PAX2 Activates WNT4 Expression during Mammalian Kidney Development

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. 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 (1Dressler G.R. Deutsch U. Chowdhury K. Nornes H.O. Gruss P. Development (Camb.). 1990; 109: 787-795Crossref PubMed Google Scholar, 2Torres M. Gomez-Pardo E. Dressler G.R. Gruss P. Development (Camb.). 1995; 121: 4057-4065Crossref PubMed Google Scholar). 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 (3Bouchard M. Souabni A. Mandler M. Neubuser A. Busslinger M. Genes Dev. 2002; 16: 2958-2970Crossref PubMed Scopus (415) Google Scholar). When the nephric duct arrives at about somite 26, a “ureteric bud” (UB) 2The abbreviations used are: UB, ureteric bud; RT, reverse transcriptase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; PBS, phosphate-buffered saline; E, embryonic day; mut, mutant. 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 (4Brophy P.D. Ostrom L. Lang K.M. Dressler G.R. Development (Camb.). 2001; 128: 4747-4756Crossref PubMed Google Scholar). Thus, homozygous Pax2 knockout mice lack normal ureteric bud outgrowth and are unable to induce metanephric kidneys (2Torres M. Gomez-Pardo E. Dressler G.R. Gruss P. Development (Camb.). 1995; 121: 4057-4065Crossref PubMed Google Scholar). A third function of PAX2 in developing kidney involves suppression of programmed cell death in ureteric bud cells. During normal development, the UB arborizes as it penetrates the metanephric mesenchyme, inducing nephrons to form at the tip of each branch. Final nephron endowment is determined by the extent of UB branching achieved at the point when new nephron formation comes to an end in the perinatal period. Mice and humans lacking one Pax2 allele have increased apoptosis of UB cells, reduced UB branching, and suboptimal final nephron number (5Torban E. Eccles M.R. Favor J. Goodyer P.R. Am. J. Pathol. 2000; 157: 833-842Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Porteous S. Torban E. Cho N.P. Cunliffe H. Chua L. McNoe L. Ward T. Souza C. Gus P. Giugliani R. Sato T. Yun K. Favor J. Sicotte M. Goodyer P. Eccles M. Hum. Mol. Genet. 2000; 9: 1-11Crossref PubMed Scopus (168) Google Scholar). Delivery of a pro-apoptotic gene (Baxα) to the developing UB reduces arborization (7Dziarmaga A. Clark P. Stayner C. Julien J.P. Torban E. Goodyer P. Eccles M. J. Am. Soc. Nephrol. 2003; 14: 2767-2774Crossref PubMed Scopus (43) Google Scholar); conversely, the branching defect seen in Pax2 heterozygous mutants can be rescued by targeted expression of an anti-apoptotic gene (Bcl2) to the UB lineage 3E. Torban, A. Dziarmaga, D. Iglesias, L. L. Chu, T. Vassilieva, M. Little, M. Eccles, M. Discenza, J. Pelletier, and P. Goodyer, unpublished data. or by administration of caspase inhibitors to pregnant mice bearing heterozygous mutant Pax2 fetuses (8Clark P. Dziarmaga A. Eccles M. Goodyer P. J. Am. Soc. Nephrol. 2004; 15: 299-305Crossref PubMed Scopus (38) Google Scholar). Once within the mesenchyme, the arborizing UB delivers signals to nearby mesenchymal cells, inducing them to cluster at the tip of each UB branch. These induced mesenchymal cells express moderately high levels of PAX2 as they undergo a profound transformation into polarized epithelia, but the precise role of PAX2 during this process is unknown (2Torres M. Gomez-Pardo E. Dressler G.R. Gruss P. Development (Camb.). 1995; 121: 4057-4065Crossref PubMed Google Scholar). Rapid cell division and coordinated cell movement result in twisting growth of an elongated “S-shaped” body with a central lumen lined by epithelial cells. Capillary ingrowth at one end allows formation of the glomerulus, and at its other end fusion with the UB branch tip affords outflow of luminal fluid into the common collecting system. Torban et al. (9Torban E. Goodyer P.R. Biochim. Biophys. Acta. 1998; 1401: 53-62Crossref PubMed Scopus (34) Google Scholar) showed that PAX2 expression is associated with an increase in E-cadherin and suppression of vimentin levels in cultured fetal kidney cells, thus proposing that PAX2 might regulate the transition from mesenchyme to epithelium. However, the PAX2 gene targets that might mediate this transition remain elusive. In 1994, Stark et al. (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar) reported that formation of S-shaped bodies in the developing kidney is critically dependent on WNT4, a member of the WNT family of secreted glycoproteins. WNT4 is expressed by the induced metanephric mesenchymal cells as they cluster and undergo transformation into epithelia (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar). Knock-out mice with homozygous inactivation of the Wnt4 gene exhibit initial outgrowth of the ureteric bud, induction of mesenchymal cell clustering, and local expression of PAX2 but no further development of S-shaped bodies (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar, 11Kispert A. Vainio S. McMahon A.P. Development (Camb.). 1998; 125: 4225-4234Crossref PubMed Google Scholar). In the absence of S-shaped body formation, UB branching comes to an abrupt halt, and the mutant Wnt4 mice are born anephric (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar). In general, WNTs are thought to transmit signals to nearby cells bearing cognate receptors, thereby organizing cell alignment and tissue patterns during development (12Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (491) Google Scholar). WNT4 was recently shown to be capable of activating the “canonical” β-catenin signaling pathway and therefore presumably regulates β-catenin-responsive gene targets, such as c-Myc, involved in cellular growth (13Lyons J.P. Mueller U.W. Ji H. Everett C. Fang X. Hsieh J.C. Barth A.M. McCrea P.D. Exp. Cell Res. 2004; 298: 369-387Crossref PubMed Scopus (119) Google Scholar). In this study, we hypothesize that a key function of PAX2 in the S-shaped body is to activate the WNT4 gene. We show that PAX2 protein is present in the distal portion of the S-shaped body and persists in the emerging proximal tubular cells of fetal kidney. Transient transfection of a PAX2 expression vector into JTC12 cells (derived from the simian proximal tubule) stimulates transcriptional activity of the human WNT4 promoter. We identify several unique recognition motifs in the WNT4 promoter that specifically bind PAX2 protein and show that JTC12 cells stably transfected with PAX2 cDNA have increased endogenous WNT4 expression. Finally, we demonstrate that endogenous WNT4 expression is reduced in hypoplastic fetal kidneys of mice bearing a heterozygous Pax2 mutation. Cell Lines, Transfections, and Luciferase Assays—The monkey proximal tubule cell line (JTC12) was grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin. Cells were incubated at 37 °C with 5% CO2 in a humidified environment. The activity of a 10.7-kb WNT4 promoter was analyzed in transient transfections of the JTC12 cell line in the presence or absence of a PAX2 expression vector. Plasmid DNA preparation, cell culture, and transient transfections using FuGENE™ 6 transfection reagent (Roche Applied Science) were carried out as described (14Stayner Cunliffe Ward Eccles M.R. J. 1998; Full Text Full Text PDF PubMed Scopus Google with cells at were with of and of human WNT4 Cells were in a and for and for were in of on three for PAX2 was on embryonic kidney as described S. Torban E. Cho N.P. Cunliffe H. Chua L. McNoe L. Ward T. Souza C. Gus P. Giugliani R. Sato T. Yun K. Favor J. Sicotte M. Goodyer P. Eccles M. Hum. Mol. Genet. 