smaller kidneys with an otherwise normal kidney morphology. In contrast to WT kidneys, CreER^T;β-cat^Δ3/+ kidneys demonstrate an absence of a nephrogenic zone (black asterisk), a paucity of nephrogenic intermediate structures, and a deficiency developing and maturing glomeruli (D). Deep cortical glomeruli are observed in both WT and CreER^T;β-cat^Δ3/+ mutant mice (black arrows). (E, F) CITED1-CYTOKERATIN dual immunofluorescence demonstrates ectopic CITED1 expression in the mesenchyme and tubule structures in E16.5 CreER^T;β-cat^Δ3/+ mice (white arrows). In contrast, CYTOKERATIN was not observed in cortical tubules or medullary tubules in kidneys from CreER^T;β-cat^Δ3/+ mice (white arrowhead). (G, H) PAX2 and NCAM co-immunofluorescence. PAX2 is expressed in renal tubules (T), and condensing mesenchyme (CM) around the tips of the UB in WT (white arrow, CM). Similar to WT mice, PAX2 is expressed in renal tubules, but is virtually absent in the cortical mesenchyme in CreER^T;β-cat^Δ3/+ mice (white asterisk). A similar pattern of NCAM expression was observed in WT and CreER^T;β-cat^Δ3/+. (I, J) In situ hybridization for Tgfβ2 demonstrates an increase in mRNA expression in tubules localized in the cortex and medullary regions in CreER^T;β-cat^Δ3/+ kidneys when compared with WT (black arrows and highlighted in inset boxes). (K, L) Immunohistochemistry demonstrates increased Tgfβ2 protein expression in glomeruli and tubules in CreER^T;β-cat^Δ3/+mice. Tgfβ2 protein expression was also observed in the condensing mesenchyme surrounding the UB (highlighted in inset boxes). (M, N) In situ hybridization for Dkk1 demonstrates an obvious increase in mRNA expression in cortex and medullary tubules (black arrow) in CreER^T;β-cat^Δ3/+ kidneys when compared with WT (black asterisk). DG, developing glomeruli; G, glomeruli; CM, condensing mesenchyme; T, tubules; NZ, nephrogenic zone.

Next, we determined whether overexpression of β-catenin at stages during which nephrogenesis and branching morphogenesis are already established increased Tgfβ2 and Dkk1 as we observed in β-cat^GOF-UB mice. In situ hybridization revealed that Tgfβ2 was rarely detected in cortical or medullary tubules in WT mice (Figure 7I). In contrast, Tgfβ2 was expressed in the vast majority of tubules in CreER^T;β-cat^Δ3/+ mice (Figure 7, I and J) in a similar pattern to that observed in β-cat^GOF-UB mice (Figure 4, K and L). Consistent with these results, Tgfβ2 protein was detected at low levels and was localized to only a small subset of tubules primarily localized in the medulla of WT mice (Figure 7K). In contrast, in CreER^T;β-cat^Δ3/+ mice high levels of Tgfβ2 protein were detected in numerous tubules localized in the cortex and medulla, deep glomerular structures, and condensing mesenchyme in the outer cortex (Figure 7, K and L, inset boxes). In mice not treated with tamoxifen, Dkk1 expression was restricted to mesenchyme in the outer renal cortex (Figure 7M). However, in CreER^T;β-cat^Δ3/+ mice, Dkk1 expression was expanded within the peripheral cortex and was misexpressed within epithelial tubules in the deep cortex (Figure 7N). Together, these studies indicate that overexpression of β-catenin at stages well after the initiation of kidney development causes renal tissue malformation associated with aberrant expression of CITED1, PAX2, Tgfβ2, and Dkk1 as observed in β-cat^GOF-UB mutant mice. Moreover, the morphologic and cellular abnormalities observed in β-catenin overexpressing mice mimic those observed in humans, suggesting a pathogenic role for β-catenin in human renal dysplasia.

