ß-Catenin Causes Renal Dysplasia Via Upregulation of Tgfß2 and Dkk1

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␤-Catenin Causes Renal Dysplasia via Upregulation of Tgf␤2 and Dkk1

Darren Bridgewater,*† Valeria Di Giovanni,*†‡ Jason E. Cain,*† Brian Cox,* Madis Jakobson,§ Kirsi Sainio,§ and Norman D. Rosenblum*†‡

*Program in Developmental and Stem Cell Biology and †Division of Nephrology, The Hospital for Sick Children, Toronto, Ontario, Canada; ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; and §Biochemistry and Developmental Biology, Institute of Biomedicine, University of Helsinki, Helsinki, Finland

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 ␤-catenin–activated genetic pathway in murine renal dysplasia. Stabilization of ␤-catenin in the ureteric cell lineage before the onset of kidney development increased ␤-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 Tgf␤2 and Dkk1. Treatment of wild-type kidney explants with TGF␤2 or DKK1 generated morphogenetic phenotypes strikingly similar to those observed in mutant kidney tissue. Stabilization of ␤-catenin after the onset of kidney development also caused dysplasia and upregulation of Tgf␤2 and Dkk1 in the epithelium. Together, these results demonstrate that elevation of ␤-catenin levels during kidney development causes dysplasia.

J Am Soc Nephrol 22: 718–731, 2011. doi: 10.1681/ASN.2010050562

Congenital renal malformation is the major cause sponses by activating canonical and noncanonical of childhood renal failure. Yet underlying patho- signaling pathways.7 Canonical WNT signaling is genic mechanisms are poorly defined and no spe- mediated by ␤-catenin, which controls cell adhe- cific treatments exist.1 Formation of the mamma- sion and gene transcription. Canonical WNT sig- lian kidney is dependent on reciprocal inductive naling inhibits phosphorylation of ␤-catenin by tissue interactions between the ureteric bud and the glycogen synthase kinase-3 (GSK-3). Conse- metanephric blastema. These tissue interactions re- quently, ␤-catenin degradation is inhibited, result- sult in growth and branching of ureteric-derived ing in cytoplasmic accumulation, nuclear translo- tubules that differentiate into collecting ducts and cation of ␤-catenin, and regulation of target gene formation of nephrons from mesenchyme-derived expression.8 structures.2 Failure of these processes results in kid- Both the ureteric cell lineage and the mesen- ney dysplasia, which is defined by a paucity of chyme cell population of committed nephron pro- nephrons, decreased branching morphogenesis, abnormal differentiation of mesenchymal and epi- Received May 28, 2010. Accepted December 12, 2010. thelial-derived tissue elements, and abnormal cor- tico-medullary patterning.3 Published online ahead of print. Publication date available at www.jasn.org. Kidney formation is regulated by secreted Correspondence: Dr. Norman D. Rosenblum, Division of Ne- growth factors including members of the WNT phrology, The Hospital for Sick Children, 555 University Avenue, family of secreted glycoproteins. Numerous WNT Toronto, Ontario, Canada M5G 1X8. Phone: (416) 813-5667; Fax: family members are required during kidney devel- (416) 813-5252; E-mail: [email protected] opment.4–6 WNT proteins elicit their biologic re- Copyright © 2011 by the American Society of Nephrology

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genitor cells require canonical WNT/␤-catenin signaling for and ␤-catGOF-UB mice containing a ROSA-26 reporter allele,19 their respective morphogenetic activities. Homozygous which expresses ␤-galactosidase under the control of CRE re- ␤-catenin deficiency targeted to metanephric mesenchyme combinase. LacZ staining in WT and ␤-catGOF-UB in E12.5 uro- cells inhibits nephron formation and decreases expression of gential tissues demonstrated Hoxb.7-Cre mediated excision in genes including Wnt4, Lim1, and Fgf8, which are required for the Wolffian duct, mesonephric tubules, and ureteric cells nephrogenesis.9 Homozygous ␤-catenin deficiency in ureteric (Figure 1, A and B). ␤-Catenin protein expression, analyzed by cells causes a near arrest of ureteric branching, decreased ex- immunofluorescence imaging of ureteric cells, was greatly in- pression of genes required for ureteric tip cell function (C-ret creased throughout the cytoplasm and nucleus in ␤-catGOF-UB and Wnt11), and bilateral renal aplasia or severe hypodyspla- mutant mice when compared with WT (Figure 1, C and D). sia.10,11 Next, we determined the effect of increased ␤-catenin levels on Abnormal canonical WNT/␤-catenin signaling has been transcriptional activity in ureteric cells. We generated implicated in the pathogenesis of human kidney diseases with ␤-catGOF-UB mice containing TCF sites linked to a LacZ re- a developmental origin. In human renal dysplasia ␤-catenin is porter gene.20 Analysis of ␤-galactosidase activity in urogential upregulated and misexpressed in ureteric cell–derived cysts.12 tissue revealed an increase in the caudal portion of the Wolf- Perturbation of ureteric branching in transgenic mice induces fian duct and ureteric cells of ␤-catGOF-UB mutants compared ␤-catenin expression and causes dysplasia, suggesting a sec- with WT littermates analyzed simultaneously (Figure 1, E and ondary pathogenic mechanism whereby elevated ␤-catenin ex- F). Increased levels of ␤-catenin in ␤-catGOF-UB mutant kid- pression contributes to the genesis of renal dysplasia.12–14 neys were confirmed using immunoblotting and protein ly- Wilms’ tumor, a polymorphic childhood tumor derived from sates generated from four E18.5 ␤-catGOF-UB mutant kidneys. mesenchyme blastemal tissue elements, is characterized by Quantitation of ␤-catenin controlled for by the expression of ␤-catenin overexpression in blastemal and mesenchymal tu- GAPDH demonstrated a 1.3-fold increase in ␤-catenin in mor tissue.15 ␤-catGOF-UB mutant kidneys compared with WT (note de- Here, we report a functional contribution of elevated levels creased loading of ␤-catGOF-UB kidney protein compared of ␤-catenin to kidney development using genetic murine with WT) (Figure 1, G and H). The relatively modest in- models of ␤-catenin stabilization. Stabilization of ␤-catenin in crease in ␤-catenin expression is likely to underestimate the the ureteric cell lineage, before the onset of kidney develop- increase in ureteric cells (Figure 1, C and D) since ureteric ment, caused renal aplasia or dysplasia caused by a severe dis- cells constitute 10% or less of all cells in the intact kidney. ruption of ureteric branching. Analysis of global gene expres- Together, these data demonstrate that genetic stabilization sion in kidneys isolated from mutant mice revealed of ␤-catenin in vivo increases ␤-catenin protein expression misexpression of Tgf␤2 and Dkk1, which inhibited ureteric and ␤-catenin–dependent transcriptional activity in ure- branching and nephrogenesis, respectively, in embryonic kid- teric cells. neys cultured ex vivo. Stabilization of ␤-catenin at stages after nephrogenesis and ureteric branching are established also dis- Overexpression of ␤-Catenin in the Ureteric Cell rupted nephrogenesis and caused misexpression of Tgf␤2 and Lineage Causes Renal Agenesis and Hypodysplasia Dkk1. These results demonstrate a pathologic role for ␤-catGOF-UB mice survived to term but died within hours of ␤-catenin in the genesis of renal dysplasia. 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 RESULTS analysis of kidney tissue from mutant mice at PN0 revealed disorganization of tissue elements, decreased number of A Genetic Murine Model of ␤-Catenin Overexpression nephrons, tubular dilation, and uninduced mesenchymal cells, in Embryonic Mouse Kidney In Vivo the hallmark features of severe dysgenesis (Figure 2, C and D). Because elevated ␤-catenin expression is associated with ab- Dilated tubules were bound by Dolichos biflorus agglutinin normal ureteric cell branching, we investigated the direct ef- (DBA), a ureteric cell-specific marker (Supplemental Figure fects of increased ␤-catenin expression on ureteric cell func- 1). Next, we examined embryologic mechanisms that underlie tion. Phosphorylation of serine/threonine residues in exon 3 of renal malformation in ␤-catGOF-UB mice. Although initial ure- the ␤-catenin allele by GSK-3␤ targets ␤-catenin for intracel- teric branches were comparable in WT and mutant mice at lular degradation.8 We used CRE-mediated recombination E11.5 (Figure 2, E and F), by E12.5, kidney tissue from targeted specifically to ureteric cells using Hoxb.7-Cre;GFP ␤-catGOF-UB mutant mice was smaller than that in WT mice (GFP, green fluorescent protein) mice16,17 and a ␤-catenin al- and appeared to consist of fewer ureteric branches (Figure 2, G ⌬ lele in which exon 3 is flanked by loxP sites (␤-cat 3 mice)18 to and H). Ureteric branching was investigated directly using generate mice (termed ␤-catGOF-UB [Gain of Function-Ure- GFP fluorescence at E12.5. In contrast to WT mice, in which teric Bud]) in which excision of exon 3 from the ␤-catenin multiple generations of ureteric branches are formed, ureteric allele is targeted to the ureteric cell lineage. To localize the branching was greatly attenuated in mutant mice (Figure 2, I spatial domain of CRE activity, we generated wild type (WT) and J). Together, these data demonstrate that stabilization of

