Human Molecular Genetics, 2000, Vol. 9, No. 1 1-11
© 2000 Oxford University Press
Primary renal hypoplasia in humans and mice with PAX2 mutations: evidence of increased apoptosis in fetal kidneys of Pax21Neu +/– mutant mice
Cancer Genetics Laboratory, Department of Biochemistry, and 1Department of Pathology, University of Otago, PO Box 56, Dunedin, New Zealand, 2Division of Pediatric Nephrology, Montreal Children’s Hospital, Montreal, Quebec, Canada, 3Medical Genetics Service, Hospital de Clinicas de Porto Alegre, Rua Ramiro Barcelos 2350, 90035-003 Porto Alegre, RS Brazil, 4Department of Pediatrics, Saga Medical School, 5-1-1, Nabeshima, Saga City 849, Japan and 5GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany
Received 17 August 1999; Revised and Accepted 28 October 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PAX2 mutations cause renal-coloboma syndrome (RCS), a rare multi-system developmental abnormality involving optic nerve colobomas and renal abnormalities. End-stage renal failure is common in RCS, but the mechanism by which PAX2 mutations lead to renal failure is unknown. PAX2 is a member of a family of developmental genes containing a highly conserved ‘paired box’ DNA-binding domain, and encodes a transcription factor expressed primarily during fetal development in the central nervous system, eye, ear and urogenital tract. Presently, the role of PAX2 during kidney development is poorly understood. To gain insight into the cause of renal abnormalities in patients with PAX2 mutations, kidney anomalies were analyzed in patients with RCS, including a large Brazilian kindred in whom a new PAX2 mutation was identified. In a total of 29 patients, renal hypoplasia was the most common congenital renal abnormality. To determine the direct effects of PAX2 mutations on kidney development fetal kidneys of mice carrying a Pax21Neu mutation were examined. At E15, heterozygous mutant kidneys were ~60% of the size of wild-type littermates, and the number of nephrons was strikingly reduced. Heterozygous 1Neu mice showed increased apoptotic cell death during fetal kidney development, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in mutant kidneys; Pax2 was shown to be biallelically expressed during kidney development. These findings support the notion that heterozygous mutations of PAX2 are associated with increased apoptosis and reduced branching of the ureteric bud, due to reduced PAX2 dosage during a critical window in kidney development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PAX2 is a transcription factor critically required during the development of the nervous and excretory systems, including the midbrain–hindbrain, spinal cord, eye, ear and urogenital tract (1–4). Like other members of the PAX gene family, PAX2 encodes a conserved 128 amino acid paired box DNA-binding domain in the N-terminal portion of the molecule (4). Although PAX2 is a transcriptional regulator, there are no proven target genes regulated by PAX2, and little is yet known of the exact role of PAX2 during development of the nervous or excretory systems.
Eight of the nine Pax genes cause phenotypic abnormalities when mutated in humans or mice, and in four of these (including abnormalities caused by PAX2 mutations) developmental abnormalities are observed in the heterozygotes, revealing haploinsufficiency (4–6). To date all PAX2 mutations in humans have occurred within the conserved paired box and octapeptide sequences contained in the 5' half of the gene (refer to the human PAX2 sequence variant database online: http://www.hgu.mrc.ac.uk/Softdata/PAX2 ) (5,7–11). The 3' half of the PAX2 gene, which covers the remaining 7 of the 12 exons (12) including alternative splices in exons 6, 10 and 12 (12–14), encodes a putative transactivation domain (15).
In previous reports, patients with PAX2 mutations have been noted to have optic nerve colobomas and renal hypoplasia [renal-coloboma syndrome (RCS)] (5,16,17). This syndrome has mostly been characterized within the last 10 years, and is associated with a number of less common features, including high frequency hearing loss, seizure and brain malformation disorders, joint and skin anomalies and vesico-ureteral reflux (5,9,11). The renal phenotype associated with RCS is frequently accompanied by end-stage renal failure, often necessitating renal transplant (5,7–11). Although it is clear that PAX2 plays a critical role during kidney development, the exact role is unknown, and our present understanding of the pathogenesis of renal failure as a result of PAX2 mutations is poor.
During embryonic life the ureteric bud emerges from the nephric duct, growing outward into and arborizing within the undifferentiated mesenchyme. Signals from each branch of the ureteric bud induce adjacent mesenchymal cells to transform into proximal tubules and glomeruli of individual nephrons which ultrafilter fluid from blood. Each nephron fuses with its parent branch of the ureteric bud, providing an outlet to the bladder. Thus, the size and functional capacity of each kidney is ultimately determined by the complexity of ureteric bud branching and the number of individual nephrons that have been induced by the time the pool of metanephric stem cells has been consumed and nephrogenesis is terminated (18).
