The canonical WNT signaling pathway plays a crucial role in patterning of the embryo during development, but little is known about the specific developmental events which are under WNT control. To understand more about how the WNT pathway orchestrates mammalian organogenesis, we studied the canonical ␤-catenin-mediated WNT signaling pathway in kidneys of mice bearing a ␤-catenin-responsive TCF/␤Gal reporter transgene. In metanephric kidney, intense canonical WNT signaling was evident in epithelia of the branching ureteric bud and in nephrogenic mesenchyme during its transition into renal tubules. WNT signaling activity is rapidly downregulated in maturing nephrons and becomes undetectable in postnatal kidney. Sites of TCF/␤Gal activity are in proximity to the known sites of renal WNT2b and WNT4 expression, and these WNTs stimulate TCF reporter activity in kidney cell lines derived from ureteric bud and metanephric mesenchyme lineages. When fetal kidney explants from HoxB7/GFP mice were exposed to the canonical WNT signaling pathway inhibitor, Dickkopf-1, arborization of the ureteric bud was significantly reduced. We conclude that restricted zones of intense canonical WNT signaling drive branching nephrogenesis in fetal kidney. nephrogenesis; ␤-catenin; branching morphogenesis THE WNT FAMILY is comprised of 19 secreted glycoproteins which act as short-range intercellular signaling molecules, recognizing one of the 10 frizzled receptors expressed at the surface of nearby target cells. The canonical signaling pathway is activated by WNTs which bind to cognate frizzled receptors heterodimerized with LRP5 or LRP6 coreceptors (2). Activated receptors recruit dishevelled protein (Dvl) and inhibit degradation of cytoplasmic ␤-catenin via the GSK3␤-axin-APC complex (11). When its degradation is blocked, cytoplasmic ␤-catenin is available to translocate to the nucleus, dimerize with partners belonging to the T-cell factor (TCF) family, and activate target genes. TCF recognition motifs have been well-studied, allowing design of vectors (e.g., TOPFlash) which drive transcription of reporter genes in response to canonical WNT signaling activity (32). In general, canonical ␤-catenin/TCF signaling is thought to activate gene targets (e.g., c-myc) involved in cell proliferation (3, 27).More than 35 years ago, Unsworth and Grobstein (33) reported that tissue from spinal cord could induce formation of renal tubules when cocultured with isolated metanephric mesenchyme. In 1994, Herzlinger et al. (12) found that WNT1-expressing NIH3T3 cells were also able to induce tubule formation in the coculture assay, suggesting that the canonical (␤-catenin-mediated) WNT signaling pathway is essential for mammalian nephrogenesis. However, the precise function of canonical WNT signaling in renal development is unknown. Surprisingly, WNT1 is not present in the developing kidney, but numerous other WNTs are transiently expressed in specific cell lineages (34). Several of these are able to activate the canonical signaling pathway in other context...

DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.No conflicts of interest were declared. pleuropulmonary blastoma (PPB) syndrome (OMIM 601200) [7,8]. Several genes have been identified as being somatically mutated in WT, including WT1 , CTNNB1 , IGF2 , TP53 , WTX , DIS3L2 , FBXW7 and MYCN [5,9]. There are overlapping distributions of molecular abnormalities at 11p15, WTX , WT1 and CTNNB1 in WT [9,10], but other genes could be contributing to the aetiology of WT [3,[11][12][13]. WT histopathology is heterogeneous and tumours associated with different types of nephrogenic rests and histologies tend to show different underlying genetic changes [14,15]. A revised model for WT ontogeny takes into account genetic data (WT1 status, 11p15 imprint control region 1 methylation status, gene expression patterns of other genes) and histological data [14]. Nevertheless, each WT is thought to be of monoclonal origin [16].

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the "S-shaped" body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.PAX2 belongs to the "paired box" family of transcription factors. Like other family members, it is thought to orchestrate the patterns of gene expression in specific cells during organ development. Homozygous inactivation of the Pax2 gene in mice causes malformation of the midbrain-hindbrain region, the optic nerve, and complete absence of the kidneys (1, 2). Although these observations clearly implicate PAX2 in the development of renal and neural tissue, little is known about its precise gene targets or the specific developmental processes in which it plays a role.Studies thus far suggest that the developmental functions of PAX2 may be multiplex, activating distinct panels of genes in different cell lineages at different stages. During development of mammalian kidney, PAX2 first appears during the caudal descent of the nephric duct where it affects the fate of a cell (3). When the nephric duct arrives at about somite 26, a "ureteric bud" (UB) 2 emerges from its wall and grows toward the adjacent lateral mesenchyme. This event is again orchestrated by PAX2 through activation of glial cell line-derived neurotropic Factor (GDNF) in the uninduced mesenchyme and activation of the GDNF receptor (RET) in UB cells (4). Thus, homozygous Pax2 knockout mice lack normal ureteric bud outgrowth and are unable to induce metanephric kidneys (2).A third function of PAX2 in developing kidney involves suppression of programmed cell death in ureteric bud cells. During ...

Human nephrons are formed during fetal life through an interaction between the branching ureteric bud and progenitor cells. The wide variation in final nephron number has been attributed to allelic variants of genes regulating ureteric bud arborization. Here, we hypothesize that dysfunctional variants of the Odd-Skipped Related 1 (OSR1) gene which compromise the renal progenitor cell pool might also limit newborn kidney size and function. We show that OSR1 is expressed in human mesenchymal stem cells, the blastemal component of Wilms tumors and CD24+/CD133+ progenitor cells isolated from the mature kidney. We identified an OSR1(rs12329305(T)) allele in 6% of normal Caucasians which alters an exon2 splice enhancer. This variant is predicted to reduce spliceosome-binding affinity and stability of the OSR1 mRNA. In cultured cells, the OSR1(rs12329305)(T) allele produced no identifiable transcript. Normal Caucasian newborns from Montreal with the OSR1(rs12329305)(T) allele had kidney volume 11.8% smaller (P= 0.006) and cord blood cystatin C levels 12.6% higher (P = 0.005) than those with wild-type genotype. Effects of the OSR1(rs12329305)(T) allele are additive with genes that alter ureteric bud branching. Kidney volume was reduced more in newborns bearing both RET(rs1800860)(A) and OSR1(rs12329305)(T) alleles (22%, P= 0.0008) and cystatin C was increased by 17% (P= 0.006) versus newborns with wild-type alleles. Although only two subjects had PAX2(rs11599825)(A) and OSR1(rs12329305)(T) alleles, kidney size was reduced by 27% and cystatin C was increased by 14% versus wild-types (P= NS).

b-Catenin/Wnt signaling is essential during early inductive stages of kidney development, but its role during postinductive stages of nephron development and maturation is not well understood. In this study, we used Pax8Cre mice to target b-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional b-catenin knockout mice formed abnormal kidneys and had reduced renal function. The kidneys were hypoplastic with a thin cortex; a superficial layer of tubules was missing. A high proportion of glomeruli had small, underdeveloped capillary tufts. In these glomeruli, well differentiated podocytes replaced parietal epithelial cells in Bowman's capsule; capillaries toward the outer aspect of these podocytes mimicked the formation of glomerular capillaries. Tracing nephrogenesis in embryonic conditional b-catenin knockout mice revealed that these "parietal podocytes" derived from precursor cells in the parietal layer of the S-shaped body by direct lineage switch. Taken together, these findings demonstrate that b-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.

Mutations in PKD1 cause dominant polycystic kidney disease (PKD), characterized by large fluid-filled kidney cysts in adult life, but the molecular mechanism of cystogenesis remains obscure. Ostrom et al. [Dev. Biol., 219, 250-258 (2000)] showed that reduced dosage of Pax2 caused increased apoptosis, and ameliorated cystogenesis in Cpk mutant mice with recessive PKD. Pax2 is expressed in condensing metanephrogenic mesenchyme and arborizing ureteric bud, and plays an important role in kidney development. Transient Pax2 expression during fetal kidney mesenchyme-to-epithelial transition, as well as in nascent tubules, is followed by marked down-regulation of Pax2 expression. Here, we show that in humans with PKD, as well as in Pkd1(del34/del34) mutant mice, Pax2 was expressed in cyst epithelial cells, and facilitated cyst growth in Pkd1(del34/del34) mutant mice. In Pkd1(del34/del34) mutant kidneys, the expression of Pax2 persisted in nascent collecting ducts. In contrast, homozygous Pkd1(del34/del34) fetal mice carrying mutant Pax2 exhibited ameliorated cyst growth, although reduced cystogenesis was not associated with increased apoptosis. Pax2 expression was attenuated in nascent collecting ducts and absent from remnant cysts of Pkd1(del34/del34)/Pax2(1Neu/+) mutant mice. To investigate whether the Pkd1 gene product, Polycystin-1, regulates Pax2, MDCK cells were engineered constitutively expressing wild-type Pkd1; Pax2 protein levels and promoter activity were both repressed in MDCK cells over-expressing Pkd1, but not in cells without transgenic Pkd1. These data suggest that polycystin-1-deficient tubular epithelia persistently express Pax2 in ADPKD, and that Pax2 or its pathway may be an appropriate target for the development of novel therapies for ADPKD.

Polycystic liver disease (PCLD) is characterized by the growth of fluid-filled cysts of biliary epithelial origin in the liver. Although the disease is often asymptomatic, it can, when severe, lead to complications requiring surgical therapy. PCLD is most often associated with autosomal dominant polycystic kidney disease (ADPKD); however, families with an isolated polycystic liver phenotype without kidney involvement have been described. The clinical presentation and histological features of polycystic liver disease in the presence or absence of ADPKD are indistinguishable, raising the possibility that the pathogenetic mechanisms in the diseases are interrelated. We ascertained two large families with polycystic liver disease without kidney cysts and performed a genomewide scan for genetic linkage. A causative gene, PCLD, was mapped to chromosome 19p13.2-13.1, with a maximum LOD score of 10.3. Haplotype analysis refined the PCLD interval to 12.5 cM flanked by D19S586/D19S583 and D19S593/D19S579. The discovery of genetic linkage will facilitate diagnosis and study of this underdiagnosed disease entity. Identification of PCLD will be instrumental to an understanding of the pathogenesis of cyst formation in the liver in isolated PCLD and in ADPKD.