Another niche for Notch.

The Notch signaling pathway patterns the developing nephron along the proximal-distal axis during renal development. In an adult acute tubular necrosis model, Kobayashi et al. now show expression of many Notch components and the activation of Notch target genes, suggesting a critical function for Notch in regenerating proximal tubules.

Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. We demonstrate that Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin.

PTIP, a novel BRCT domain-containing protein interacts with Pax2 and is associated with active chromatin.

The Pax gene family encodes transcription factors essential for organ and tissue development in higher eukaryotes. Pax proteins are modular with an N-terminal DNA binding domain, a C-terminal transcription activation domain, and a transcription repression domain called the octapeptide. How these domains interact with the cellular machinery remains unclear. In this report, we describe the isolation and characterization of a novel gene and its encoded protein, PTIP, which binds to the activation domain of Pax2 and other Pax proteins. PTIP binds to Pax2 in vitro, in the yeast two-hybrid assay and in tissue culture cells. The binding of PTIP to Pax2 is inhibited by the octapeptide repression domain. The PTIP protein contains five BRCT domains, first identified in BRCA1 and other nuclear proteins involved in DNA repair/recombination or cell cycle control. Pax2 and PTIP co-localize in the cell nucleus to actively expressed chromatin and the nuclear matrix fraction. For the first time, these results point to a link between Pax transcription factors and active chromatin.

Kidney development in cadherin-6 mutants: delayed mesenchyme-to-epithelial conversion and loss of nephrons.

During nephrogenesis, dynamic changes in the expression of cell adhesion molecules are evident as epithelial structures differentiate from the induced mesenchyme. The cadherins are thought to play an important role in the metanephric mesenchyme, when cells aggregate to form the renal vesicle, a polarized epithelial structure which eventually fuses with the ureteric bud to generate a continuous nascent nephron. We have generated and analyzed mice with a targeted mutation in the gene encoding cadherin-6 (Cad-6), a type II cadherin expressed during early stages of nephrogenesis. These mice are viable and fertile, and they complete both early and late aspects of nephrogenesis. However, upon closer examination in vitro and in vivo, a fraction of the induced metanephric mesenchyme in Cad-6 mutant kidneys fails to form a fully polarized epithelium on schedule. Moreover, a significant number of the renal vesicles in Cad-6 mutant kidneys apparently fail to fuse to the ureteric bud. These alterations in epithelialization and fusion apparently lead to a loss of nephrons in the adult. These studies support the idea that cadherins play an essential role in the formation of epithelial structures and underscore the importance of timing in orchestrating the morphogenesis of complex epithelial tissues.

Reduced Pax2 gene dosage increases apoptosis and slows the progression of renal cystic disease.

The murine cpk mouse develops a rapid-onset polycystic kidney disease (PKD) with many similarities to human PKD. During kidney development, the transcription factor Pax2 is required for the specification and differentiation of the renal epithelium. In humans, Pax2 is also expressed in juvenile cystic kidneys where it correlates with cell proliferation. In this report, Pax2 expression is demonstrated in the cystic epithelium of the mouse cpk kidneys. To assess the role of Pax2 during the development of polycystic kidney disease, the progression of renal cysts was examined in cpk mutants carrying one or two alleles of Pax2. Reduced Pax2 gene dosage resulted in a significant inhibition of renal cyst growth while maintaining more normal renal structures. The inhibition of cyst growth was not due to reduced proliferation of the cystic epithelium, rather to increased cell death in the Pax2 heterozygotes. Increased apoptosis with reduced Pax2 gene dosage was also observed in normal developing kidneys. Thus, increased cell death is an integral part of the Pax2 heterozygous phenotype and may be the underlying cause of Pax gene haploinsufficiency. That the cystic epithelium requires Pax2 for continued expansion underscores the embryonic nature of the renal cystic cells and may provide new insights toward growth suppression strategies.

Pax2 in development and renal disease.