During normal kidney development PAX2 is expressed throughout the branching ureteric bud, Wolffian and Mullerian ducts. PAX2 is subsequently expressed in each focus of induced nephrogenic mesenchyme and its derivatives, the early epithelial structures of the emerging nephron (1,3,19,20). The importance of PAX2 to nephrogenesis is evident in mice with targeted disruption or spontaneous mutations of the Pax2 gene (19,21). Heterozygous mutants have a phenotype very similar to the human PAX2 mutation syndrome (RCS) and are able to reproduce. Homozygous Pax2 mutant mice lack kidneys, ureters, vas deferens, epidydimis, seminal vesicles, uterus, oviducts and vagina and have developmental defects of the eyes, ears and central nervous system; these defects are lethal in the perinatal period (19,21).
To gain insight into the cause of renal abnormalities in patients with PAX2 mutations and to understand how these abnormalities lead to renal failure, we initially focused on the renal phenotype in a series of patients with RCS. A new PAX2 mutation was identified in this study, which was a novel stop codon mutation in PAX2 exon 7 in nine members of a large Brazilian pedigree spanning three generations. We also identified the common G619 insertion mutation in exon 2 of PAX2 in a sporadic Japanese patient. The renal phenotypes in a total of 29 RCS patients were then compared, identifying renal hypoplasia as the most common congenital renal abnormality. To characterize the etiology of the renal hypoplasia, fetal kidneys of mice carrying a Pax21Neu mutation (22) were analyzed. This mutation is identical to the G619 insertion mutation in some humans (7,9). The fetal kidney size of heterozygous mutants was reduced to 60% of that of wild-type littermates, closely resembling renal hypoplasia in humans. Heterozygous 1Neu mice showed reduced branching and increased apoptotic cell death in fetal kidney collecting ducts, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in the mutant kidneys, and Pax2 was biallelically expressed during kidney development. The extent of the apoptosis correlated well temporally and spatially with the known pattern of PAX2 expression, tapering off when PAX2 expression levels are known to decline. Our findings support the notion that heterozygous PAX2 mutations lead to increased apoptosis and reduced branching of the ureteric bud during a critical window in kidney development.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Renal abnormalities in 29 patients with PAX2 mutations
To further characterize the renal phenotype associated with RCS, renal features were compared in a total of 29 patients with PAX2 mutations, including 10 new patients from two families. The 10 new patients are described below. A three generation Brazilian kindred (Fig. 1) was evaluated for optic nerve and kidney defects consistent with diagnosis of RCS. The associated phenotypic abnormalities are listed in Table 1. Polymerase chain reaction and single-strand conformation polymorphism analysis (PCR–SSCP) revealed a variant pattern in exon 7 that was present in five of the affected members of the family, but was not present in 18 unaffected members (Fig. 2A). Independently derived mutant alleles from the exon 7 PCR products contained a C
T substitution at position 1289 (Fig. 2B). Normal alleles were also identified in DNA from these individuals, whereas unaffected patients did not contain the mutation. In exon 7 the nucleotide substitution at this position resulted in the disruption of a Cac8I restriction endonuclease site due to the change from a GCNNGC recognition motif to GTNNGC. Restriction digestion with Cac8I of exon 7 PCR products from one of the affected Brazilian family members showed sequences resistant to digestion as well as sequences which could be digested by Cac81 (Fig. 2C). Unaffected family members contained only sequences that digested with Cac81. Additionally, a Japanese patient was examined who had sporadic occurrence of optic nerve colobomas, renal anomalies and bilateral cryptorchidism (see Table 1, patient X2003 for phenotypic details). As above, each exon of the PAX2 gene was amplified by PCR to identify a PAX2 mutation. A variant SSCP pattern was observed for exon 2 (data not shown), and sequencing of this PCR product revealed a guanosine nucleotide insertion at position 619 (Fig. 2D).
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A list of the renal abnormalities identified in all 29 patients with PAX2 mutations, including the patients in this study, is given in Table 2. The most common renal abnormalities in the 29 patients were renal failure with histological abnormalities, proteinuria and renal hypoplasia (small kidneys). Renal failure, histological abnormalities and proteinuria are features commonly seen in association with other disease processes, and are not specific for RCS. These abnormalities were probably secondarily acquired since birth. On the other hand, renal hypoplasia, although seen in association with a number of congenital conditions, is caused by failure of embryonic growth of the kidney, and would therefore be consistent with an abnormality arising primarily from the PAX2 mutation. Small kidneys were found in 17/29 patients (59%), and in 10 of these patients the sizes of the kidneys were measured by ultrasound and graphed as a percentage of the mean normal size of kidneys for individuals at that age (Fig. 3). Renal hypoplasia was observed even in the youngest patients in this study, consistent with it being a congenital abnormality associated with RCS.
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Detailed analysis of the cause of the renal hypoplasia associated with RCS would have required characterization of fetal kidneys from these patients. This was not possible in human subjects, but a suitable mouse model of RCS (Pax21Neu) was available in which the mutation and syndrome were identical to those described in some humans (22). We therefore undertook a detailed study of the kidney phenotype in heterozygous Pax21Neu mutant fetal mice.
Renal hypoplasia in Pax21Neu heterozygous mouse embryos
Homozygous Pax21Neu mice have renal agenesis, bilateral optic nerve colobomas, abnormalities of ear development and missing midbrain–hindbrain regions (22). These animals die within 24 h of birth. However, heterozygous 1Neu mice, like their human counterparts, are viable and have no gross midbrain–hindbrain phenotype. Their main characteristics are abnormalities of optic nerve development and hypoplastic kidneys (22). Fetal mice carrying a heterozygous Pax2 mutation were obtained by crossing heterozygous Pax21Neu males with C57BL/6 females, and the fetuses were genotyped. Microscopic analysis of hematoxylin–eosin-stained sections from each embryo of three litters revealed that ~60% of the animals had hypoplastic kidneys with maximal cross-sectional surface area ranging from 30 to 75% of that of wild-types. A representative litter is presented in Figures 4 and 5. The largest heterozygous mutant kidneys overlapped in size with the smallest kidneys from wild-type littermates. Unilateral renal agenesis was encountered in ~1% and cystic abnormalities were additionally observed in ~1% of animals (data not shown).
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The nephrogenic zones of fetal [embryonic day (E) 15–16)] Pax21Neu/+ kidneys were thin and contained fewer nephrons in comparison with those of normal littermates. The number of early epithelial structures derived from induced metanephric mesenchyme (vesicles, comma- and S-shaped bodies) was reduced to 30–40% of normal (Fig. 5B). The number of mature glomeruli was even more sharply reduced (20% of the wild-type). Interestingly, early tubular structures and glomeruli in Pax2 mutant kidneys appeared to be of normal size and morphology (Fig. 4).
Renal hypoplasia in Pax21Neu/+ mice is associated with enhanced apoptosis of the ureteric epithelium
To gain insight into the mechanism of renal hypoplasia in Pax2 mutant mice, we examined the patterns of apoptotic cell death and cell proliferation in mutant and wild-type fetal kidneys. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to investigate whether the Pax2 mutation in heterozygous Pax21Neu mutant mice was associated with increased apoptosis. An increase in TUNEL-positive staining was observed in the kidneys of E15–16 Pax21Neu/+ mutant mice compared with wild-type littermates (Fig. 6A and B). In contrast, the rate of cell proliferation, as analyzed by BrdU uptake into DNA in the developing kidneys of mutant Pax21Neu/+ mice, was equivalent to that of wild-type mice from E15 through to postnatal day 6 (Fig. 6C and D). Similarly, PCNA staining was unchanged in E15 1Neu embryos compared with their wild-type littermates (data not shown). These results suggest that heterozygosity for the 1Neu mutation is associated with an increase in the rate of apoptotic cell death but not with a difference in the pattern of fetal kidney cell proliferation.
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Apoptotic cell death in the mutant kidneys was examined in greater detail to identify the stages of development in which apoptosis was increased and the cell types involved. Although there were consistently more TUNEL-positive cells (P < 0.001) in E15–16 Pax21Neu/+ mutant kidneys than in those of wild-type littermates, this difference was less noticeable at later stages, and was confined to the collecting ducts (see below). In total kidneys of E18, newborns (1-day-old) and 5- to 6-day-old pups the numbers of TUNEL-positive cells were not significantly different in mutant and wild-type kidneys (Fig. 7A). By counting apoptotic cells in specific renal structures of Pax21Neu/+ mutant kidneys a significant increase (P = 0.0013) in TUNEL-positive staining was identified in the collecting ducts and renal pelvis in fetal (E15–16) Pax21Neu/+ mutants (Fig. 7B). This difference was still detectable in E18 but not in 5- to 6-day-old mice. TUNEL staining was also increased in the renal cortex in E15–16 Pax21Neu/+ mutant fetal kidneys (Fig. 7C), which mostly corresponded to ureteric buds. Increased apoptosis was not observed in glomeruli, or proximal or distal tubules at either E15–16 or later in development (data not shown). The rate of apoptosis in fetal 1Neu kidneys was not determined prior to E15 because successfully mated females did not have vaginal plugs, and excessive numbers of randomly pregnant female mice would have been required to obtain the figures for apoptotic rates.
