SUMMARYThe Wilms' tumor suppressor 1 (WT1) gene encodes a DNA-and RNA-binding protein that plays an essential role in nephron progenitor differentiation during renal development. To identify WT1 target genes that might regulate nephron progenitor differentiation in vivo, we performed chromatin immunoprecipitation (ChIP) coupled to mouse promoter microarray (ChIP-chip) using chromatin prepared from embryonic mouse kidney tissue. We identified 1663 genes bound by WT1, 86% of which contain a previously identified, conserved, high-affinity WT1 binding site. To investigate functional interactions between WT1 and candidate target genes in nephron progenitors, we used a novel, modified WT1 morpholino loss-of-function model in embryonic mouse kidney explants to knock down WT1 expression in nephron progenitors ex vivo. Low doses of WT1 morpholino resulted in reduced WT1 target gene expression specifically in nephron progenitors, whereas high doses of WT1 morpholino arrested kidney explant development and were associated with increased nephron progenitor cell apoptosis, reminiscent of the phenotype observed in Wt1 -/-embryos. Collectively, our results provide a comprehensive description of endogenous WT1 target genes in nephron progenitor cells in vivo, as well as insights into the transcriptional signaling networks controlled by WT1 that might direct nephron progenitor fate during renal development.

Understanding the mechanisms that regulate nephron progenitors during kidney development should aid development of therapies for renal failure. MicroRNAs, which modulate gene expression through post-transcriptional repression of specific target mRNAs, contribute to the differentiation of stem cells, but their role in nephrogenesis is incompletely understood. Here, we found that the loss of miRNAs in nephron progenitors results in a premature depletion of this population during kidney development. Increased apoptosis and expression of the pro-apoptotic protein Bim accompanied this depletion. Profiling of miRNA expression during nephrogenesis identified several highly expressed miRNAs (miR10a, miR-106b, miR-17-5p) in nephron progenitors that are either known or predicted to target Bim. We propose that modulation of apoptosis by miRNAs may determine congenital nephron endowment. Furthermore, our data implicate the pro-apoptotic protein Bim as a miRNA target in nephron progenitors. Kidney development begins with the outgrowth of the ureteric bud from the Wolffian duct into the metanephric mesenchyme. 1,2 The metanephric mesenchyme condenses as a tight "cap" of nephron progenitors around the tip of the ureteric bud and the ureteric bud branches to form the collecting system. Nephron progenitors have the capacity to selfrenew to generate the full complement of nephrons and to differentiate into the multiple cell types required to form the nephron. This process continues in an iterative fashion during nephrogenesis such that the most immature cells are present in the subcapsular cortex of the developing kidney, termed the nephrogenic zone.MicroRNAs (miRNAs) are a group of endogenous, small noncoding RNAs that function by causing the post-transcriptional repression of their respective target mRNAs. The first suggestion that miRNAs are critical in stem cell populations came from the observation that embryos that are null for Dicer, an enzyme required for the production of

Paracrine signaling between podocytes and glomerular endothelial cells through vascular endothelial growth factor A (VEGFA) maintains a functional glomerular filtration barrier. Heparan sulfate proteoglycans (HSPGs), located on the cell surface or in the extracellular matrix, bind signaling molecules such as VEGFA and affect their local concentrations, but whether modulation of these moieties promotes normal crosstalk between podocytes and endothelial cells is unknown. Here, we found that the transcription factor Wilms' Tumor 1 (WT1) modulates VEGFA and FGF2 signaling by increasing the expression of the 6-O-endosulfatases Sulf1 and Sulf2, which remodel the heparan sulfate 6-O-sulfation pattern in the extracellular matrix. Mice deficient in both Sulf1 and Sulf2 developed age-dependent proteinuria as a result of ultrastructural abnormalities in podocytes and endothelial cells, a phenotype similar to that observed in children with WT1 mutations and in Wt1 ϩ/Ϫ mice. These kidney defects associated with a decreased distribution of VEGFA in the glomerular basement membrane and on endothelial cells. Collectively, these data suggest that WT1-dependent sulfatase expression plays a critical role in maintaining the glomerular filtration barrier by modulating the bioavailability of growth factors, thereby promoting normal crosstalk between podocytes and endothelial cells.

The molecular signals that regulate growth and branching of the ureteric bud during formation of the renal collecting system are largely undefined. Members of the bone morphogenetic protein (BMP) family signal through the type I BMP receptor ALK3 to inhibit ureteric bud and collecting duct cell morphogenesis in vitro. We investigated the function of the BMP signaling pathway in vivo by generating a murine model of ALK3 deficiency restricted to the ureteric bud lineage (Alk3 UBϪ/Ϫ mice). At the onset of branching morphogenesis, Alk3 UBϪ/Ϫ kidneys are characterized by an abnormal primary (1Њ) ureteric bud branch pattern and an increased number of ureteric bud branches. However, during later stages of renal development, Alk3 UBϪ/Ϫ kidneys have fewer ureteric bud branches and collecting ducts than wild-type kidneys. Postnatal Alk3 UBϪ/Ϫ mice exhibit a dysplastic renal phenotype characterized by hypoplasia of the renal medulla, a decreased number of medullary collecting ducts, and abnormal expression of ␤-catenin and c-MYC in medullary tubules. In summary, normal kidney development requires ALK3-dependent BMP signaling, which controls ureteric bud branching.