Pax genes are associated with a variety of developmental mutations in mouse and man that are gene dosage sensitive, or haploinsufficient. The Pax2 gene encodes a DNA binding, transcription factor whose expression is essential for the development of the renal epithelium. Both gain and loss of function mutants in the mouse demonstrate a requirement for Pax2 in the conversion of metanephric mesenchymal precursor cells to the fully differentiated tubular epithelium of the nephron. However, Pax2 expression is down-regulated as cells leave the mitotic cycle. Humans carrying a single Pax2 mutant allele exhibit renal hypoplasia, vesicoureteric reflux, and optic nerve colobomas. Conversely, persistent expression of Pax2 has been demonstrated in a variety of cystic and dysplastic renal diseases and correlates with continued proliferation of renal epithelial cells. Thus, Pax2 misexpresssion may be a key determinant in the initiation and progression of renal diseases marked by increased or deregulated cell proliferation.

Kidney development branches out.

For more than 40 years now, the developing kidney has served as a model paradigm for epithelial-mesenchymal interactions. The principles of inductive signaling, epithelial cell differentiation, and pattern formation are now being addressed with modern genetic and biochemical tools. In addition to the mammalian kidney organ culture model, both zebrafish and Xenopus laevis demonstrate great potential for investigating the molecular mechanisms of kidney organogenesis within a whole organism. In this review, the papers presented in this special issue are discussed with respect to recent progress in the renal development field. Coincidentally, it has become increasingly clear that progress made in renal development can impact our understanding of the genetic basis of disease.

WT1 and PAX-2 podocyte expression in Denys-Drash syndrome and isolated diffuse mesangial sclerosis.

Denys-Drash syndrome is a rare disorder of urogenital development characterized by the association of early onset glomerulopathy caused by diffuse mesangial sclerosis, gonadal dysgenesis leading to pseudohermaphroditism in males, and a high risk of developing Wilms' tumor. The syndrome is caused by dominant negative point mutations in the WT1 gene that encodes a tumor suppressor transcription factor normally expressed in podocytes. Mutations usually affect the zinc fingers of the WT1 protein. The basic defect is unknown in most cases of isolated diffuse mesangial sclerosis, a disease characterized by the same glomerular changes as in Denys-Drash syndrome but possibly transmitted as an autosomal recessive trait. Here we show that the distribution of WT1 is abnormal in most patients with Denys-Drash syndrome : WT1 nuclear staining of podocytes is decreased or absent. This finding is consistent with the decreased DNA binding capacity of the mutated protein. One target gene of WT1 is PAX2, the expression of which is down-regulated in podocytes during early stages of nephrogenesis. We demonstrate that WT1 mislocalization is associated with abnormal podocyte expression of PAX2 protein and RNA. We suggest that persistent expression of PAX2 is likely to result from the loss of WT1 dependent transcriptional repression and may participate in the pathological mechanisms leading to glomerular dysfunction. Abnormal distribution of WT1 and PAX2 was also observed in isolated diffuse mesangial sclerosis suggesting that a defect in WT1 could also be operative in isolated diffuse mesangial sclerosis. Primary involvement of PAX2 is an alternative hypothesis because persistent expression of PAX2 in transgenic mice is associated with the occurrence of early and severe glomerulopathy.

Expression of Grb7 growth factor receptor signaling protein in kidney development and in adult kidney.

Grb7, a signaling protein whose physiological function is unknown, binds receptor tyrosine kinases important for normal kidney development. By investigating and correlating Grb7 gene expression with that reported for Grb7-binding receptors, we provide clues to Grb7 function(s). RT-PCR and immunoblot were used to demonstrate Grb7 gene and protein expression in the mature kidney. Additional RT-PCR studies detected gene expression in all microdissected adult nephron segments examined, except glomeruli, and in the mouse metanephric kidney from embryonic day 11 (E11) through to day 17 (E17). In situ hybridization at E14 demonstrated the following cellular pattern of localization: Grb7 mRNA in metanephric epithelia of mesenchymal and ureteric bud origin; no expression in the undifferentiated mesenchyme; and little expression in podocyte-destined cells or primitive glomeruli. Grb7 mRNA was also present in the epithelia of the lung and gut at E14. Thus Grb7 may have a basic function in growth factor signaling in terminally differentiated epithelia along the nephron and in developing epithelia in the kidney, lung, and gut. It is localized in a pattern permissive for a role in Her2 and Ret receptor signaling.

TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs.

The Tcf family of transcription factors, in association with beta-catenin, mediate Wnt signaling by transactivating downstream target genes. Given the function of wnt genes in neural development and organogenesis, Tcf transcription factors must be integral to the development of many embryonic tissues. In fact, the role of Tcf genes in axis formation in Xenopus and in segment polarity in Drosophila is well established. In this report, we have identified two isoforms of the mouse Tcf-4 gene. Tcf-4 expressing cells showed nuclear localization of beta-catenin. Although Tcf-4 RNA was widely distributed throughout embryogenesis, high levels of Tcf-4 expression were particularly evident in the developing CNS and limb buds. In extended streak stage embryos (E7.5), Tcf4 expression was detected in anterior endoderm. E8.5 embryos had Tcf-4 expression in rostral neural plate and in alternating rhombomeres of the hindbrain. By E9.5 and thereafter, expression in the hindbrain disappeared and strong expression was detected in the diencephalon. Strikingly Tcf-4 expression in the forebrain was undetected in Small eye mutant embryos indicating that Pax-6 is required for Tcf-4 expression in the forebrain. In developing limbs, Tcf-4 is readily detected starting at E10.5 and is limited to mesenchymal cells surrounding the areas of chondrification. These data indicate a function for Tcf-4 in neural and limb development, two tissues where Wnt signaling plays an essential role.

The RET-glial cell-derived neurotrophic factor (GDNF) pathway stimulates migration and chemoattraction of epithelial cells.

Embryonic development requires cell migration in response to positional cues. Yet, how groups of cells recognize and translate positional information into morphogenetic movement remains poorly understood. In the developing kidney, the ureteric bud epithelium grows from the nephric duct towards a group of posterior intermediate mesodermal cells, the metanephric mesenchyme, and induces the formation of the adult kidney. The secreted protein GDNF and its receptor RET are required for ureteric bud outgrowth and subsequent branching. However, it is unclear whether the GDNF-RET pathway regulates cell migration, proliferation, survival, or chemotaxis. In this report, we have used the MDCK renal epithelial cell line to show that activation of the RET pathway results in increased cell motility, dissociation of cell adhesion, and the migration towards a localized source of GDNF. Cellular responses to RET activation include the formation of lamellipodia, filopodia, and reorganization of the actin cytoskeleton. These data demonstrate that GDNF is a chemoattractant for RET-expressing epithelial cells and thus account for the developmental defects observed in RET and GDNF mutant mice. Furthermore, the RET-transfected MDCK cells described in this report are a promising model for delineating RET signaling pathways in the renal epithelial cell lineage.

Differential expression and function of cadherin-6 during renal epithelium development.

The cadherin gene family encodes calcium-dependent adhesion molecules that promote homophilic interactions among cells. During embryogenesis, differential expression of cadherins can drive morphogenesis by stimulating cell aggregation, defining boundaries between groups of cells and promoting cell migration. In this report, the expression patterns of cadherins were examined by immunocytochemistry and in situ hybridization in the embryonic kidney, during the time when mesenchymal cells are phenotypically converted to epithelium and the pattern of the developing nephrons is established. At the time of mesenchymal induction, cadherin-11 is expressed in the mesenchyme but not in the ureteric bud epithelium, which expresses E-cadherin. The newly formed epithelium of the renal vesicle expresses E-cadherin near the ureteric bud tips and cadherin-6 more distally, suggesting that this primitive epithelium is already patterned with respect to progenitor cell types. In the s-shaped body, the cadherin expression patterns reflect the developmental fate of each region. The proximal tubule progenitors express cadherin-6, the distal tubule cells express E-cadherin, whereas the glomeruli express P-cadherin. Ultimately, cadherin-6 is down-regulated whereas E-cadherin expression remains in most, if not all, of the tubular epithelium. Antibodies generated against the extracellular domain of cadherin-6 inhibit aggregation of induced mesenchyme and the formation of mesenchyme-derived epithelium but do not disrupt ureteric bud branching in vitro. These data suggest that cadherin-6 function is required for the early aggregation of induced mesenchymal cells and their subsequent conversion to epithelium.

The molecular basis of embryonic kidney development.