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Table 1. Renal phenotype in ␤-catGOF-UB mice Mutants/WT Aplasia Dysplasia Perinatal ␤-catGOF-UB 11/36 (30.5%) 5/11 (45%) 6/11 (54.5%) Embryonic ␤-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.

␤-catenin in the ureteric cell lineage results in arrested ureteric branching by E12.5.

Effect of ␤-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/␤-catenin signaling. We determined the effects of increased ␤-catenin expression on these processes using renal tissue isolated from ␤-catGOF-UB mice. To function in cell adhesion, cadherins must be associated with the actin cytoskeleton.23 ␤-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 ϭ 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 ␤-catGOF-UB and WT mice (P ϭ 0.78) (Figure 3, F through H). However, apoptosis in the metanephric mesenchyme was increased 4.9-fold (P ϭ 0.015) in ␤-catGOF-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 ␤ Figure 1. Conditional overexpression of -catenin in ureteric metanephric mesenchyme cell survival.25 Together, these re- cells results in severe renal defects. (A, B) Whole mount X-gal sults indicate a primary effect of increased ␤-catenin expres- staining in E12.5 urogenital ridges resected from WT (A) and sion on ureteric cell proliferation. However, a modest increase ␤-catGOF-UB mice (B) containing a ROSA-26 reporter allele. LacZ ␤ GOF-UB in cell proliferation is unlikely to account for the severe disrup- staining in Wt and -cat in E12.5 urogential ridges dem- ␤ GOF-UB onstrated Hoxb.7-Cre mediated excision in Wolffian duct (WD), tion of branching morphogenesis observed in -cat mesonephric tubules (MT), and ureteric bud (UB) tissue. (C, D) mice. ␤-catenin immunofluoresence in E12.5 WT and ␤-catGOF-UB kidney tissue. (C) In WT kidney ␤-catenin is localized in the UB Overexpression of ␤-Catenin Causes Abnormal plasma membrane consistent with a role in adherens junctions Regulation of Gene Expression in the Embryonic (white arrow). ␤-Catenin expression was not observed in the Kidney cytoplasm or nucleus in WT UBs. (D) In ␤-catGOF-UB mutant mice During normal kidney development, ␤-catenin functions ␤-catenin localized to the ureteric cell plasma membrane as well within ureteric cells to control the expression of a network of 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 ␤-catGOF-UB;TCF mice demonstrates activity in indicating increased transcriptional activity. (G, H) Western anal- the WD and UB. The X-gal staining in ␤-catGOF-UB;TCF mice is ysis and corresponding densitometry for ␤-catenin and GAPDH in noticeably more intense when compared with WT littermates, thus WT and ␤-catGOF-UB mutant kidneys.

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genes, each of which is required for branching morphogenesis.26 We hypothesized that abnormally high levels of ␤-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 ␤-catGOF-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 ␤-catenin expression, itself, versus pathogenic changes associated more generally with hypodysplasia. In triplicate experiments, we compared mRNA species isolated from WT and ␤-catGOF-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 Ͻ 0.003 (Supplemental Tables 1 and 2). We performed a gene ontology analysis to identify func- tionally related groups of differentially expressed genes. A complete list of the functional categories and their accompa- nying 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 ␤-catGOF-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 ␤ ⌬3/ϩ Figure 2. Arrested branching morphogenesis in Hoxb.7; -cat would predict a more modest effect on renal development than ␤ GOF-UB mutant mice. (A, B) Urinary systems from P0 WT and -cat that observed in ␤-catGOF-UB mice4,29,30 and suggest that addi- mice, demonstrating severe renal malformations in ␤-catGOF-UB mice tional mechanisms contribute to this severe phenotype. In- (white arrows). (C, D) H&E stained kidney sections from P0 WT and ␤-catGOF-UB mice, demonstrating a lack of cortical medullary pattern- deed, although treatment of WT embryonic kidney explants ing, cystic tubules (C), and a paucity of nephrogenic structures. (E with exogenous GDNF induced a 1.5-fold increase in the num- through H) H&E stained cross sections of mouse embryos at E11.5 ber of ureteric branches, no difference in ureteric branching GOF-UB and E12.5 demonstrates a paucity of ureteric bud (UB) tissue and was observed in GDNF-treated ␤-cat embryonic kidney nephrogenic elements as early as E12.5 in ␤-catGOF-UB mice. White explants (Supplemental Figure 2). arrow, MM; black arrow, UB tissue. (I, J) GFP fluorescence demon- Identification of upregulated genes in ␤-catGOF-UB mice us- strating arrested branching morphogenesis in ␤-catGOF-UB mice. ing a gene ontology analysis revealed an enrichment of genes MM, metanephric mesenchyme. involved in cell morphogenesis, cell motility, muscle cell development, vasculogenesis, and negative regulation of signal transduction (Table 4). Inspection of upregulated genes for

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from ␤-catGOF-UB and WT kidneys (P ϭ 0.038) (Figure 4J). The spatial expression of Tgf␤2 and Dkk1 was next determined by in situ hybridization. In E12.5 WT mice, Tgf␤2 mRNA transcripts were detected at low levels in the metanephric mesenchyme and ureteric cells (Figure 4K). In contrast, Tgf␤2 mRNA transcripts were expressed at high levels in the ureteric cells of ␤-cat- GOF-UB mice (Figure 4L). We hypothesized Tgf␤2 protein is generated in the UB and then secreted to the mesenchyme. Consis- tent with our hypothesis, examination of Tgf␤2 protein expression by immunohistochemistry revealed marked up regulation of Tgf␤2 protein in the metanephric mesenchyme of ␤-catGOF-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 ␤-catGOF-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 Tgf␤2 and Dkk1 mRNA expression in ureteric cells in ␤-catGOF-UB mice. Figure 3. ␤-catenin stabilization modulates cellular events during ureteric branching. ␤ (A, B) Analysis of adherens junctions (white arrows) by transmission electron micros- TGF 2 Inhibits Ureteric Branching copy demonstrates the presence of junctional complexes in ␤-catGOF-UB tissue indis- and Expands the Population of tinguishable from WT. (C through E) Qualitative and quantitative analysis of cell Committed Nephrogenic Precursor proliferation in E12.5 WT and ␤-catGOF-UB kidney tissue using an in situ BrdU incorpo- Cells ration assay. Quantitative analysis of BrdU incorporation (red color) demonstrated a We investigated the functional contribu- 1.24-fold increase (% BrdU-positive ureteric cells, WT [43.2%] versus ␤-catGOF-UB tion of increased Tgf␤2 expression to renal GOF-UB [53.7%], *P ϭ 0.03) in ␤-cat ureteric cell proliferation when compared with WT. dysplasia using an in vitro culture model of (F through K) Qualitative and quantitative analysis of ureteric bud and mesenchymal murine renal development. Embryonic apoptosis by TUNEL analysis. TUNEL-positive cells (brown color, arrowhead) are rarely kidney explants isolated at E12.5 and cul- detected in the ureteric bud (black arrow) in WT (F, G) and ␤-catGOF-UB (I, J) mutant tured for 48 hours are characterized by mice (% TUNEL-positive ureteric cells, WT [0.33 Ϯ 0.19%] versus ␤-catGOF-UB [0.40 Ϯ 0.16%], P ϭ 0.78). Apoptosis in the metanephric mesenchyme was increased 4.9-fold multiple ureteric branches, each of which is (number TUNEL-positive cells per mm2, 0.1 Ϯ 0.02 versus 0.55 Ϯ 0.12, *P ϭ 0.015) adjacent to a two- to three-cell-thick layer in ␤-catGOF-UB mice when compared with that in WT (J, K). of condensed cap mesenchyme. This condensed mesenchyme is distinct from unin- those that regulate branching morphogenesis and that were duced mesenchyme, which exists at a greater distance from not categorized within the ontology analysis identified effec- ureteric branch tips and is loosely organized (Figure 5A). tors in signaling pathways controlled by TGF␤ (Tgf␤2 and Treatment with recombinant TGF␤2 (50 ng/ml) for 48 hours Tgf␤r2) and WNT proteins. Interestingly, those genes in the resulted in decreased ureteric branching (Figure 5, B versus A), WNT pathway encoded WNT inhibitors including Dickkopf 1 and condensation of metanephric mesenchyme cells through- (Dkk1), WNT inhibitory factor 1 (Wif1), and secreted frizzled- out almost the entire explant, even at sites distant from ureteric related protein (sFrp) (Table 5). Because previous studies dem- branch tips (Figure 5B). Next, we determined if the condensed onstrated that Dkk1 and Tgf␤2 inhibit ureteric branching and appearance of the mesenchyme indeed reflected induction of nephrogenesis, respectively,31,32 we proceeded with further these cells by investigating the expression of PAX2, neural cell analysis of these effectors. Upregulation of Tgf␤2 and Dkk1 was adhesion molecule (NCAM), and CITED1, markers of in- verified using quantitative real-time PCR on mRNA isolated duced mesenchyme cells. In untreated kidneys, PAX2 was ex-

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pressed in ureteric cells, the condensing mesenchyme around the tips of the ureteric bud, and in developing nephrogenic structures (Figure 5E). Remarkably, in TGF␤2- 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 (Fig- ure 5E). In contrast, in TGF␤2-treated explants, NCAM was coexpressed with PAX2 in cells distant from ureteric branch tips (Figure 5F). Dur- ing 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, TGF␤2-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 ␤-catGOF-UB mutant mice. Analysis of marker expression in renal tissue from mutant mice revealed striking similarities to TGF␤2-treated kidney explants (Figure 5, C through L). Kidney tissue isolated from␤-catGOF-UB micewascharacterizedbyag- gregation 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 ␤-catGOF-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 Figure 4. ␤ ␤ -catenin stabilization in ureteric cells increases Tgf 2 and DKK1 expression. ␤ GOF-UB (A, B) Ureteric bud–specific (UB-specific) green fluorescent protein fluorescence to -cat versus WT kidneys (Supple- identify the UB branching pattern in WT and ␤-catGOF-UB mice. Ureteric tissue is mental Figure 3, A and B). Similarly, ␣ marked with a white arrow. WT and ␤-catGOF-UB mice were pooled to generate biologic analysis of -smooth muscle actin pro- triplicates. (C) Heat-map representation of differentially expressed genes between WT tein demonstrated no evidence of ectopic and ␤-catGOF-UB demonstrates a low level of variability of differentially expressed expression in E13.5 mutant kidney tissue transcripts among sample replicates. Each lane represents individual biologic repli- (Supplemental Figure 3, C and D). To- cates normalized to each of three WT samples. (D through I) In situ hybridization for gether, these studies indicate that TGF␤2 GOF-UB Gdnf, Ret, and Wnt11 in E12.5 WT and ␤-cat kidneys. Gdnf was expressed in a inhibits ureteric branching and induces ␤ GOF-UB mosaic pattern around the ureteric tips in -cat mice and Ret expression was metanephric mesenchyme cells to initiate ␤ GOF-UB limited to the tips of the UB tips in WT and -cat (black arrows). (H, I) In contrast a nephrogenic program even in the ab- to WT, Wnt11 was nearly absent in ␤-catGOF-UB ureteric tips (black arrows). (J) Valida- sence of signaling by adjacent ureteric tion of microarray data by quantitative real-time PCR confirmed a significant upregu- ␤ GOF-UB lation of Tgf␤2 (WT versus mutant: 0.013 Ϯ 0.009 versus 0.067 Ϯ 0.35, P ϭ 0.03) and cells as observed in -cat mice. Dkk1 (0.9 Ϯ 0.05 versus 1.41 Ϯ 0.16. P ϭ 0.038). (K through P) Spatial localization of the candidate genes. (K, L) Tgf␤2 mRNA is upregulated in E12.5 UB cells in ␤-catGOF-UB WNT-Dependent Nephrogenesis Is Dis- GOF-UB mutants. (M, N) Immunohistochemistry demonstrates a marked increase in Tgf␤2 rupted in ␤-cat Mouse Kidney protein expression in the metanephric mesenchyme (MM) in ␤-catGOF-UB mutants. In contrast to our results demonstrating that (O,P) Dkk1 is markedly upregulated in E12.5 UB cells (UB) in ␤-catGOF-UB mutants. No Tgf␤2 promotes nephrogenesis, ␤-catGOF-UB changes in Tgf␤2 and Dkk1 mRNA were observed in MM cells. kidney tissue is characterized by nephron deficiency. We investigated this apparent para-

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Table 2. Gene ontology analysis of selected functional categories of genes decreased in kidney tissue of ␤-catGOF-UB mice GO-ID P Description Genes in test set Ϫ 1822 1.25 ϫ 10 8 Kidney development GDNF SPRY1 BMP2 SALL1 PAX8 PBX1 BMP7 SLIT2 SHH Ϫ 1656 2.08 ϫ 10 7 Metanephros development SPRY1 BMP2 SALL1 PAX8 PBX1 GDNF SHH Ϫ 1657 4.22 ϫ 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 Ͻ 0.003) in structures (Figure 6G), DKK1-treated ex- kidney tissue of ␤-catGOF-UB mice plants demonstrated a marked reduction Probe set Gene title Gene symbol Fold change in these nephron structures (Figure 6H). 1450428 LIM homeobox protein 1 Lhx1 4.92 Remarkably, exogenous DKK1 resulted 1450782 Wingless-related MMTV integration site 4 Wnt4 3.25 in an increase in the metanephric mesen- 1418208 Paired box gene 8 Pax8 2.83 chyme cell apoptosis (Figure 6, I and J). 1426155 Odd-skipped related 2 (Drosophila) Osr2 2.64 Together, these data demonstrate an in- 1440650 Slit homolog 2 (Drosophila) Slit2 2.46 terruption of WNT-dependent signaling 1448738 Calbindin-28K Calb1 2.30 in ␤-catGOF-UB mice and suggest a re- 1456258 Empty spiracles homolog 2 (Drosophila) Emx2 2.30 quirement for WNT signaling in cell sur- 1421106 Jagged 1 Jag1 2.30 vival. 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 Temporal Stabilization of -Catenin 1431225 Sprouty homolog 1 (Drosophila) Spry1 1.52 after the Onset of Renal 1431225 Cbp/p300-interacting transactivator Cited2 1.52 Development Causes Renal Dysplasia 1431225 Sal-like 1 (Drosophila) Sall1 1.41 Overexpression of ␤-catenin is observed in 1431225 Glypican 3 Gpc3 1.32 human and murine renal dysplasia.12–14 Analyses of ␤-catenin expression in some dox by examining the expression of the WNT-dependent genetic genetic mouse models indicate that upregulation begins af- pathway that is required for nephrogenesis using in situ hybrid- ter the onset of tissue malformation.14 These findings sug- ization in WT and mutant kidney tissue. Wnt9b, Wnt4, and Lim1 gest that ␤-catenin may participate in a secondary pathway act sequentially to control nephrogenesis.5,6,33 Wnt9b is the pri- of tissue injury in renal dysplasia. To investigate this possi- mary inductive signal required for the formation of the re- bility, we generated mice in which ␤-catenin overexpression nal vesicle and acts upstream of Wnt4.5 Wnt9b was ex- was timed to occur after the onset of kidney development. pressed in a normal pattern in both WT and ␤-catGOF-UB We determined whether these result in similar cellular and kidney tissue (Figure 6, A and B). In WT mice, Wnt4 is molecular consequences to those observed in mice with expressed in pretubular aggregates (Figure 6C). In contrast, ␤-catenin overexpression starting at the onset of kidney de- in ␤-catGOF-UB mutant kidneys, Wnt4 expression was mis- velopment. Stabilization of ␤-catenin after the initiation of expressed in mesenchyme cells lining ureteric branches renal morphogenesis was achieved by breeding transgenic (Figure 6D). Lim1 is expressed in ureteric cells, pretubular mice containing a ubiquitous Cre recombinase fused to the aggregates, and renal vesicles in WT mice and is activated by ligand-binding domain of the estrogen receptor (CreERT)36 ⌬ Wnt46,9,33,34(Figure 6E). In ␤-catGOF-UB mutants, Lim1 was to ␤-cat 3 mice. Pregnant mice, 14.5-days postcoitus, were observed in the ureteric tissue but was absent from the met- treated with a single intraperitoneal injection of tamoxifen anephric mesenchyme (Figure 6F). and pups were isolated after 48 hours. Histologic examina- Our finding that Dkk1 is upregulated in mutant kidney tion of E16.5 CreERT;␤-cat⌬3/ϩ embryonic kidney tissue re- tissue was particularly interesting given the disruption in vealed reduced kidney size (Figure 7, A and C). Because WNT signaling. Consistent with the disruption of WNT- nephrons are formed in an axis from the interior to the periph- dependent nephrogenesis, our global gene expression anal- ery of the kidney, nephrons formed during the early stages of ysis indicated upregulation of Dkk1. DKK1 inhibits canon- kidney development are located in the kidney interior that will ical WNT signaling by binding to the frizzled coreceptor, become the medulla. Indeed, glomeruli were observed in the LRP5/6, thereby blocking ligand-receptor interactions.35 deep cortex, which likely formed before the stage at which ta- We determined the effects of DKK1 on nephrogenesis using moxifen was administered to CreERT;␤-cat⌬3/ϩ mice (Figure 7, recombinant DKK1 in embryonic kidney explant cultures. C and D). In contrast, in mutant mice the peripheral nephro- Kidney explants isolated from E11.5 mice were incubated in genic zone was devoid of condensing mesenchyme and devel- the presence or absence of 1 ␮g/ml DKK1. In contrast to oping nephrogenic intermediate structures and contained an untreated kidneys, which were characterized by multiple increased mass of loosely organized stromal cells (Figure 7, C nephron structures identified by brush boarder positive and D). To further investigate the differentiated state of the