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Pax21Neu/+ mutant kidneys contain transcripts of both PAX2 alleles and express a reduced level of PAX2 protein
Recent evidence by Nutt et al. (23) suggests that another PAX gene, PAX5, is normally expressed stochastically from only one allele in developing lymphocytes. This observation raises the question as to whether PAX2 expression in developing kidney is also monoallelic. Accordingly, heterozygous mutant kidney would be comprised of a mixture of normal and null mutant cells. Apoptotic cell death might occur in those cells expressing the mutant allele, and viable collecting duct cells could consist of those randomly expressing normal amounts of PAX2 protein from the wild-type allele. Alternatively, if PAX2 is expressed from both alleles, then nephrons of heterozygous Pax21Neu kidneys should express PAX2 protein with perhaps a slight reduction in intensity.
To test the hypothesis that renal hypoplasia and the increased rate of apoptosis in Pax21Neu/+ mutant fetal kidneys was associated with monoallelic Pax2 transcription, we analyzed allelic patterns of Pax2 expression. The insertion mutation was used to discriminate the mutant and wild-type alleles (Fig. 8a) in mRNA from heterozygous 1Neu mutant mouse kidneys following RT–PCR. We reasoned that if Pax2 transcription were monoallelic in the developing kidney, cells randomly expressing the mutant allele might have a selective disadvantage and represent a diminishing portion of the kidney as development progressed. However, we found that levels of mutant and wild-type Pax2 mRNA (alleles) were equivalent at all stages of development between fetal (E12) and postnatal (day +1) kidneys (Fig. 8b), suggesting that there was no selection for the favored expression of one allele in mutant kidneys.
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In contrast to the above reasoning, cells monoallelically transcribing the mutant Pax2 allele might successfully proliferate, but these cells would not express the normal PAX2 protein. If there were large numbers of cells in the ureteric buds in the mutant kidneys not expressing immunoreactive Pax2 protein, then this would be consistent with the notion that they express Pax2 monoallelically from the mutant allele. We would be able to detect the resultant mosaicism (i.e. cells staining positively and negatively for PAX2 protein) by performing immunohistochemistry with a polyclonal anti-murine Pax2 antibody (24) that reacts with C-terminal epitopes present in wild-type Pax2 protein, but which are absent in the truncated mutant Pax2 protein. Immunohistochemical staining for Pax2 protein was observed in all identifiable nuclei of ureteric bud and collecting duct cells of heterozygous 1Neu fetal kidneys (Fig. 8c), but the intensity of Pax2 staining was uniformly less than that in fetal kidneys of wild-type littermates analyzed simultaneously. In comparison, strong staining for Pax2 was observed in all portions of wild-type ureteric buds and collecting ducts (Fig. 8d). This pattern of staining was observed in each of three wild-type and three mutant embryos analyzed simultaneously. These results suggest that all the cells of the ureteric buds expressed the wild-type Pax2 allele. To examine PAX2 allelic expression patterns directly in clonal cell populations we analyzed transcription in Wilms’ tumors and in cloned renal carcinoma cells using a polymorphism in human PAX2 (Fig. 8e). RT–PCR–SSCP was used to discriminate between the two PAX2 alleles (Fig. 8f, lanes 1–3). Biallelic transcription of PAX2 in fetal kidney (Fig. 8f, lane 7) was not unexpected since monoallelic transcription of PAX2 in one cell (if present) would be statistically matched by monoallelic transcripion of the opposite allele in another cell. However, Wilms’ tumors are predominantly comprised of clonal blastemal and epithelial tissues, derived originally from embryonic kidney (25). Biallelic PAX2 transcription was observed in mRNA isolated from three Wilms’ tumors (Fig. 8f, lanes 4–6). In addition, biallelic (albeit slightly unbalanced) PAX2 transcription was observed in clonally expanded renal carcinoma cells (Fig. 8f, lanes 8 and 9). The small imbalance of PAX2 allelic expression observed in the cells may be due to alterations in the number of copies of each PAX2 allele in the cells, and was an indication that the RT–PCR–SSCP technique was sensitive enough for this purpose.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this paper we have further characterized the renal phenotype associated with RCS. End-stage renal failure is commonly associated with this syndrome, and one goal of this research was to determine how PAX2 mutations could cause renal failure. This was addressed firstly by characterizing the renal phenotype in RCS patients. Renal hypoplasia was the most common congenital abnormality in a series of 29 patients, including 10 new patients reported here.