inhibitor, blocked the stimulatory effect of BMP7 on mIMCD-3 cell morphogenesis but had no effect on BMP7-dependent inhibition in a three-dimensional culture model. To identify mechanisms by which BMP7-dependent inhibitory signaling suppresses p38 MAPK activity, we measured p38 MAPK activity in ligand independent mIMCD-3 models of enhanced and suppressed Smad signaling. Basal activity of p38 MAPK was decreased in mIMCD-3 cells and in embryonic kidney tissue expressing a constitutively active activin-like kinase receptor, but was increased in mIMCD-3 cells stably expressing a dominant negative form of Smad1. We conclude that BMP7 stimulates renal epithelial cell morphogenesis via p38 MAPK and that p38 MAPK activity is negatively regulated by Smad1.Renal branching morphogenesis, defined as growth and branching of epithelial tubules during embryogenesis, is dependent on reciprocal inductive tissue interactions between the mesenchymal metanephric blastema and the epithelial ureteric bud and its daughter collecting ducts. These interactions are mediated, in part, by secreted growth factors and their cognate signaling effectors. Bone morphogenetic protein (BMP) 1 -7, a member of the transforming growth factor (TGF)-␤ superfamily, modulates renal branching morphogenesis, consistent with its spatial expression pattern during branching morphogenesis (1) and the arrested branching phenotype observed in Bmp7 null mice (2, 3). The response of ureteric bud and collecting duct cells to BMP7 is complex and distinct from that of other members of the TGF-␤ superfamily. BMP7 exerts dose-dependent and opposite effects on ureteric bud morphogenesis in embryonic kidney explants treated with BMP7-agarose beads and in the murine inner medullary collecting duct (mIMCD-3) cell culture model (4, 5). In contrast, BMP2, a related TGF-␤ superfamily member expressed during renal embryogenesis, only inhibits ureteric bud and collecting duct morphogenesis in a monophasic dose-dependent manner (4). These findings suggest that BMP7 acts via distinct intracellular signaling pathways to exert stimulatory and inhibitory effects.BMPs initiate intracellular signaling after binding to cell surface type I (activin-like kinase (ALK)) and type II serine/ threonine kinases. Upon ligand binding, the type II receptor, BMPRII, transphosphorylates and activates the ALK receptor. ALK receptors signal via Smad proteins and mitogen-activated protein kinases (MAPK). Activation of the ALK receptor leads to association and phosphorylation of a receptor-activated Smad protein. Phosphorylation induces the receptor-activated Smad to dissociate from the receptor, stimulates the assembly of a heteromeric complex between the phosphorylated receptoractivated Smad and the co-Smad, Smad 4, and induces nuclear accumulation of this complex (6). We have demonstrated that BMPs inhibit renal epithelial cell morphogenesis after binding ALK receptors and activating receptor-dependent Smads (7,8). In the case of BMP7, high doses activate Smad1 and induce formation of Smad1-Smad4 mo...

The kidney is a model developmental paradigm of vertebrate organogenesis. As in many other organs, kidney development involves reciprocal inductive tissue interactions between multiple cell lineages. The most well defined of these interactions occurs between the ureteric bud and the nephrogenic mesenchyme. A population of mesenchymal cells distinct from nephrogenic precursors and termed stromal cells, have been relatively understudied. Yet existing knowledge indicates that stromal cells are critical regulators in the normal and diseased kidney. This commentary reviews current knowledge regarding the origin and functional roles of the stromal cell population during kidney development. Gaps in our current understanding of renal stromal cells and future directions needed to advance this expanding field of study are highlighted. Developmental Dynamics 243:853-863,

Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is a tightly regulated process controlled by growth factor-dependent tissue interactions. Previously, using in vitro models of branching morphogenesis, we demonstrated that BMP2 signals via its intracellular effectors, SMAD1 and SMAD4, to control UB cell proliferation and branching in a manner modulated by Glypican-3 (GPC3), a cell surface heparan sulfate proteoglycan. Here, we used loss-of-function genetic mouse models to investigate the functions of Bmp2 and Gpc3-Bmp2 interactions in vivo. Progressively greater increases in UB cell proliferation were observed in Bmp2+/-, Smad4+/-, and Bmp2+/-; Smad4+/- mice compared to Wt. This increased cell proliferation was accompanied by a significant increase in UB branching in Smad4+/- and Bmp2+/-;Smad4+/- mice compared to Wt. Reduction of Gpc3 gene dosage also increased UB cell proliferation, an effect that was enhanced in Gpc3+/-;Bmp2+/- mice to an extent greater than the sum of that observed in Gpc3+/- and Bmp2+/- mice. Reduction of both Gpc3 and Bmp2 gene dosage enhanced cell proliferation in the metanephric mesenchyme compared to Wt, an effect not observed in either Bmp2+/- or Gpc3+/- mice. Phosphorylation of SMAD1, a measure of SMAD1 activation, was progressively decreased in Gpc3+/- and Gpc3+/-;Bmp2+/- mice compared to Wt, suggesting that Gpc3 stimulates Bmp2-dependent SMAD signaling in vivo. These results demonstrate that BMP2-SMAD signaling, modulated by GPC3, inhibits renal branching morphogenesis in vivo.

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