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Table 4. Gene ontology analysis of selected functional categories of genes increased in kidney tissue of ␤-catGOF-UB mice GO-ID P Description Genes in test set Ϫ 9653 2.83 ϫ 10 10 Morphogenesis MYO7A IGFBP6 TBX20 HOXD13 TGFB2 OSR1 GATA6 SOX17 SLIT3 CNTN4 TBX18 GAP43 SOX2 COL8A2 COL8A2 TGFBR2 IGF1 DKK1 NFIB Ϫ 7148 5.74 ϫ 10 5 Cell morphogenesis MYO7A IGFBP6 TGFB2 ACTN2 SLIT3 NTRK2 CNTN4 GAP43 Ϫ 1570 8.22 ϫ 10 4 Vasculogenesis PTPRJ NTRK2 ZFPM2 WARS2 SOX17 Ϫ 3528 2.87 ϫ 10 3 Segmentation SFRP1 ZFHX1B MAFB PCDH8 TBX18 Ϫ 30111 3.16 ϫ 10 3 Regulation of Wnt receptor signaling pathway DKK2 DKK1 MDFIC WIF1

tected at low levels and was localized to Table 5. Selected mRNA transcripts significantly increased (P Ͻ 0.003) in only a small subset of tubules primarily ␤ GOF-UB kidney tissue of -cat mice localized in the medulla of WT mice (Fig- Probe set Gene title Gene symbol Fold change ure 7K). In contrast, in CreERT;␤-cat⌬3/ϩ 1425425 Wnt inhibitory factor 1 Wif1 6.96 mice high levels of Tgf␤2 protein were 1449350 Odd-skipped related 1 (Drosophila) Osr1 1.74 detected in numerous tubules localized 1458232 Dickkopf homolog 1 (Xenopus laevis) Dkk1 1.74 in the cortex and medulla, deep glomer- 1423250 Transforming growth factor, beta 2 Tgfb2 1.52 ular structures, and condensing mesen- 1455851 Bone morphogenetic protein 5 Bmp5 1.52 chyme in the outer cortex (Figure 7, K 1426397 Transforming growth factor, beta receptor II Tgfbr2 1.41 and L, inset boxes). In mice not treated 1418876 Secreted frizzled-related sequence protein 1 Sfrp1 1.41 1418876 Forkhead box D1 Foxd1 1.32 with tamoxifen, Dkk1 expression was re- 1443221 Wilms tumor homolog Wt1 1.23 stricted to mesenchyme in the outer renal cortex (Figure 7M). However, in CreERT; ␤-cat⌬3/ϩ mice, Dkk1 expression was ex- cells in the nephrogenic zone, we defined the expression pat- panded within the peripheral cortex and was misexpressed tern of PAX2 and CITED1, which are misexpressed in TGF␤2- within epithelial tubules in the deep cortex (Figure 7N). ⌬ ϩ treated WT kidney explants. In CreERT;␤-cat 3/ renal tissue, Together, these studies indicate that overexpression of CITED1 was misexpressed in nonaggregated cortical mes- ␤-catenin at stages well after the initiation of kidney devel- enchyme and in epithelial tubules also located in the cortex. opment causes renal tissue malformation associated with CITED1-expressing tubules did not coexpress cytokeratin, a aberrant expression of CITED1, PAX2, Tgf␤2, and Dkk1 as marker of ureteric cells, suggesting that they are derived from observed in ␤-catGOF-UB mutant mice. Moreover, the mor- mesenchyme. This expression pattern was in sharp contrast to phologic and cellular abnormalities observed in ␤-catenin the restricted expression of CITED1 in cap mesenchyme cells overexpressing mice mimic those observed in humans, sug- in WT mice (Figure 7, E and F). In contrast to WT mice, gesting a pathogenic role for ␤-catenin in human renal dys- PAX2 was virtually absent from the cortical mesenchyme in plasia. CreERT;␤-cat⌬3/ϩ mice (Figure 7, G and H). Taken together, these results indicate that upregulation of ␤-catenin abro- gates nephron formation in the presumptive nephrogenic zone and causes misexpression of genes that control early DISCUSSION stages of the mesenchymal to epithelial transformation. These deleterious effects are more severe than those ob- Renal dysplasia is a complex disorder characterized by variable served in mice in which stabilization of ␤-catenin is targeted phenotype and severity. At the level of histopathology, renal to the Six-2–positive subpopulation of metanephric mesen- dysplasia is characterized by a core set of features including chyme cells, which are committed to forming nephrons at reduction in the number of nephrons and collecting ducts, the time of ␤-catenin stabilization.9 disorganization of tissue elements, and abnormal patterning of Next, we determined whether overexpression of cortical and medullary tissues.3 The degree of nephron and ␤-catenin at stages during which nephrogenesis and collecting duct deficiency, the focal versus diffuse nature of the branching morphogenesis are already established increased dysplastic phenotype, and the degree of epithelial cyst forma- Tgf␤2 and Dkk1 as we observed in ␤-catGOF-UB mice. In situ tion varies widely among cases. These variations are consis- hybridization revealed that Tgf␤2 was rarely detected in cor- tent with the observed variation of clinical phenotypes rang- tical or medullary tubules in WT mice (Figure 7I). In con- ing from aplasia, diffuse dysplasia with variable degrees of trast, Tgf␤2 was expressed in the vast majority of tubules in cystic transformation, and focal dysplasia. The recent iden- CreERT;␤-cat⌬3/ϩ mice (Figure 7, I and J) in a similar pat- tification of gene mutations in affected individuals is begin- tern to that observed in ␤-catGOF-UB mice (Figure 4, K and ning to provide insight into the primary molecular mecha- L). Consistent with these results, Tgf␤2 protein was de- nisms that control renal tissue malformation.37 Yet the

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interactions and at stages during which these interactions are well-established. Analysis of pathogenic mechanisms that could underlie ␤-catenin function revealed novel changes in gene expression involving TGF␤ signaling effectors (Tgf␤2) and WNT inhibitors (Dkk1). Our data demonstrate that these effectors inhibit ureteric branching and nephrogenesis in cultures of embryonic kidney explants. Overexpression of ␤-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 re- Figure 5. Expanded and ectopic metanephric mesenchyme (MM) cell induction in duced but not totally lost (Figure 4, D ␤ GOF-UB ␤-catGOF-UB mutants. (A, B, E, F, I, J) Analysis of TGF␤2-treated E12.5 kidney explants through I). Yet -cat mutant kid- and (C, D, G, H, K, L) kidney tissue isolated from E12.5 WT and ␤-catGOF-UB mice. (A neys manifested a complete arrest of through D) H&E stained kidney tissue in untreated and TGF␤2-treated kidney explants branching morphogenesis by E12.5, which (A, B) and WT and ␤-catGOF-UB mutants (C, D) (black arrows). TGF␤2-treated (B) and is more consistent with a complete loss of ␤-catGOF-UB kidneys (D) demonstrate an expansion of mesenchymal aggregates the GDNF/RET signaling.38,39 Moreover, around the tips of the UB (black arrows). (E through H) Dual-label immunofluorescence total deficiency of Wnt11 observed in imaging of PAX2 and NCAM demonstrating expanded and ectopic NCAM and PAX2 ␤-catGOF-UB mutant mice causes renal hyp- ␤ ␤ GOF-UB expression in TGF 2-treated kidney explants (E, F) and -cat mice (white arrow) oplasia, characterized by a modest decrease (G, H). (I through L) Expression of CITED1, a nephrogenic lineage–specific marker, in the number of ureteric branch tips, not confirms an expansion of induced mesenchyme in TGF␤2-treated kidney explants 4,29 (white arrow) (I, J). (K, L) CITED1 and NCAM co-immunofluorescence in WT and severe hypodysplasia. Thus, we posited ␤-catGOF-UB mice confirms an expanded domain of cells expressing CITED1 adjacent that the relative severity of the renal phe- ␤ GOF-UB to the UB (white arrows). notype in -cat mice was due to either a combinatorial effect of decreased variable severity and phenotype observed in individuals expression shared by many genes important to renal devel- with the same mutation3 suggests that additional mecha- opment and/or transcriptional effects directly related to the nisms control renal tissue maldevelopment. increased dose of ␤-catenin. Previously, we reported elevated ␤-catenin expression in A global gene expression analysis revealed upregulation human renal dysplastic tissue with different underlying eti- of numerous genes involved in morphogenesis and WNT ologies.12 This observation, combined with our recognition signaling (Table 4). Particularly notable among these genes that ␤-catenin expression is increased in models of murine was Tgf␤2 and inhibitors of WNT signaling. TGF␤2isex- renal dysplasia in which gene expression is manipulated in pressed in the basement membrane surrounding ureteric the ureteric lineage,12,14 led us to hypothesize that ␤-catenin cells during kidney development and decreases ureteric may be elevated as a secondary manifestation during the branching and nephrogenesis when applied to embryonic morphogenesis of renal dysplasia. Here, we investigated the kidney explant cultures,40 and is upregulated in human fetal functional contribution of ␤-catenin to the pathogenesis of dysplastic tissue.41 Furthermore, Tgf␤2 heterozygous mice renal tissue malformation. ␤-Catenin stabilization in the are characterized by increased ureteric branch length and ureteric cell lineage resulted in renal aplasia or dysplasia. nephron number, consistent with a physiologic inhibitory Disruption of renal development was observed whether effect of Tgf␤2 in vivo.31 Combined, these results support a ␤-catenin stabilization was present at the initiation of renal role for TGF␤2 inhibiting ureteric branching in ␤-catGOF-UB development, or during intermediate stages of kidney de- mutant mice. velopment. These findings indicated that nephrogenesis Histologic and molecular analysis of ␤-catGOF-UB mutant and branching morphogenesis are vulnerable to increased kidneys demonstrated an increased population of commit- ␤-catenin expression during initial ureteric-mesenchymal ted mesenchyme progenitors. Consistent with this observa-

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tion, treatment of WT embryonic kidney explants with TGF␤2 caused a similar effect. Yet we rarely detected nephrogenic structures beyond the renal cap mesenchyme stage in ␤-catGOF-UB mutant or TGF␤2-treated WT mouse kidneys, suggesting a block in the nephrogenic program. Abro- gation 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 ␤-catenin, Tgf␤2, and Dkk occurs at two distinctly different matura- tional stages. That is, we find upregulation of these effectors when ␤-catenin is overexpressed at the onset of kidney development and well after the onset of nephrogensis and branching morphogensis. During embryogenesis, Tgf␤2 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 Tgf␤2-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 ␤-catenin overexpression during renal embryogenesis. Our model predicts that overexpression of ␤-catenin in ureteric cells during embryogen- Figure 6. Disruption in nephrogenesis is Dkk1-dependent. (A esis causes increased expression of TGF␤2 and Dkk1 through F) Analysis of genes necessary for nephrogenesis. Wnt9b ␤ localizes to the ureteric bud (UB) in E12.5 ␤-catGOF-UB mice in a (Figure 8). In this context, Tgf 2 acts in an autocrine man- pattern similar to WT (A, B). In contrast to WT, Wnt4 is ectopically ner to inhibit ureteric branching and acts in a paracrine expressed in the induced mesenchymal aggregates surrounding manner to induce the formation of an ectopic population of the ureteric tips in ␤-catGOF-UB mice (black arrow) (C, D). Lim1 mesenchyme cells committed to a nephrogenic fate. Pro- mRNA is expressed in UB cells in both WT and ␤-catGOF-UB gression of nephrogenesis from these cells, as well as those mutants. In contrast to WT, Lim1 mRNA expression is practically induced by adjacent ureteric cells, is inhibited by the simul- absent from the metanephric mesenchyme. (E, F). Resected E11.5 taneous paracrine actions of DKK1. Together, these signal- kidney explants were treated with DKK1 for 96 hours. CYTOKERATIN ing events act in addition to the primary defect that led to (red) demonstrates a similar ureteric branch pattern in untreated ␤-catenin overexpression to generate a dysplastic pheno- and DKK1-treated samples. In contrast, a noticeable decrease in type. Our model has important implications for future re- brush boarder–positive structures (proximal tubules, green, white search aimed at further establishing the functional roles of arrows) is observed in DKK1-treated samples (G, H). (I, J) Analysis ␤ of activated caspase-3 expression (apoptotic marker) in DKK1- TGF and Dkk signaling in renal dysplasia and in the devel- treated explants. Activated caspase-3 expression is increased opment of therapeutic strategies aimed at treating these dis- within metanephric mesenchyme in DKK1-treated kidney ex- orders. The concept that renal dysplasia is a multistage pro- plants. n, nephrogenic structure. cess controlled by signaling pathways that are triggered by a

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CONCISE METHODS

Mice Hoxb7-Cre:EGFP mice45 were crossed with mice containing loxP sites flanking exon 3 of the ␤-catenin allele (␤-cat⌬3/⌬3)18 to generate ␤-catenin gain-of-function mutant mice, termed ␤-catGOF-UB. TCF-␤-galactosidase re- porter20 and ROSA26 reporter mice19 were crossed to ␤-cat⌬3/⌬3 to generate ␤-cat⌬3/ϩ; ROSA or ␤-cat⌬3/ϩ;TCF mice. These mice were subsequently crossed to Hoxb7-Cre: EGFP mice. PCR genotyping was performed as described.18,20 ␤-Galatosidase staining was performed as described previously.46 Tamox- ifen-inducible mice (CreERT) were crossed to ␤-cat⌬3/⌬3 mice. Pregnant females were in- jected 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 Hospi- tal for Sick Children Research Institute Ani- mal Care Committee.

Histology, Immunofluorescent Microscopy, Immunohistochemistry, Figure 7. Tamoxifen-induced overexpression of ␤-catenin in E14.5 embryos results in and Western Analysis renal dysplasia. (A, D) H&E stained sections from resected E16.5 kidneys from WT and Whole kidney tissue was fixed in 4% paraformalde- ⌬ ϩ ⌬ ϩ CreERT;␤-cat 3/ mice. CreERT;␤-cat 3/ mice demonstrate slightly smaller kidneys hyde for 24 hours at 4°C with agitation. Paraffin- with an otherwise normal kidney morphology. In contrast to WT kidneys, CreERT;␤- embedded embryos were analyzed by histology after ⌬ ϩ cat 3/ kidneys demonstrate an absence of a nephrogenic zone (black asterisk), a generating 4-␮m tissue sections and stained with he- paucity of nephrogenic intermediate structures, and a deficiency developing and matoxylin and eosin or Dolicus bifloris aggulatinin T maturing glomeruli (D). Deep cortical glomeruli are observed in both WT and CreER ; (Vector Labs). Immunofluorescence was performed ␤ ⌬3/ϩ -cat mutant mice (black arrows). (E, F) CITED1-CYTOKERATIN dual immunoflu- on 4% paraformaldehyde fixed tissue sections using orescence demonstrates ectopic CITED1 expression in the mesenchyme and tubule ⌬ ϩ antibodies specific for ␤-catenin (Upstate, Lake structures in E16.5 CreERT;␤-cat 3/ mice (white arrows). In contrast, CYTOKERATIN Placid, NY; 1:200 dilution), PAX2 (Covance, Berkley, was not observed in cortical tubules or medullary tubules in kidneys from CreERT;␤- ⌬ ϩ CA; 1:200 dilution), NCAM (Sigma, St Louis; 1:100 cat 3/ mice (white arrowhead). (G, H) PAX2 and NCAM co-immunofluorescence. PAX2 is expressed in renal tubules (T), and condensing mesenchyme (CM) around the dilution), pan cytokeratin (Sigma; 1:200 dilu- tips of the UB in WT (white arrow, CM). Similar to WT mice, PAX2 is expressed in renal tion), and CITED1 (Neomarkers, Fremont, ⌬ ϩ tubules, but is virtually absent in the cortical mesenchyme in CreERT;␤-cat 3/ mice CA,; 1:200 dilution and anti–active caspase-3 (white asterisk). A similar pattern of NCAM expression was observed in WT and (BD Transduction Labs; 1:200 dilution). Whole ⌬ ϩ CreERT;␤-cat 3/ . (I, J) In situ hybridization for Tgf␤2 demonstrates an increase in mount immunofluoresence was performed as mRNA expression in tubules localized in the cortex and medullary regions in CreERT; described48 using anti–pan cytokeratin (1:200) ␤ ⌬3/ϩ -cat kidneys when compared with WT (black arrows and highlighted in inset and anti–Brush Boarder antibody (Gift from ␤ boxes). (K, L) Immunohistochemistry demonstrates increased Tgf 2 protein expression Aaro Miettinen, University of Helsinki). Alexa T ␤ ⌬3/ϩ ␤ in glomeruli and tubules in CreER ; -cat mice. Tgf 2 protein expression was also 568 goat anti-mouse and Alexa 488 goat anti- observed in the condensing mesenchyme surrounding the UB (highlighted in inset rabbit were used as secondary antibodies (Invit- boxes). (M, N) In situ hybridization for Dkk1 demonstrates an obvious increase in mRNA ⌬ ϩ rogen; 1:500 dilution). Immunohistochemistry expression in cortex and medullary tubules (black arrow) in CreERT;␤-cat 3/ kidneys ␤ when compared with WT (black asterisk). DG, developing glomeruli; G, glomeruli; CM, was performed using anti-Tgf 2 (Santa Cruz). condensing mesenchyme; T, tubules; NZ, nephrogenic zone. The reaction was visualized using AP-conjugated secondary antibody (Cedarlane) and AP primary insult provides a new foundation for developing substrate to develop color reaction (Promega). WT and ␤-catGOF-UB molecular therapies aimed at attenuating the activities of these mutant whole kidneys were homogenized in T-PER tissue protein pathways. extraction reagent (Thermo Scientific) supplemented with Protease