DISCUSSION

Renal dysplasia is a complex disorder characterized by variable phenotype and severity. At the level of histopathology, renal dysplasia is characterized by a core set of features including reduction in the number of nephrons and collecting ducts, disorganization of tissue elements, and abnormal patterning of cortical and medullary tissues.³ The degree of nephron and collecting duct deficiency, the focal versus diffuse nature of the dysplastic phenotype, and the degree of epithelial cyst formation varies widely among cases. These variations are consistent with the observed variation of clinical phenotypes ranging from aplasia, diffuse dysplasia with variable degrees of cystic transformation, and focal dysplasia. The recent identification of gene mutations in affected individuals is beginning to provide insight into the primary molecular mechanisms that control renal tissue malformation.³⁷ Yet the variable severity and phenotype observed in individuals with the same mutation³ suggests that additional mechanisms control renal tissue maldevelopment.

Previously, we reported elevated β-catenin expression in human renal dysplastic tissue with different underlying etiologies.¹² This observation, combined with our recognition that β-catenin expression is increased in models of murine renal dysplasia in which gene expression is manipulated in the ureteric lineage,^12,14 led us to hypothesize that β-catenin may be elevated as a secondary manifestation during the morphogenesis of renal dysplasia. Here, we investigated the functional contribution of β-catenin to the pathogenesis of renal tissue malformation. β-Catenin stabilization in the ureteric cell lineage resulted in renal aplasia or dysplasia. Disruption of renal development was observed whether β-catenin stabilization was present at the initiation of renal development, or during intermediate stages of kidney development. These findings indicated that nephrogenesis and branching morphogenesis are vulnerable to increased β-catenin expression during initial ureteric-mesenchymal interactions and at stages during which these interactions are well-established. Analysis of pathogenic mechanisms that could underlie β-catenin function revealed novel changes in gene expression involving TGFβ signaling effectors (Tgfβ2) and WNT inhibitors (Dkk1). Our data demonstrate that these effectors inhibit ureteric branching and nephrogenesis in cultures of embryonic kidney explants.

Overexpression of β-catenin in the Wolffian duct and ureteric cell lineages resulted in downregulation of genes that play critical roles during renal development. Yet subsequent analysis suggested that the degree of downregulation did not account for the severity of the renal phenotype. Indeed, analysis of the spatial expression of Ret indicated that its expression was mildly increased. Gdnf mRNA expression was reduced but not totally lost (Figure 4, D through I). Yet β-cat^GOF-UB mutant kidneys manifested a complete arrest of branching morphogenesis by E12.5, which is more consistent with a complete loss of the GDNF/RET signaling.^38,39 Moreover, total deficiency of Wnt11 observed in β-cat^GOF-UB mutant mice causes renal hypoplasia, characterized by a modest decrease in the number of ureteric branch tips, not severe hypodysplasia.^4,29 Thus, we posited that the relative severity of the renal phenotype in β-cat^GOF-UB mice was due to either a combinatorial effect of decreased expression shared by many genes important to renal development and/or transcriptional effects directly related to the increased dose of β-catenin.

A global gene expression analysis revealed upregulation of numerous genes involved in morphogenesis and WNT signaling (Table 4). Particularly notable among these genes was Tgfβ2 and inhibitors of WNT signaling. TGFβ2 is expressed in the basement membrane surrounding ureteric cells during kidney development and decreases ureteric branching and nephrogenesis when applied to embryonic kidney explant cultures,⁴⁰ and is upregulated in human fetal dysplastic tissue.⁴¹ Furthermore, Tgfβ2 heterozygous mice are characterized by increased ureteric branch length and nephron number, consistent with a physiologic inhibitory effect of Tgfβ2 in vivo.³¹ Combined, these results support a role for TGFβ2 inhibiting ureteric branching in β-cat^GOF-UB mutant mice.