The development of the mature mammalian kidney begins with the invasion of metanephric mesenchyme by ureteric bud. Mesenchymal cells near the bud become induced and convert to an epithelium which goes on to generate the functional filtering unit of the kidney, the nephron. The collecting duct system is elaborated by the branching ureter, the growth of which is dependent upon signals from the metanephric mesenchyme. The process of reciprocal induction between ureter and mesenchyme is repeated many times over during development and is the key step in generating the overall architecture of the kidney. Genetic studies in mice have allowed researchers to begin to unravel the molecular signals that govern these early events. These experiments have revealed that a number of essential gene products are required for distinct steps in kidney organogenesis. Here we review and summarize the developmental role played by some of these molecules, especially certain transcription factors and growth factors and their receptors. Although the factors involved are far from completely known a rough framework of a molecular cascade which governs embryonic kidney development is beginning to emerge.

Pax-2, kidney development, and oncogenesis.

The development of a complex tissue from a few simple precursor cells requires the precise activation and repression of tissue-specific genes that determine cell lineages, tissue patterning, and cellular proliferation. In the kidney, a number of recently identified genes are critical for normal development. Among these, the Pax-2 gene encodes a transcription factor that is expressed in the ureter bud, in the induced kidney mesenchyme, and in the progenitor cells of the glomerular and tubular epithelium. Although the differentiation of the renal epithelium requires Pax-2 function, failure to suppress the gene in mature epithelium is detrimental to normal renal function. Recent, data suggest that the Wilms' tumor-suppressor gene WT1 can down-regulate Pax-2 expression, consistent with high levels of Pax-2 in Wilms' tumors. Additional studies suggest that reactivation of this developmental regulator can contribute to a variety of other renal diseases.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

Mapping of Pax-2 transcription activation domains.

Pax genes encode transcription factors known to play crucial roles during the development of specific embryonic tissues. In humans and mice, several abnormalities have been linked to deficiencies in Pax gene dosage, indicating that normal development is particularly sensitive to the level of Pax gene expression. Despite these facts, relatively little is known about how these proteins act as transcriptional regulators. In this study we define the transactivation domains of murine Pax-2, an essential factor in kidney organogenesis. Within the COOH terminus of Pax-2, amino acids 279-373 are essential for transactivation. However, this region alone is insufficient for full transactivation when fused to the paired domain alone or to a heterologous DNA binding domain. Mutation or deletion of the conserved octapeptide sequence results in increased transactivation by Pax proteins. The octapeptide-mediated repression is also seen within a heterologous context using the GAL4 DNA binding domain. Thus transactivation by Pax-2 relies upon several regions within the COOH terminus and is down-modulated by the octapeptide element.

The PAX2 tanscription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations.

Human dysplastic kidneys are developmental aberrations which are responsible for many of the very young children with chronic renal failure. They contain poorly differentiated metanephric cells in addition to metaplastic elements. We recently demonstrated that apoptosis was prominent in undifferentiated cells around dysplastic tubules (Winyard, P.J.D., J. Nauta, D.S. Lirenman, P. Hardman, V.R. Sams, R.A. Risdon, and A.S. Woolf. 1996. Kidney Int. 49:135-146), perhaps explaining the tendency of some of these organs to regress. In contrast, apoptosis was rare in dysplastic epithelia which are thought to be ureteric bud malformations. On occasion, these tubules form cysts which distend the abdominal cavity (the multicystic dysplastic kidney) and dysplastic kidneys may rarely become malignant. We now demonstrate that dysplastic tubules maintain a high rate of proliferation postnatally and that PAX2, a potentially oncogenic transcription factor, is expressed in these epithelia. In contrast, both cell proliferation and PAX2 are downregulated during normal maturation of human collecting ducts. We demonstrate that BCL2, a protein which prevents apoptosis in renal mesenchymal to epithelia] conversion, is expressed ectopically in dysplastic kidney epithelia. We propose that dysplastic cyst formation may be understood in terms of aberrant temporal and spatial expression of master genes which are tightly regulated in the normal program of human nephrogenesis.

Identification of novel Pax-2 binding sites by chromatin precipitation.

The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci.