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...
The molecular mechanisms that set congenital nephron number are unknown. However, humans with modest suboptimal nephron number may be at increased risk for essential hypertension, and those with more severe nephron deficits at birth may develop progressive renal insufficiency. A model of branching morphogenesis during fetal kidney development in which the extent of ureteric bud arborization is dependent on suppression of programmed cell death has been proposed. This study shows that the increased apoptosis and reduced ureteric bud branching of heterozygous Pax2 mutant mice is associated with 40% decrease in nephron number at birth. This leads to postnatal glomerular hypertrophy and long-term renal insufficiency in the absence of glomerulosclerosis. To determine whether restoration of antiapoptotic factors alone is sufficient to rescue the nephron deficit in these mice, a BCL2 transgene that is under the control of the PAX2 promoter was targeted to the ureteric bud. The transgene suppressed programmed cell death in the ureteric bud lineage, increased nephron number to 90% of that of wild-type littermates at birth, and normalized renal function at 1 yr. These observations lend strong support to the hypothesis that factors that control ureteric bud apoptosis are powerful determinants of congenital nephron endowment.
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 ...
Abstract. In humans, PAX2 haploinsufficiency causes renalcoloboma syndrome (RCS) involving eye abnormalities, renal hypoplasia, and renal failure in early life. The authors previously showed that heterozygous mutant Pax2 mice have smaller kidneys with fewer nephrons, associated with elevated apoptosis in the ureteric bud (UB). However, PAX2 may have a variety of developmental functions such as effects on cell fate and differentiation. To determine whether apoptosis alone is sufficient to cause a UB branching deficit, the authors targeted a pro-apoptotic gene (Bax␣) to the embryonic kidney under the control of human PAX2 regulatory elements. The exogenous PAX2 promoter directed Bax␣ gene expression specifically to the developing kidney UB, eye, and mid/hindbrain. At E15.5PAX2Promoter-Bax␣ fetal mice exhibited renal hypoplasia, elevated UB apoptosis, and retinal defects, mimicking the phenotype observed in RCS. The kidneys of E15.5 PAX2Promoter-Bax␣ fetal mice were 55% smaller than those of wild-type fetal mice, and they contained 70% of the normal level of UB branching. The data indicate that loss of Pax2 anti-apoptotic activity is sufficient to account for the reduced UB branching observed in RCS and suggest that elevated UB apoptosis may be a key process responsible for renal hypoplasia. The authors propose a morphogenic unit model in which cell survival influences the rate of UB branching and determines final nephron endowment.In humans and mice, heterozygous PAX2 mutations cause kidney, eye, and central nervous system abnormalities, constituting a syndrome called renal-coloboma syndrome (RCS) (1-3). The kidney abnormalities in patients with RCS involve primary renal hypoplasia, which together with optic nerve colobomas represent the major presenting features of RCS (3). Mutations in PAX2 have also been identified in patients with isolated primary renal hypoplasia, including oligomeganephronia (renal hypoplasia with glomerular hypertrophy) (4,5). Analyses of renal biopsies from children with either RCS or oligomeganephronia show that renal hypoplasia is secondary to mechanisms leading to a paucity of nephrons (3-5). This observation points to a deficit in ureteric bud (UB) branching in patients carrying PAX2 mutations, as UB branching is a prerequisite for nephron formation. Although PAX2 mutations lead to renal hypoplasia, the mechanism by which reduced PAX2 dosage leads to a decreased extent of UB branching and lack of nephrons in patients with RCS is not clear.Mammalian kidney development begins early in embryonic life, when the UB emerges from the nephric duct. UB growth is elicited by trophic signals (e.g., glial cell-derived neurotrophic factor [GDNF] and Wnt2b) from the adjacent mesenchyme, which activate specific receptors on the UB cell surface (6,7). As the UB penetrates the mesenchyme, it begins to arborize, inducing individual nephrons at the tip of each of its branches as it grows. Signals from each branch tip recruit adjacent mesenchymal cells to condense at its lateral aspect and undergo rapid ...
Abstract. In renal-coloboma syndrome (RCS), null mutations of the PAX2 gene cause renal hypoplasia due to a congenital deficit of nephrons; affected individuals may develop renal insufficiency in childhood. During normal kidney development, PAX2, is expressed at high levels throughout the arborizing ureteric bud (UB); recent observations suggest that one of its key roles is to suppress apoptosis in this collecting duct lineage. The authors hypothesized that increased UB cell apoptosis due to PAX2 haploinsufficiency must directly influence the rate of branching morphogenesis in developing kidney and the number of nephrons that can be formed before birth, when nephrogenesis in humans comes to an end. If so, the authors reasoned that caspase inhibitors might be used to suppress unwanted UB cell apoptosis during kidney development in Pax2 1Neu mutant mice and rescue the genetic UB branching defect. E17.5 kidneys from Pax2 1Neu mutant mice had smaller (Ϫ25%) longitudinal cross-sectional area and 3.5-fold increase in collecting duct cell apoptosis versus wild-type littermates; mutant E13.5 kidney explants allowed to arborize for 50 h in vitro had 18% fewer terminal branches than wild-types. However, exposure to the caspase inhibitor, Z-VAD-fmk (25 M), significantly increased terminal branch number in mutant explants (23%). It also increased branching in wild-type explants, apparently reflecting an effect of Z-VAD-fmk on basal apoptosis induced by ex vivo culture conditions. Similarly, when pregnant mice were injected daily with Z-VAD-fmk (10 g/g weight from E10.5 to E17.5), apoptosis of Pax2 1Neu fetal collecting duct cells was suppressed to 40% of untreated mutants; by E14, terminal branch number was increased to 152% that of untreated litters. These studies support the hypothesis that PAX2 normally optimizes the rate of branching morphogenesis in fetal kidney by suppressing UB apoptosis. Furthermore, it suggests that caspase inhibitors can rescue the branching defect caused by PAX2 mutations.During normal kidney development, interactions between the arborizing ureteric bud (UB) and the metanephric mesenchyme (MM) generate the nephrons of each kidney. This process begins at 5 to 6 wk fetal age, when the UB emerges from the nephric duct, grows laterally into the undifferentiated mesenchyme, and begins to arborize. Each terminal branch of the UB signals adjacent MM cells to form the proximal portion of a nephron, which then fuses to the common collecting system. By about 1 mo before birth, when nephrogenesis comes to a halt, final nephron number has been determined by how many UB branching events have occurred in the intervening period. This crop of fetal nephrons will constitute the individual's nephron endowment for life.Nephron number varies widely from 0.3 to 1.3 million/ kidney among normal humans (1,2). Children born at the lower end of this spectrum may have increased risk of hypertension or susceptibility to acquired renal disease later in life (3). In families with the autosomal dominant renal-coloboma syndrom...
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