Of the 10 new patients identified with RCS, nine were from a large three generation Brazilian family transmitting a novel CT substitution at position 1289 of PAX2, resulting in a change from an arginine codon in exon 7 to a stop codon. The mutation identified in the Brazilian family is the most 3' mutation so far identified in PAX2, and is the first report of a PAX2 mutation that leads directly to the introduction of a stop codon. If expressed, the protein resulting from this mutant allele is predicted to be truncated midway through the partial homeodomain, and would result in loss of the entire transactivation domain. PAX2 protein lacking the transactivation domain is unlikely to be fully functional, as studies have shown that this region in the closely related PAX5 protein is required for transcriptional activation of target genes (15). In the 10th patient, a Japanese boy, we identified a G619 insertion mutation of PAX2 exon 2. The G619 insertion mutation has previously been documented in other patients (7,9). This patient also presented with bilateral cryptorchidism, which has not previously been described in RCS and may be just a chance association. However, Pax2 is expressed in the developing urogenital ridge, vas deferens and epidydimis (1,19,26,27), and these structures are important in the maturation of the genital tract. It is possible that PAX2 mutations cause less obvious abnormalities of the reproductive system in other patients with RCS.
Four of the ten new patients in this report had obvious renal hypoplasia; three others had clinical evidence of renal insufficiency. When we reviewed clinical data on the 19 previously reported cases of proven human PAX2 mutations, we noted 13/19 with obvious renal hypoplasia and all had evidence of renal insufficiency or dysfunction. In at least two of the three cases where renal biopsy had been performed in childhood, there was striking atrophy of the proximal and distal tubules, but nephron number could not be assessed (8,9,16). Renal hypoplasia was pronounced even in the youngest patients in this study (patients 2 and 3), who were 5 and 6 years old, respectively, suggesting that this is a congenital anomaly.
Further studies to determine how PAX2 mutations cause renal hypoplasia in human subjects were impractical because it was not possible to obtain fetal kidneys from patients with RCS. However, Pax21Neu mutant mice (22) harbor a mutation that is identical to a mutation in approximately one-third of families with RCS (7,9). These mice transmit a single base pair insertion amid a string of seven guanidine residues (positions 613–619) in the second exon of Pax2, which produces a frameshift and presumably a null allele (22). Pax21Neu mice have optic nerve and kidney abnormalities, similar to humans with RCS. Although the homozygous mutants die within 24 h of birth and are phenotypically similar to ‘knockout’ mice generated by targeted homologous recombination (19,21), heterozygotes survive and reach adulthood. The heterozygous mutant mice therefore afford a useful animal model in which to analyze fetal kidney defects associated with Pax2 mutations.
During normal kidney development Pax2 is first detected along the nephrogenic cord at the sites from which ureteric buds will emerge (1,19). Intense Pax2 expression persists in cells of the ureteric buds as they invade the metanephric blastema to each side and begin to undergo dichotomous branching (1,3). Inspection of fetal Pax21Neu/+ mouse kidneys demonstrated renal hypoplasia at an early (E15) stage of development. This is apparently due to a decrease in the rate of new nephron induction since the total number of early epithelial structures (at the tips of ureteric buds) and glomeruli (representing more advanced nephrons) is strikingly reduced in mutant kidneys. The nephrons that are formed, though reduced in number, appear to have normal morphology. It can be inferred, therefore, that arborization of the ureteric bud is less complex in patients with heterozygous PAX2 mutations than in normal individuals. It should be pointed out that even a modest reduction in the efficiency of ureteric bud branching would reduce final kidney size substantially, since the dichotomous branching process is repeated many times during renal development (28,29).
The downstream gene targets of PAX2 are largely unknown, but appear to influence growth and branching morphogenesis of the ureteric bud. For that reason we examined the pattern of cell proliferation and apoptotic cell death in embryonic kidneys of heterozygous Pax21Neu mutant mice. Cell division, as assessed by PCNA immunohistochemistry and BrdU uptake, was normal. However, apoptosis in the medullary segments of the collecting duct (as assessed by TUNEL assay) was strikingly increased at the time when Pax2 is maximally expressed in kidney development, and in cells which normally express Pax2 (1). Indeed, the amount and localization of the apoptosis correlated temporally and spatially with the known expression patterns of PAX2 (1,3), except that apoptosis was not observed in mesenchyme-derived epithelial structures, whereas PAX2 is expressed in the differentiating mesenchyme and early mesenchyme-derived structures. E15 mutant mice exhibited the greatest levels of apoptosis and, at later stages when overall PAX2 expression levels have declined (1), differences in the rate of apoptosis in collecting ducts was less marked between heterozygous Pax21Neu mutant and wild-type kidneys. Apoptosis is known to occur as part of normal kidney development (30), but the rate of apoptosis that we observed in E15 collecting ducts was ~9-fold higher than in wild-type offspring at the same age. Since apoptotic cells are rapidly cleared by phagocytosis, we cannot ascertain whether cell death is extensive enough to compromise arborization of the ureteric bud. Conceivably, our observation of increased apoptosis may reflect reduced signaling by trophic factors influencing branching morphogenesis as well as cell survival.
It is unclear why the apoptosis in the heterozygous mutant kidneys was predominantly confined to Wollfian duct-derived structures such as ureteric buds and collecting ducts, even though Pax2 is also expressed in mesenchyme-derived structures such as comma- and S-shaped bodies. One possible explanation arises from the observation that Pax8 is co-expressed with Pax2 in the differentiating mesenchyme-derived structures (31), but not in ureteric buds or collecting ducts. It is possible that, as has been described for other Pax genes (32), expression of Pax8 was able to compensate for heterozygous Pax2 mutations in the mesechyme-derived structures of kidneys from Pax2 mutant mice, and that this ultimately rescued these cells from apoptosis.
Our data do not determine whether PAX2 directly regulates genes in apoptotic pathways during kidney development. However, PAX2, PAX5 and PAX8 have all been reported to inhibit p53 transcription (33), which can promote apoptosis (34). Furthermore, transgenic mice overexpressing wild-type p53 have altered differentiation of the ureteric bud, and have small kidneys (35). In Pax5 knockout mice the B cells fail to differentiate and undergo apoptotic cell death at an early stage (36). Similarly, large-scale apoptosis of photoreceptor precursors occurs in the eye discs of Drosophila with the eyeless mutation (ey2) (homologous to the mammalian Pax6 gene) (37). These authors also found no difference in BrdU uptake studies in wild-type and mutant eye discs (37). As in our studies, this suggests that the mutation affects cell survival rather than proliferation. Pax3 mutant mice were shown to have enhanced apoptosis in the somitic mesoderm during embryonic development (32), and treatment of rhabdomyosarcoma cells lines with PAX3 antisense oligonucleotides has been shown to result in apoptosis (38). Unlike the above studies reporting apoptosis in homozygous Pax mutant animals, our studies were carried out on heterozygous mutant animals.
Torres et al. (19) speculated that Pax2 +/– mice might develop renal hypoplasia due to a decreased rate of cell proliferation in renal calyces and upper ureters. However, our results suggest that the cause of the small kidneys in RCS is due to decreased cell survival in these structures rather than decreased proliferation. The fact that the collecting system is derived from the Wolffian duct, and that we observed apoptosis at the earliest time-point examined (E15), suggests that apoptosis could have occurred in the Wolffian duct while the ureteric bud was sprouting from it. This notion would be consistent with, and may help to explain, the observed degeneration and lack of caudal extension of the Wolffian duct in Pax2 null mutant mice (19). In Pax2 mutants the lack of development, or dysplasia, of the epidydimis, vas deferens, seminal vesicles and ureters, each of which are derived from the Wolffian duct (31), could also be caused by enhanced apoptosis in the Wolffian duct and its derivatives.