Histologic and molecular analysis of β-cat^GOF-UB mutant kidneys demonstrated an increased population of committed mesenchyme progenitors. Consistent with this observation, treatment of WT embryonic kidney explants with TGFβ2 caused a similar effect. Yet we rarely detected nephrogenic structures beyond the renal cap mesenchyme stage in β-cat^GOF-UB mutant or TGFβ2-treated WT mouse kidneys, suggesting a block in the nephrogenic program. Abrogation of the nephrogenic program is likely explained by concomitant expression of Dkk1, an inhibitor of WNT signaling. Nephrogenesis is dependent on the sequential expression of Wnt9b in ureteric cells,⁵ Wnt4 in the condensing mesenchyme,^6,34 and Lim1 in renal vesicles.³³ Our data demonstrate reduced and disorganized expression Wnt4, and Lim1, findings that are consistent with misexpression of Dkk1 and our demonstration that administration of DKK1 to WT embryonic kidney explants reduces nephron formation.

Our results demonstrate that upregulation of β-catenin, Tgfβ2, and Dkk occurs at two distinctly different maturational stages. That is, we find upregulation of these effectors when β-catenin is overexpressed at the onset of kidney development and well after the onset of nephrogensis and branching morphogensis. During embryogenesis, Tgfβ2 induces mesenchymal cells to express molecular markers characteristic of the transition from mesenchyme to epithelium.⁴² The importance of these actions is demonstrated by the dysplastic renal phenotype in Tgfβ2-deficient mice.⁴³ Members of the DKK family of WNT signaling inhibitors are expressed at low levels during kidney embryogenesis (www.gudmap.org), consistent with the critical functions performed by WNT family members. Indeed, treatment of kidney tissue explants with recombinant DKK1 inhibits renal development³² and Figure 6, G and H. In human disease, DKK family members are increased along with WNT genes and their effectors.⁴⁴ Because DKK genes are WNT targets, DKK expression may serve to negatively regulate WNT signaling within an autoregulatory signaling cascade.

Our results provide the basis for a model that predicts the overlapping and distinct effects of β-catenin overexpression during renal embryogenesis. Our model predicts that overexpression of β-catenin in ureteric cells during embryogenesis causes increased expression of TGFβ2 and Dkk1 (Figure 8). In this context, Tgfβ2 acts in an autocrine manner to inhibit ureteric branching and acts in a paracrine manner to induce the formation of an ectopic population of mesenchyme cells committed to a nephrogenic fate. Progression of nephrogenesis from these cells, as well as those induced by adjacent ureteric cells, is inhibited by the simultaneous paracrine actions of DKK1. Together, these signaling events act in addition to the primary defect that led to β-catenin overexpression to generate a dysplastic phenotype. Our model has important implications for future research aimed at further establishing the functional roles of TGFβ and Dkk signaling in renal dysplasia and in the development of therapeutic strategies aimed at treating these disorders. The concept that renal dysplasia is a multistage process controlled by signaling pathways that are triggered by a primary insult provides a new foundation for developing molecular therapies aimed at attenuating the activities of these pathways.

Figure 8.

Effects of β-catenin overexpression during kidney embryogenesis. Overexpression of β-catenin in ureteric cells during embryogenesis increases Tgfβ2 and Dkk1. Tgfβ2 acts in an autocrine manner to inhibit ureteric branching. In addition, Tgfβ2 acts in a paracrine manner to induce the formation of an ectopic population of mesenchyme cells committed a nephrogenic fate. Progression of nephrogenesis within this ectopic population of cells, as well as those induced by adjacent ureteric cells, is inhibited by the simultaneous paracrine actions of DKK1. CM, condensing mesenchyme; MM, metanephric mesenchyme; RV, renal vesicle; WD, Wolffian duct; UB, ureteric bud; UBM, ureteric branching morphogenesis.