2000; 9: 1-11Crossref PubMed Scopus (168) Google Scholar). were and for in activity was using in for at a with serum, were incubated with of at by with the as described by the were incubated with reagent were with for and with was on of embryonic kidney. were in for and in for were with for at and incubated with a of at were for at to with as the were with a of at °C and with the proximal tubule of JTC12 cells, were cultured on in Dulbecco's modified Eagle's medium supplemented with 10% and they 60% The cells were for on in several in PBS, cells were incubated with L. for at 37 were in and with In Wnt4 cDNA into vector was by McMahon The Wnt4 and were by the vector with and and and of the was with of of transcription 100 DTT, of of and of 100 DTT, and of (Roche Applied of the was and the final was incubated for at 37 DNA was with of (Roche Applied Science) at 37 °C for The were by in the presence of and with of was on a of the in a of 100 was used for in on each and heterozygous were in and in were on and incubated with μg/ml of by three in were incubated for at in 100 of with by at °C in 100 of and 100 μg/ml 100 μg/ml 10% with the were in at for and in the at °C by in at °C and at were three for in at and with μg/ml A in at 37 were in and three at in by in were in the with and for were from The of the are as and and recognition are were in PAX2 recognition for and these are shown in for and for for A for and for to the WNT4 promoter were by with the of DNA using The Pax2 were using a The in were incubated with at for in a and of In the protein was also with the of number for at and incubated with the for at were for at with to the of the binding were on a in and at at The were and to at Transfection of JTC12 Cells and JTC12 cells were transfected with a PAX2 expression or with an vector the PAX2 using the transfection FuGENE™ 6 (Roche Applied cells were in medium μg/ml for were in cell were on 10% and with number by with protein were incubated in to the and to was and for of mice were and was from each of of the and were identified as described was from kidney with to the was with to the were carried out using the We used the for reverse transcription with were using the by the the contained and reverse and the to the DNA during to for or were for each DNA was with in We with glyceraldehyde-3-phosphate were for were as initial at °C for to by of °C for of for specific and at The of the were by on the and by was using the The was determined for and as the of the Wnt4 gene in to the gene are as induction of gene mRNA expression as determined by the the the number of to the of Wnt4 and the gene PAX2 in the S-shaped and in have Pax2 mRNA expression in induced mesenchymal cells as they cluster at the tip of each ureteric bud branch (2Torres M. Gomez-Pardo E. Dressler G.R. Gruss P. Development (Camb.). 1995; 121: 4057-4065Crossref PubMed Google Scholar). show that PAX2 protein expression is in tubular cells of the distal of S-shaped bodies However, PAX2 protein is in epithelial cells at the proximal of the S-shaped body, which to of the PAX2 expression is in tubules that for the proximal tubule L. The JTC12 cell derived from simian renal proximal also for L. and was used for the in of PAX2 and WNT4 PAX2 the WNT4 in PAX2 activates WNT4 promoter activity in a 10.7-kb 5′-flanking sequence of the human WNT4 gene A. E. P. 2002; PubMed Scopus Google Scholar) was into a vector and with a human expression vector into cultured JTC12 cells Luciferase activity in cell was In the presence of transcriptional activity of the 10.7-kb WNT4 promoter was increased of in the WNT4 the WNT4 promoter we the 10.7-kb 5′-flanking sequence of the human WNT4 gene A. E. P. 2002; PubMed Scopus Google Scholar) with The recognition for PAX2 by and appears to from gene to gene a sequence of about a of (4Brophy P.D. Ostrom L. Lang K.M. Dressler G.R. Development (Camb.). 2001; 128: 4747-4756Crossref PubMed Google Scholar). with to this were shown to specifically bind PAX2 The first A in was identified to in to the transcriptional contained of the of the PAX2 sequence but was for study it also exhibited with the recognition A at to and third at to PAX2 recognition sequence were identified further and in and These motifs contained and 6 of the PAX2 motifs and within a 0.66-kb of DNA to that was completely duplicated further to Thus, PAX2 motifs and were identified on DNA but these were of in the cassette. The three unique motifs and are in and with PAX2 recognition motifs reported by the three recognition motifs exhibited specific high PAX2 a of shift was with the and in PAX2 of PAX2 protein with each in seen as in and The in the presence of PAX2 and but The of Pax2 binding was in assays with of and mutant A and of PAX2 to was out by of was at and were also with PAX2 protein and A and as of a in the recognition of A reduces When of and were formation was completely and mutant were unable to formation in the as the Thus, PAX2 each of the three in PAX2 WNT4 in JTC12 cells are derived from the monkey proximal tubule lineage and express the proximal tubular L. The cells express levels of endogenous by the of PAX2 on endogenous WNT4 JTC12 cells were stably transfected with a human PAX2 expression vector and in were and for PAX2 protein by seen in the expressed about as PAX2 protein as the cell WNT4 mRNA levels were in JTC12 cells and in the by for mRNA seen in WNT4 mRNA levels were about 7-fold in the with cells. WNT4 in the we that fetal kidneys from have reduced PAX2 protein and moderately renal the of nephrons is normal S. Torban E. Cho N.P. Cunliffe H. Chua L. McNoe L. Ward T. Souza C. Gus P. Giugliani R. Sato T. Yun K. Favor J. Sicotte M. Goodyer P. Eccles M. Hum. Mol. Genet. 2000; 9: 1-11Crossref PubMed Scopus (168) Google Scholar). the in of WNT4 by PAX2 an pathway for mammalian kidney development in we endogenous Wnt4 expression in fetal kidney of heterozygous mutant mice and mice were with were as described (8Clark P. Dziarmaga A. Eccles M. Goodyer P. J. Am. Soc. Nephrol. 2004; 15: 299-305Crossref PubMed Scopus (38) Google and fetal kidneys were and for in with a Wnt4 seen in A and Wnt4 mRNA expression is but is to the mesenchyme adjacent to ureteric bud branch in normal fetal kidney. In contrast, the Wnt4 is in mesenchyme of fetal kidneys and in were for transcription factor expressed in induced renal mRNA expression was in and kidneys Wnt4 mRNA was in mutant and fetal kidneys by and for mRNA in the In with in Wnt4 mRNA in mutant kidney was of that in kidneys from of the during plays a central role in the caudal descent of the nephric the of ureteric and the branching of the collecting system. PAX2 mRNA is also in metanephric mesenchymal cells as they cluster at UB (2Torres M. Gomez-Pardo E. Dressler G.R. Gruss P. Development (Camb.). 1995; 121: 4057-4065Crossref PubMed Google and its function is as unknown. show that PAX2 protein expression is this appears in distal portions of the developing S-shaped body as and is in the fetal proximal tubule. PAX2 is involved in the process metanephric mesenchyme is into polarized in the of the proximal tubule as it suggest that these are also critically dependent on expression of the secreted growth In mice with homozygous inactivation of the Wnt4 the UB metanephric mesenchyme and mesenchymal cells to cluster and express PAX2 at its (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar, A. Vainio S. L. McMahon A.P. Development (Camb.). PubMed Google Scholar). However, to the S-shaped body (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar, A. Vainio S. L. McMahon A.P. Development (Camb.). PubMed Google Scholar). This PAX2 of WNT4 during this demonstrate reduced Wnt4 mRNA in the and that Pax2 is of WNT4 expression in the developmental as is that WNT4 is involved in cell movement during development. through the WNT4 murine as they the J. M. 2003; PubMed Scopus Google Scholar). In WNT4 in cell of WNT4 regulates during through the protein pathway J. J. Dev. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). WNT4 signaling this pathway might a role in the growth of the S-shaped body and proximal tubule. the other is also that WNT4 may have a of through the β-catenin signaling pathway (13Lyons J.P. Mueller U.W. Ji H. Everett C. Fang X. Hsieh J.C. Barth A.M. McCrea P.D. Exp. Cell Res. 2004; 298: 369-387Crossref PubMed Scopus (119) Google Scholar). This pathway to WNT activation of at the of cells X. M. K. X. Development (Camb.). 