The effects of PAX2 haploinsufficiency on the renal phenotype are similar to the developmental effects of haploinsufficiency for other PAX genes. PAX3 mutations cause an autosomal dominant defect in epidermal pigmentation (39), PAX6 mutations cause autosomal dominant aniridia (40,41) and PAX8 mutations cause autosomal dominant hypothyroidism (42). In each case, inactivation of one allele is sufficient to interfere with normal organ development. With regard to the mechanism of haploinsufficiency, Nutt et al. (23) have hypothesized that monoallelic Pax gene expression may be related to the semi-dominant effects of Pax gene mutations. We analyzed the allelic expression pattern of Pax2 in 1Neu mutant kidneys both by RT–PCR and by immunostaining. In Pax21Neu/+ mutant kidneys, immunostaining of Pax2 was detected in virtually every cell in the ureteric buds. Taken together with our RT–PCR data, these observations suggest that Pax2 is biallelically transcribed in wild-type fetal kidney, and that ureter and collecting duct abnormalities produced by Pax2 haploinsufficiency probably result from sensitivity to reduced gene dosage in each cell. We cannot rule out the possibility that Pax2 is monallelically expressed at embryonic stages earlier than E15, or in stem cells which were not amenable to study by immunohistochemistry in E15 kidney.
In summary, the murine Pax21Neu mutation adds much to our understanding of the human disease. We have shown that renal hypoplasia is the most common congenital renal anomaly in humans and mice with PAX2 mutations, and that in mice this phenotype is associated with significantly enhanced apoptosis. Furthermore, the Pax2 gene was found to be biallelically expressed in mutant mice; therefore, it seems unlikely that the enhanced apoptosis was due to random stochastic inactivation of Pax2 expression in cells monoallelically expressing Pax2. We conclude from this work that haploinsufficiency of Pax2 leads to small kidneys and renal failure because reduced Pax2 dosage compromises the survival of ureteric bud-derived epithelial cells, and that this is associated with reduced branching of the ureteric bud at an early stage of development. Further studies are required to determine whether PAX2 is a primary determinant of cell survival, or whether this effect is the consequence of a default pathway of apoptosis in cells in which development is disrupted.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PCR–SSCP and detection of PAX2 mutations
Genomic DNA from each individual was extracted from peripheral blood (collected with informed consent) using a DNA extraction kit (Promega, Madison, WI). Fragments spanning exons 1–12 of PAX2 were amplified from genomic DNA using PCR primers in the introns flanking the exons (12). The PCR products were labeled by incorporation of [32P]dCTP in reactions containing 100 ng DNA, 62.5 µM dNTPs, 1 µCi [
-32P]dCTP (3000 Ci/mmol), 20 pmol of each primer, reaction buffer (50 mM KCl, 10 mM Tris pH 8.3, 1.0–3.0 mM MgCl2) and Pwo (Boehringer Mannheim, Mannheim, Germany) or AmpliTaq Gold (Perkin Elmer, Branchburg, NJ) DNA polymerase, and electrophoresed in non-denaturing 6 or 12% polyacrylamide gels as described previously (9) to reveal single-strand conformation variants.
Subcloning and DNA sequencing
PCR products were directly sequenced using [
-33P]ATP-radiolabeled kinased primers and a cycle sequencing kit (Gibco BRL, Rockville, MD). For subcloning of exon 2 and exon 7 PCR products, mutant and normal alleles of PAX2 were amplified using Pwo DNA polymerase (Boehringer Mannheim), and subcloned into EcoRV digested pBluescript II (Stratagene, La Jolla, CA). Positive clones were identified by blue/white selection, and plasmid DNA was isolated using Wizard mini-preps kits (Promega). Sequencing reactions were carried out on plasmid DNA as described previously (9).
Mouse breeding, genotyping and embryo preparation
Wild-type and 1Neu mouse colonies were bred and maintained at Neuherberg, Germany, and at the University of Otago, New Zealand. Timed matings of male mice heterozygous for the Pax21Neu mutant allele (22) were carried out with female C57BL/6 mice, and the embryos were genotyped. Genotyping was performed as described (22), or by PCR amplification of genomic DNA extracted from tail slices by using primers 2F and 2R (Fig. 8). PCR amplification reactions were as previously described (9). The presence of a 167 bp fragment in addition to a 166 bp fragment when the PCR products were electrophoresed on a 6% polyacrylamide gel indicated the presence of the mutant Pax2 allele. Fetal kidneys (E15–18) were dissected from heterozygous Pax21Neu mutant or wild-type mice. The expression of the mutant Pax21Neu allele was demonstrated in fetal kidney RNA from Pax21Neu/+ mutant mice by RT–PCR amplification of total kidney RNA using primers 1F and 3R, spanning exons 1–3 of Pax2. Nested PCR amplification of the RT–PCR product from exons 1–3 was carried out using primers 2F and 2R. PCR products were electrophoresed on 6% polyacrylamide gels. Primer sequences were based on the murine Pax2 sequence (1) and were as follows: 1F, 5'-CCA CCG TCC CTC CCT TTT CTC CT-3'; 2F, 5'-GGG CAC GGG GGT GTG AAC CAG-3'; 2R, 5'-CTG CCC AGG ATT TTG CTG ACA CAG CC-3'; 3R, 5'-CTG TGT CAT TGT CAC AGA TGC CCT CG-3'.