CONCISE METHODS

Mice

Hoxb7-Cre:EGFP mice⁴⁵ were crossed with mice containing loxP sites flanking exon 3 of the β-catenin allele (β-cat^Δ3/Δ3)¹⁸ to generate β-catenin gain-of-function mutant mice, termed β-cat^GOF-UB. TCF-β-galactosidase reporter²⁰ and ROSA26 reporter mice¹⁹ were crossed to β-cat^Δ3/Δ3 to generate β-cat^Δ3/+;ROSA or β-cat^Δ3/+;TCF mice. These mice were subsequently crossed to Hoxb7-Cre:EGFP mice. PCR genotyping was performed as described.^18,20 β-Galatosidase staining was performed as described previously.⁴⁶ Tamoxifen-inducible mice (CreER^T) were crossed to β-cat^Δ3/Δ3 mice. Pregnant females were injected intraperitoneally with a single dose of tamoxifen (3 mg/40 g body wt) (Sigma T5648) for 48 hours. Tamoxifen was prepared as described previously.⁴⁷ Mouse experiments complied with ethical standards of the Hospital for Sick Children Research Institute Animal Care Committee.

Histology, Immunofluorescent Microscopy, Immunohistochemistry, and Western Analysis

Whole kidney tissue was fixed in 4% paraformaldehyde for 24 hours at 4°C with agitation. Paraffin-embedded embryos were analyzed by histology after generating 4-μm tissue sections and stained with hematoxylin and eosin or Dolicus bifloris aggulatinin (Vector Labs). Immunofluorescence was performed on 4% paraformaldehyde fixed tissue sections using antibodies specific for β-catenin (Upstate, Lake Placid, NY; 1:200 dilution), PAX2 (Covance, Berkley, CA; 1:200 dilution), NCAM (Sigma, St Louis; 1:100 dilution), pan cytokeratin (Sigma; 1:200 dilution), and CITED1 (Neomarkers, Fremont, CA,; 1:200 dilution and anti–active caspase-3 (BD Transduction Labs; 1:200 dilution). Whole mount immunofluoresence was performed as described⁴⁸ using anti–pan cytokeratin (1:200) and anti–Brush Boarder antibody (Gift from Aaro Miettinen, University of Helsinki). Alexa 568 goat anti-mouse and Alexa 488 goat anti-rabbit were used as secondary antibodies (Invitrogen; 1:500 dilution). Immunohistochemistry was performed using anti-Tgfβ2 (Santa Cruz). The reaction was visualized using AP-conjugated secondary antibody (Cedarlane) and AP substrate to develop color reaction (Promega). WT and β-cat^GOF-UB mutant whole kidneys were homogenized in T-PER tissue protein extraction reagent (Thermo Scientific) supplemented with Protease inhibitor cocktail (Thermo Scientific). For immunoblotting, primary anatibodies were directed against β-catenin (Upstate; 1:1000) and GAPDH (Abcam; 1:1000).

Global Genome Expression Analysis in Mouse Kidney Tissue

Eighteen β-cat^GOF-UB mutant kidneys and 9 WT kidneys were microdissected at E12.5. Mutant kidneys were divided into three random pools (n = 3) consisting of six kidneys each, and WT samples were divided into three pools (n = 3) consisting of three kidneys each. RNA quantity (1-μg total RNA) was sufficient such that only one cycle of amplification was required. Microarray data were processed using GCOS (v1.4, Affymetrix). All chips were scaled to a target value of 500 before expression analysis. Two WT replicates were normalized to a third WT replicate and these were used as baselines to normalize and compare the three replicate β-cat^GOF-UB samples. Comparisons were made in all combinations to create a matrix of 3 × 3 crosswise comparisons (9 in total). Probe sets with present calls in all replicates in either the β-cat^GOF-UB or WT samples were kept and all others were removed from the data set. Probe sets with a significant change call in 6 of 9 comparisons were considered significantly changed in the β-cat^GOF-UB versus WT samples. To minimize the false positives, a threshold signal log ratio (SLR) value was determined by estimating the background error using the distribution of SLR for probe sets with no significant change calls.

Probe sets were mapped to their gene symbol identifiers from annotation tables supplied by Affymetrix (http://www.affymetrix.com). Lists of gene symbols were entered into the BINGO plugin (v2.0⁴⁹) for Cytoscape (v 2.4⁵⁰) and compared with Gene Ontology (GO) annotation tables from MGI (http://www.informatics.jax.org/) to calculate the enrichment of terms and their P value, which was then adjusted by Benjamini Hochberg correction for false discovery rate.⁵¹ All reported enrichments were at a significance of 0.05 or less.