2004; PubMed Scopus Google Scholar). and other to of β-catenin and its to the β-catenin with transcription factor of the family to regulate expression of genes R. Cell Res. 15: PubMed Scopus Google Scholar). cell in the developing is by signaling M. M. Mol. 2004; PubMed Scopus Google Scholar). WNT4 signaling development X. X. L. L. Genes Dev. 2004; PubMed Scopus Google Scholar) and the of on growth X. M. Mol. 2004; PubMed Scopus Google Scholar). levels of β-catenin signaling activity have identified in the distal S-shaped body and of the ureteric bud during kidney development S. M. S. P. D. S. U. S. A. 2003; PubMed Scopus Google Scholar, M. D. Goodyer P. J. Am. Soc. Nephrol. 2004; 15: Google Scholar). Thus, WNT4 expression might signaling during formation of the S-shaped is the that WNT4 also a to the adjacent ureteric bud WNT4 is reported to activate the β-catenin signaling pathway in cultured kidney cells derived from the UB lineage (13Lyons J.P. Mueller U.W. Ji H. Everett C. Fang X. Hsieh J.C. Barth A.M. McCrea P.D. Exp. Cell Res. 2004; 298: 369-387Crossref PubMed Scopus (119) Google Scholar). WNT4 in of cells during development C. A. T. J. McMahon McMahon A.P. Genes Dev. 2000; 14: Google Scholar). Wnt4 to form S-shaped but this is by an abrupt to further UB branching (10Stark K. Vainio S. Vassileva G. McMahon A.P. Nature. 1994; 372: 679-683Crossref PubMed Scopus (907) Google Scholar). PAX2 to within the WNT4 demonstrate that PAX2 activates transcriptional activity of the 5′-flanking sequence of the human WNT4 several unique PAX2 recognition A novel sequence A in about of the transcriptional showed to the PAX2 recognition by et al. (4Brophy P.D. Ostrom L. Lang K.M. Dressler G.R. Development (Camb.). 2001; 128: 4747-4756Crossref PubMed Google Scholar) and to the used in and form a within the gene family on PAX2 and binding in other gene targets W. D. H. J. Mol. PubMed Google Scholar). Two other motifs within the 5′-flanking sequence of the human WNT4 gene motifs and in were to the PAX2 recognition (4Brophy P.D. Ostrom L. Lang K.M. Dressler G.R. Development (Camb.). 2001; 128: 4747-4756Crossref PubMed Google Scholar). motifs and were in to each other on and DNA and within a 0.66-kb to that was completely duplicated at an to in the 5′-flanking this event the of these PAX2 motifs other elements of the in WNT4 expression. is the WNT4 gene several different recognition motifs for each of the motifs or exhibited specific high binding to PAX2 protein in In each the was to of PAX2 recognition and of other transcription be involved in the expression of WNT4 in derived portions of the renal PAX2 is expressed at other in the developing kidney ureteric where WNT4 expression is transcription are to a with PAX2 and for the of WNT4 expression. The of PAX2 recognition might the of this PAX2 WNT4 in in PAX2 and the WNT4 promoter are clearly during in renal development. Wnt4 mRNA expression was reduced in the mesenchymal bodies of heterozygous mutant fetal heterozygous mutant fetal kidneys are hypoplastic S. Torban E. Cho N.P. Cunliffe H. Chua L. McNoe L. Ward T. Souza C. Gus P. Giugliani R. Sato T. Yun K. Favor J. Sicotte M. Goodyer P. Eccles M. Hum. Mol. Genet. 2000; 9: 1-11Crossref PubMed Scopus (168) Google but the in WNT4 expression is to formation of normal S-shaped bodies during development. This is normal S-shaped bodies were also in fetal kidneys of heterozygous Wnt4 Thus, the developmental of S-shaped bodies to a of PAX2 and in homozygous Wnt4 S-shaped body formation renal development, PAX2 is expressed in the distal portion of the emerging S-shaped body and proximal tubule. We suggest that it to novel elements in the WNT4 activating transcription during the mesenchyme to WNT4 expression in the S-shaped body and proximal tubule and influence its growth and PAX2 activation of WNT4 a to ureteric bud cells and further of branching nephrogenesis.

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