Microscopic analysis
Heterozygous PAX2 mutant E15 embryos and their wild-type littermates were incubated in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) for 16–18 h and stored at 4°C in 70% ethanol prior to embedding in paraffin. Serial 5 µm sagital sections of both kidneys from each mutant and wild-type embryo were prepared on Superfrost/Plus slides (Fisher, Fairtown, NJ). Sections were stained with hematoxylin–eosin and analyzed by bright-field microscopy: the number of glomeruli and early epithelial structures (renal condensates, comma- and S-shaped bodies) in the nephrogenic layer was counted in serial cross-sections from kidneys of both 1Neu and wild-type embryos. To compare the maximal cross-sectional area of heterozygous and wild-type kidneys, serial cross-sections were visualized by photomicroscopy and projected on a television screen. The outline of each renal image was traced onto paper, cut out and weighed to estimate relative surface area. The maximal cross-sectional area for each kidney was plotted in arbitrary units for inter-kidney comparisons.
PAX2 immunostaining
Polyclonal anti-mouse PAX2 antibody was purchased from Zymed (San Francisco, CA). Mouse sections were deparaffinized and incubated with primary antibody (10 µg/ml) for 30 min at room temperature in PBS supplemented with 20 mM glycine and 1% bovine serum albumin. PAX2 antibody was detected with Vecstatin ABC Rabbit IgG kit (Vector Laboratories, Burlingame, CA) as described by the manufacturer, followed by incubation with DAB substrate (Sigma, St Louis, MO). Sections were washed in PBS, counterstained with hematoxylin, dehydrated and mounted with Permount (Fisher). Wild-type and mutant tissues were stained simultaneously; each analysis was repeated three times.
Detection of apoptosis (TUNEL staining)
The TUNEL assay was performed on paraffin-embedded tissue sections as described (43) with modifications. The paraffin sections were dewaxed, rehydrated, partially digested with proteinase K (15 µg/ml; Boehringer Mannheim) in Tris–HCl (10 mM, pH 7.6) at room temperature for 15 min. After the endogenous peroxidase was inactivated with 2% H2O2 for 10 min at room temperature, the sections were incubated for 90 min at 37°C with TdT (Boehringer Mannheim) in TdT reaction buffer (1 mM dCTP, 1 mM dGTP, 1 mM TTP, 1 mM biotin-14-dATP, 140 mM sodium cacodylate, 1 mM cobalt chloride, 30 mM Tris–HCl, pH 6.4). The sections were then washed three times in PBS, incubated with extravidin peroxidase (1:250 dilution in PBS; Sigma) for 60 min at room temperature, rinsed three times in PBS, stained with diaminobenzidine (Boehringer Mannheim) for 5 min at room temperature, and counterstained with 1% methyl green.
BrdU-incorporation studies and PCNA immunostaining
BrdU incorporation studies were performed exactly as described (35). For PCNA immunostaining mouse sections were deparaffinized, hydrated, and endogenous peroxidase activity was quenched by incubating in methanol containing 0.3% H2O2. Slides were incubated in 2.5 µg/ml PCNA monoclonal antibody (Oncogene Research, Cambridge, MA) at room temperature for 30 min followed by two brief washes in PBS. PCNA antibody was detected with Vecstatin Mouse IgG kit (Vector Laboratories) as described by the manufacturer followed by incubation with DAB substrate (Sigma).
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ACKNOWLEDGEMENTS |
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We thank Drs John Schofield, Lillias Anderson and Michelle French for technical assistance, and John Harris for providing an anti-BrdU antibody. We are also grateful to Drs M. Murphy and M. Packer for comments. This work was supported by grants from the Cancer Society of New Zealand, the Health Research Council of New Zealand, the New Zealand Lottery Grants Board, and the Medical Research Council of Canada (no. MT-12954). E.T. held FCAR-FRSQ and Lloyd-Harris McGill Major Fellowship awards during the course of this work.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +64 3 4797878; Fax: +64 3 4797738; Email: meccles@otago.ac.nz
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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