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BASIC SCIENCE |
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*Department of Human Genetics, McGill University, Montreal, Quebec, Canada; Department of Paediatrics, Montreal Children’s Hospital, McGill University, Montreal, Quebec, Canada; Department of Pathology, University of Otago, Dunedin, New Zealand; Centre for Research in Neuroscience, Montreal General Hospital, McGill University, Montreal, Quebec, Canada; and ¶Department of Biochemistry, McGill University, Quebec, Canada.
Correspondence to Dr. Michael Eccles, Department of Pathology, University of Otago, Dunedin 9015, New Zealand. Phone: +64-3479-7878; Fax: +64-3479 7139;
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Abstract |
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) 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 E15.5 PAX2Promoter-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. E-mail: meccles@otago.ac.nz |
Introduction |
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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 phenotypic transformation into polarized epithelial cells of the nephron. Each emerging nephron fuses with the UB, allowing egress of filtered fluid. The extent of UB arborization, which has been achieved by the time nephrogenesis ends in the perinatal period, determines the final number of nephrons constituting the individual’s nephron endowment for life.
This complex process is orchestrated by key developmental transcription factors. Among the earliest of these is PAX2, which is expressed in the nephric duct and uninduced mesenchyme before formation of the metanephros, throughout the arborizing UB and finally in the condensing mesenchyme after induction (8). With completion of nephrogenesis, expression of the renal PAX2 gene is rapidly downregulated. In 1996, a strain of mice (Pax21Neu) was identified with RCS due to a spontaneous Pax2 mutation identical to the most common mutation in humans with RCS (9). In fetal (Pax21Neu +/-) mutant mice, we showed that the renal hypoplasia is associated with reduced nephron number and elevated apoptosis in the UB epithelium (3,10). Furthermore, a direct role for Pax2 in survival of UB cells is supported by our observation that mIMCD-3 collecting duct cells undergo apoptosis when transfected with an antisense Pax2 expression construct (10). In contrast, Pax2 null mutant mice have bilateral renal agenesis; although the caudal portion of the nephric duct initially forms, it then degenerates (9,11).
Although mutations in Pax2 are associated with elevated levels of UB apoptosis, it is not known whether apoptosis alone accounts for suboptimal UB arborization in RCS. Indeed, relatively high basal levels of apoptosis are known to occur during development of the fetal kidney and other organs, but the significance of this apoptosis is not understood (12,13). Normally, Pax2 is expressed in cells of the mesenchymal lineage as well as in the ureteric bud, yet we observed no significant increase in mesenchymal cell apoptosis in Pax2 mutant mice (3). Pax2 activates GDNF in metanephric mesenchyme (14), and we found effects of Pax2 on E-cadherin expression (15). Arguably, therefore, Pax2 haploinsufficiency disturbs key steps in cell differentiation, and the apoptosis in (Pax21Neu +/-) mutant mice is the consequence of cells not achieving their developmental fate. It is, however, important to know whether apoptosis occurring in the UB is sufficient to directly cause renal hypoplasia or whether additional mesenchyme-related functional deficits are also required. Our hypothesis is that the enhanced susceptibility of UB cells to programmed cell death in Pax2 haploinsufficiency directly compromises the rate of UB arborization and has a direct role in causing renal hypoplasia.
To examine the latter hypothesis, we have targeted an apoptosis-inducing transgene to the developing UB of fetal kidneys. We show that transgenic fetal mice expressing the pro-apoptotic PAX2Promoter-Bax transgene in embryonic kidney, eye, and brain under the control of exogenous PAX2 regulatory elements have elevated UB apoptosis and renal hypoplasia due to reduced UB branching. These data demonstrate that enhanced UB cell apoptosis during renal development compromises UB arborization, mimicking the RCS phenotype caused by PAX2 mutations. We propose the existence of an elemental UB morphogenic unit with an intrinsic mechanism for timing iterative branching events. In this model, susceptibility of the UB to apoptosis regulates UB arborization during kidney development.
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Materials and Methods |
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Transgene Construction cDNA was then ligated downstream from the promoter, using NotI and an end-filled SpeI site in the IRES vector. The GenBank accession number for the 4.2-kb ApaI/NcoI fragment of the human PAX2 promoter and flanking sequences is AF515729.
Transient Transfections and Cell Culture
The activity and tissue specificity of a 4.2-kb upstream region of the PAX2 gene was analyzed in transient transfections of mIMCD-3, HEK293, COS-7, and NIH 3T3 cell lines. Plasmid DNA preparation, cell culture, and transient transfections using FuGene 6 (Roche) were carried out as described (16), with modifications. Briefly, cells at 50 to 70% confluency were co-transfected with 0.4 µg of pRSV-
-gal and 0.8 µg of human PAX2Promoter-pGL2Basic. Cells were harvested 48 h after transfection, lysed in a passive lysis buffer (Promega), and assayed for firefly luciferase and -gal (Galacto-Star, TROPIX). Transfections were performed in replicates of six on three separate occasions.
Generation of Transgenic Mice
XhoI linearized plasmid DNA from the PAX2Promoter-Bax construct was diluted to 2 ng/µl and microinjected into the male pronuclei of C57B/6J X C3H fertilized eggs. Injected eggs were then transferred into the oviducts of pseudopregnant females. Transgenic fetal mice were identified by PCR amplification of the IRES region from DNA obtained from tails (animals which came to term) or hind limb (embryos). Transgenic mice were viable and fertile. PCR primers used for genotyping transgenic mice were, Bax3'F: 5'-TAATCTTGAAGTCTCCATCCG-3', and IRES3'R: 5'-CAGATCAGATCCCATACAATG-3', which amplified a 500-bp fragment from the PAX2Promoter-Bax construct.
-Galactosidase Detection
Fetal age was determined by timed pregnancy and morphologic analysis according to Kaufman’s Atlas of Mouse Development. Whole-mount histochemistry for -galactosidase was carried out as described (17); embryos were stained overnight. After whole-mount staining, embryos were washed and fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in paraffin wax, and sectioned at 7 µm.
Maximal Kidney Cross-Sectional Area Determination, Glomerular Counting, and TUNEL Staining to Determine the Number of Cells Undergoing Apoptosis
The cross-sectional area was determined in kidney sections showing the maximal cross-sectional area in comparison to adjacent sections. To do this, whole embryos were paraffin-embedded in the same orientation before sagittal serial sectioning. Sections were stained with nuclear red, and the section containing the maximal cross-sectional area was measured for each kidney (n = 9 wild-type kidneys; n = 10 Bax transgenic kidneys). Photographs of each section showing the maximal cross-sectional area were taken at 4x.
For determination of the number of glomeruli in wild-type and Bax transgenic kidneys, the glomeruli, S-shaped bodies, and comma-shaped bodies were counted in sections with the maximal cross-sectional area (n = 8 kidneys from 5 wild-type fetal mice; and n = 10 kidneys from 6 Bax transgenic fetal mice). The sections used for the glomerular counts were the same sections as those used to determine the maximal cross-sectional area above. In addition, glomeruli were counted with the same methodology in one kidney each from two additional PAX2Promoter-Bax transgenic animals, yielding counts of 13 and 13 glomeruli, respectively.
For determination of the number of cells undergoing apoptosis in wild-type and Bax transgenic kidneys, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining was performed as described (3). The number of cells that stained positive for TUNEL in the UB, which was observed to contain the majority of TUNEL-positive staining, were counted. Two sections from each kidney, near to the center of the kidney, were used for counting (n = 5 kidneys from 4 wild-type fetal mice; n = 5 kidneys from 4 Bax transgenic fetal mice). In two of the PAX2Promoter-Bax transgenic fetal mice, there was only one kidney present. The area in mm2 occupied by the UB in the sections was measured, and the data were expressed as a ratio of TUNEL-positive apoptotic cells/mm2.