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Cytoscape (v 2.450) and compared with Gene Ontology (GO) anno- tation 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 ␮l. Primers for TGF␤2, DKK family members, Pax2, E-cadherin, and ␤-2-microglobin were designed using Primer 3 software and verified using the University of California, Figure 8. Effects of ␤-catenin overexpression during kidney em- Santa Cruz (UCSC) genome bioinformatics web site (http:// bryogenesis. Overexpression of ␤-catenin in ureteric cells during genome.ucsc.edu). The annealing temperature was restricted to 59 to embryogenesis increases Tgf␤2 and Dkk1. Tgf␤2 acts in an auto- 60°C and the length of the PCR product was set between 100 and 200 crine manner to inhibit ureteric branching. In addition, Tgf␤2 acts bp. Specificity of the amplification was carried out by agarose gel in a paracrine manner to induce the formation of an ectopic electrophoresis. Relative levels of mRNA expression were carried out population of mesenchyme cells committed a nephrogenic fate. using the standard curve method. Individual expression values were Progression of nephrogenesis within this ectopic population of normalized by comparison to ␤-2-microglobin. 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 Electron Microscopy vesicle; WD, Wolffian duct; UB, ureteric bud; UBM, ureteric Kidneys were fixed in 2% glutaraldehyde for 24 hours, rinsed with 0.1 branching morphogenesis. M sodium cocadylate, and fixed for 1 hour in 0.2% tannic acid fol- lowed by graded fixation in 1% osmium tetroxide and in 1% osmium inhibitor cocktail (Thermo Scientific). For immunoblotting, primary tetroxide/1.25% potassium ferrocyanide. After dehydration, samples ␤ anatibodies were directed against -catenin (Upstate; 1:1000) and were embedded in SPURR resin, sectioned, collected on copper grids, GAPDH (Abcam; 1:1000). and stained for electron microscopy. Images were obtained using a FEI Tecnai 20 transmission electron microscope. Global Genome Expression Analysis in Mouse Kidney Tissue Eighteen ␤-catGOF-UB mutant kidneys and 9 WT kidneys were micro- In Situ Hybridization dissected at E12.5. Mutant kidneys were divided into three random Nonradioactive in situ hybridization was performed using DIG-la- pools (n ϭ 3) consisting of six kidneys each, and WT samples were beled cRNA probes encoding Ret, Gdnf, Wnt11, Wnt4, Lim1, Dkk1, divided into three pools (n ϭ 3) consisting of three kidneys each. RNA and Foxd1 on paraffin-embedded kidney tissue fixed with 4% PFA for 52 quantity (1-␮g total RNA) was sufficient such that only one cycle of 24 hours at 4°C as described previously. amplification was required. Microarray data were processed using GCOS (v1.4, Affymetrix). All chips were scaled to a target value of 500 In Situ TUNEL and BrdU Incorporation Assays before expression analysis. Two WT replicates were normalized to a Terminal deoxynucleotidyl transferase–mediated (TdT-mediated) third WT replicate and these were used as baselines to normalize and dUTP nick end labeling (TUNEL) was performed using 4% PFA fixed compare the three replicate ␤-catGOF-UB samples. Comparisons were paraffin-embedded tissue sections. Briefly, tissue sections were depar- made in all combinations to create a matrix of 3 ϫ 3 crosswise com- affinized, rehydrated, and enzyme-digested (10 ␮g/ml Proteinase K in parisons (9 in total). Probe sets with present calls in all replicates in PBS for 15 minutes). Labeling was performed according to manufac- either the ␤-catGOF-UB or WT samples were kept and all others were turer’s instructions with a 10-minute color reaction and counterstain- removed from the data set. Probe sets with a significant change call in ing with hematoxylin. Slides were then dehydrated and mounted with 6 of 9 comparisons were considered significantly changed in the Permount. ␤-catGOF-UB versus WT samples. To minimize the false positives, a Cell proliferation was assayed in paraffin-embedded kidney tissue threshold signal log ratio (SLR) value was determined by estimating by incorporation of 5-bromo-2-deoxyuridine (BrdU; Roche Molecu- the background error using the distribution of SLR for probe sets with lar Biochemicals, Mannheim, Germany), as described.53 Pregnant fe- no significant change calls. males received an intraperitoneal injection of BrdU (100 mg/g of body Probe sets were mapped to their gene symbol identifiers from an- wt) 2 hours before sacrifice. BrdU-positive cells were identified using notation tables supplied by Affymetrix (http://www.affymetrix.com). an anti-BrdU peroxidase–conjugated antibody as described (Boehr- Lists of gene symbols were entered into the BINGO plugin (v2.049) for inger, Mannheim, Germany). Immunoreactivity was visualized using

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aminoethyl carbazole horseradish peroxidase chromogen/substrate 8. Huelsken J, Behrens J: The Wnt signalling pathway. J Cell Sci 115: solution (Zymed Laboratories, USA). 3977–3978, 2002 9. Park JS, Valerius MT, McMahon AP: Wnt/beta-catenin signaling regulates nephron induction during mouse kidney development. Devel- Treatment of Cultured Kidney Explants opment 134: 2533–2539, 2007 Mouse embryonic kidneys were surgically resected from E11.5 or 10. Marose TD, Merkel CE, McMahon AP, Carroll TJ: Beta-catenin is E12.5 pregnant mice, transferred onto 1.0-␮m polyethylene tereph- necessary to keep cells of ureteric bud/Wolffian duct epithelium in a thalate–track-etched (PET–track-etched) membrane (Falcon) and precursor state. Dev Biol 314: 112–126, 2008 11. 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