Real-Time Reverse Transcriptase-PCR (RT-PCR)

We validated the microarray data using real-time PCR amplification using the Applied Biosystems 7900HT fast RT-PCR system. cDNA was generated using first-strand cDNA synthesis (Invitrogen). The real-time PCR reaction contained 3 ng of each cDNA sample, SYBR green PCR Master Mix (Applied Biosystems) and 300 nM of each primer to a total volume of 25 μl. Primers for TGFβ2, DKK family members, Pax2, E-cadherin, and β-2-microglobin were designed using Primer 3 software and verified using the University of California, Santa Cruz (UCSC) genome bioinformatics web site (http://genome.ucsc.edu). The annealing temperature was restricted to 59 to 60°C and the length of the PCR product was set between 100 and 200 bp. Specificity of the amplification was carried out by agarose gel electrophoresis. Relative levels of mRNA expression were carried out using the standard curve method. Individual expression values were normalized by comparison to β-2-microglobin.

Electron Microscopy

Kidneys were fixed in 2% glutaraldehyde for 24 hours, rinsed with 0.1 M sodium cocadylate, and fixed for 1 hour in 0.2% tannic acid followed by graded fixation in 1% osmium tetroxide and in 1% osmium tetroxide/1.25% potassium ferrocyanide. After dehydration, samples were embedded in SPURR resin, sectioned, collected on copper grids, and stained for electron microscopy. Images were obtained using a FEI Tecnai 20 transmission electron microscope.

In Situ Hybridization

Nonradioactive in situ hybridization was performed using DIG-labeled cRNA probes encoding Ret, Gdnf, Wnt11, Wnt4, Lim1, Dkk1, and Foxd1 on paraffin-embedded kidney tissue fixed with 4% PFA for 24 hours at 4°C as described previously.⁵²

In Situ TUNEL and BrdU Incorporation Assays

Terminal deoxynucleotidyl transferase–mediated (TdT-mediated) dUTP nick end labeling (TUNEL) was performed using 4% PFA fixed paraffin-embedded tissue sections. Briefly, tissue sections were deparaffinized, rehydrated, and enzyme-digested (10 μg/ml Proteinase K in PBS for 15 minutes). Labeling was performed according to manufacturer's instructions with a 10-minute color reaction and counterstaining with hematoxylin. Slides were then dehydrated and mounted with Permount.

Cell proliferation was assayed in paraffin-embedded kidney tissue by incorporation of 5-bromo-2-deoxyuridine (BrdU; Roche Molecular Biochemicals, Mannheim, Germany), as described.⁵³ Pregnant females received an intraperitoneal injection of BrdU (100 mg/g of body wt) 2 hours before sacrifice. BrdU-positive cells were identified using an anti-BrdU peroxidase–conjugated antibody as described (Boehringer, Mannheim, Germany). Immunoreactivity was visualized using aminoethyl carbazole horseradish peroxidase chromogen/substrate solution (Zymed Laboratories, USA).

Treatment of Cultured Kidney Explants

Mouse embryonic kidneys were surgically resected from E11.5 or E12.5 pregnant mice, transferred onto 1.0-μm polyethylene terephthalate–track-etched (PET–track-etched) membrane (Falcon) and cultured in DMEM/F12 nutrient mixture F12 (HAM) with l-glutamine and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid supplemented with 50 μg/ml transferrin (Sigma). Mouse kidney explants were cultured in the presence or absence of 50 ng/ml recombinant TGFβ2 (R&D Systems) for 48 hours or 1 μg/ml recombinant human DKK1 for 96 hours (R&D Systems) or 50 ng/ml GDNF for 48 hours (R&D Systems).

Statistical Analysis

Mean differences were examined using t test (two-tailed) and a Prism 3.0 statistics program. A difference of 5% was interpreted as being statistically significant. Group comparisons for quantitative PCR using human tissue were performed by one-way ANOVA with Tukey's multiple comparison post test.

DISCLOSURES

None.