Bax Immunostaining and Western Blot
Embryonic 7-µm mouse sections were deparaffinized, rehydrated, and boiled twice for 5 min in 10 mM citrate buffer. Endogenous peroxidase activity was quenched using 3% H202 (in methanol) for 15 min at room temperature. After 30-min incubation with blocking serum, sections were incubated with primary, polyclonal rabbit anti-Bax antibody (Santa Cruz Biotechnology) at 4°C overnight, followed by staining using a Vectastain ABC universal kit (Vector Laboratories) as described by the manufacturer, and incubation with DAB (3). Sections were counterstained in 0.5% methyl green for 1 min each, dehydrated, and mounted with Permount.
Western blot analysis to detect Bax protein was carried out using a polyclonal rabbit anti-Bax antibody (Santa Cruz Biotechnology), followed by detection with a peroxidase goat anti-rabbit IgG (Perkin Elmer). To detect actin in Western blots, a monoclonal mouse anti-actin antibody (Oncogene) was used, followed by detection with a peroxidase goat anti-mouse IgM (Calbiochem). The secondary antibody-peroxidase conjugate was detected using chemiluminescence as described by the manufacturer (Calbiochem).
Dolichos Biflorus Agglutinin (DBA) Staining
For counting of UB branch tips, E15.5 kidneys were microdissected from transgenic fetal mice. Each kidney was fixed in 200 µl of 4% formaldehyde in phosphate-buffered saline (PBS) for 48 h. The fixative was removed, and the kidneys were washed 4 x 10 min at room temperature in 500 µl of PBS-T (PBS + 1% Triton X-100). After washing, kidneys were incubated in 200 µl of Dolichos Biflorus Agglutinin-FITC (DBA-FITC) in PBS (1:100 dilution) overnight at 4°C, as described (18). The DBA-FITC was removed, and kidneys were washed for 10 min at room temperature in 500 µl of PBS-T. Kidneys were then incubated at 4°C overnight in PBS-T or until ready to photograph. For photographing, whole kidneys were placed on a slide under a cover slip with no mounting solution. Images were obtained, and the total number of UB terminal ends per kidney was counted directly on the computer screen.
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Results |
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cDNA and used to generate PAX2Promoter-Bax transgenic mice. This expression construct also carried a lacZ reporter gene flanked by a 5' internal ribosome entry site (IRES) sequence, allowing independent translation of -galactosidase under the transcriptional control of the PAX2 promoter (Figure 1A). In cell culture transfection experiments, the 4.2-kb hPAX2 promoter was able to drive high levels of reporter gene expression in specific kidney epithelial cell lines, particularly murine inner medullary collecting duct cells (mIMCD-3) (Figure 1B). High conservation of sequence identity between the human PAX2 promoter and the murine Pax2 promoter was observed between 3290 and 4158 (19) and also in a 400-bp region, which contained 89% identity between positions 383 and 782 (Figure 1A). In mice, the 400-bp sequence had previously been shown to direct expression of Pax2 in the Wolffian duct, UB, and collecting ducts during development (17). Transgenic mice were generated by pronuclear injection and germline integration of the PAX2Promoter-Bax construct (Figure 1C). Eleven PAX2Promoter-Bax transgenic embryos were analyzed at E13.5 or E15.5. In addition, six founder PAX2Promoter-Bax transgenic mice were born. No significant skewing of Mendelian ratios was observed in 45 offspring from three of these founders, suggesting that there is little or no embryonic lethality as a result of the presence of the transgene.
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-Galactosidase Reporter and Bax during Kidney Development in PAX2Promoter-Bax Transgenic Mice-galactosidase staining was identified in the kidneys and mid/hindbrain of wholemount E15.5 embryos (Figure 2A). A more detailed analysis of tissue-specificity and temporal regulation of the transgene showed that the transgene was expressed in the developing urogenital tract, including Wolffian duct, UB, and collecting ducts, and in the developing eye of E15.5 embryos (Figure 2, B through D). High levels of expression of the transgene were observed in the fetal hindbrain, eyes, and collecting ducts, mimicking the pattern of strongest endogenous Pax2 expression (8,20).
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expression was detected in the UB and collecting ducts of E15.5 transgenic fetal mice (Figure 2F). Endogenous Bax expression was present in the UB and collecting ducts in E15.5 wild-type kidneys but was at a lower level in wild-type kidneys than in the PAX2Promoter-Bax transgenic fetal mice, as shown by immunohistochemistry (Figure 2G). When mIMCD-3 cells transfected with the PAX2Promoter-Bax transgene were analyzed by Western blot using the anti-Bax antibody, there was a greater level of Bax expression in the transfected cells than in nontransfected cells (Figure 3), again suggesting that the Bax transgene was relatively strongly expressed under the control of the PAX2 promoter.
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gene in the UB of PAX2Promoter-Bax transgenic fetal mice, we analyzed the pattern and number of cells undergoing apoptosis in the fetal kidneys of transgenic and wild-type mice. In transgenic E15.5 kidneys, high levels of apoptosis were evident in UB and collecting duct epithelium, whereas apoptosis levels in the UB and collecting ducts of wild-type fetal mice were very low (Figure 4,A and B). The number of apoptotic cells per mm2 in the transgenic fetal kidney ureteric bud epithelia was more than tenfold higher than that in wild-type kidneys (Figure 4C). By eye, apoptosis levels did not appear to be elevated in other regions of the kidneys of PAX2Promoter-Bax transgenic fetal mice, including in the condensing mesenchyme or glomeruli.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Excessive levels of randomly occurring programmed cell death in fetal kidney cells might lead to small kidneys, but not necessarily to reduced nephron number. However, our studies show that apoptosis targeted to Pax2-expressing cells caused small kidneys with a reduced number of UB branches. We propose the existence of a UB "morphogenic unit," which is affected by PAX2 mutations in RCS as illustrated by our model of UB branching morphogenesis. In this model, each UB branching event is accompanied by local interactions with mesenchyme, suppressing further UB branching (Figure 8). As each new branch lengthens, the specialized UB tips grow beyond a putative zone where UB branching is inhibited, allowing the next cycle of branching to occur. In this model, stochastic depletion of cells during extension of the UB branch would slow its linear growth rate, delaying escape from the inhibitory zone and delaying the next round of branching. Assuming that there is a finite temporal window for UB branching during renal organogenesis, the balance of survival and pro-apoptotic signals would act as a timing mechanism for the rate of UB arborization during fetal life and ultimately determine final nephron number (Figure 8).
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Further support for the morphogenic unit model arises from our observation that, in contrast to the effects of excessive apoptosis on nephron number, inhibition of apoptosis in wild-type and (Pax21Neu +/-) mutant fetal kidneys leads to increased UB branching and increased nephron number. Ex vivo treatment of either wild-type or (Pax21Neu +/-) mutant fetal kidneys for 2 d with the caspase inhibitor Z-VAD-fmk, increased UB branching in DBA-stained kidney explants compared with untreated explants (Clark et al., unpublished observation). Taken together, these results suggest that the hypoplastic renal phenotype in RCS is determined primarily by loss of Pax2 anti-apoptotic function in UB cells.
As well as its role in kidney development, Pax2-mediated cell survival may also be important for the development of other organs, including the eye. Expression of the PAX2Promoter-Bax transgene in embryonic retina was associated with an immature stage of eye development, with attachment of the inner layer of the optic cup to the posterior lens cells, and poor development of the vitreous body (Figure 2, D and E). These data suggest that there may be a role for Pax2-mediated cell survival in eye development.
In conclusion, we have examined the role of apoptosis in organ patterning by targeting Bax expression to specific cell lineages during development. The studies presented here illuminate a functional relationship between apoptosis, patterning in the UB, and branching morphogenesis, whereby increased susceptibility to apoptosis in the UB lineage during fetal kidney development is sufficient to recapitulate the nephron deficit of RCS. Our results predict that potential therapies, involving inhibition of apoptosis during development may be used to treat RCS, or primary renal hypoplasia, if detected in utero. Consequently, nephron rescue therapies, if directed to the developing kidney, might fully restore the normal nephron complement in affected patients.
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Acknowledgments |
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Footnotes |
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References |
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