Congenital reduction in nephron number (renal hypoplasia) is a predisposing factor for chronic kidney disease and hypertension. Despite identification of specific genes and pathways in nephrogenesis, determinants of final nephron endowment are poorly understood. Here, we report that mice with germ-line p53 deletion (p53？/？) manifest renal hypoplasia; the phenotype can be recapitulated by conditional deletion of p53 from renal progenitors in the cap mesenchyme (CMp53？/？). Mice or humans with germ-line heterozygous mutations in Pax2 exhibit renal hypoplasia. Since both transcription factors are developmentally expressed in the metanephros, we tested the hypothesis that p53 and Pax2 cooperate in nephrogenesis. In this study, we provide evidence for the presence of genetic epistasis between p53 and Pax2: a) p53？/？ and CMp53？/？embryos express lower Pax2 mRNA and protein in nephron progenitors than their wild-type littermates; b) ChIP-Seq identified peaks of p53 occupancy in chromatin regions of the Pax2 promoter and gene in embryonic kidneys; c) p53 binding to Pax2 gene is significantly more enriched in Pax2 -expressing than non-expressing metanephric mesenchyme cells; d) in transient transfection assays, Pax2 promoter activity is stimulated by wild-type p53 and inhibited by a dominant negative mutant p53; e) p53 knockdown in cultured metanephric mesenchyme cells down-regulates endogenous Pax2 expression; f) reduction of p53 gene dosage worsens the renal hypoplasia in Pax2+/？ mice. Bioinformatics identified a set of developmental renal genes likely to be co-regulated by p53 and Pax2. We propose that the cross-talk between p53 and Pax2 provides a transcriptional platform that promotes nephrogenesis, thus contributing to nephron endowment.
References
[1]
Costantini F, Kopan R (2010) Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev Cell 18: 698–712.
[2]
Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM, et al. (2008) Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia. Developmental Biology 313: 234–245.
[3]
Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, et al. (2008) Six2 Defines and Regulates a Multipotent Self-Renewing Nephron Progenitor Population throughout Mammalian Kidney Development. Cell Stem Cell 3: 169–181.
[4]
Karner CM, Das A, Ma Z, Self M, Chen C, et al. (2011) Canonical Wnt9b signaling balances progenitor cell expansion and differentiation during kidney development. Development 138: 1247–1257.
[5]
Boyle SC, Kim M, Valerius MT, McMahon AP, Kopan R (2011) Notch pathway activation can replace the requirement for Wnt4 and Wnt9b in mesenchymal-to-epithelial transition of nephron stem cells. Development 138: 4245–4254.
[6]
Mugford JW, Sipila P, McMahon JA, McMahon AP (2008) Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney. Dev Biol 324: 88–98.
[7]
Mugford JW, Yu J, Kobayashi A, McMahon AP (2009) High-resolution gene expression analysis of the developing mouse kidney defines novel cellular compartments within the nephron progenitor population. Dev Biol 333: 312–323.
[8]
Reidy KJ, Rosenblum ND (2009) Cell and molecular biology of kidney development. Semin Nephrol 29: 321–337.
[9]
Chi N, Epstein JA (2002) Getting your Pax straight: Pax proteins in development and disease. Trends in Genetics 18: 41–47.
[10]
Muratovska A, Zhou C, He S, Goodyer P, Eccles MR (2003) Paired-Box Genes are Frequently Expressed in Cancer and Often required for Cancer Cell Survival. Oncogene 22: 7989–7997.
[11]
Bouchard M, Souabni A, Mandler M, Neubüser A, Busslinger M (2002) Nephric lineage specification by Pax2 and Pax8. Genes and Dev 16: 2958–2970.
[12]
Narlis M, Grote D, Gaitan Y, Boualia SK, Bouchard M (2007) Pax2 and pax8 regulate branching morphogenesis and nephron differentiation in the developing kidney. J Am Soc Nephrol 18: 1121–1129.
[13]
Bouchard M, Pfeffer P, Busslinger M (2000) Functional equivalence of the transcription factors Pax2 and Pax5 in mouse development. Development 127: 3703–3713.
[14]
Schwarz M, Cecconi F, Bernier G, Andrejewski N, Kammandel B, et al. (2000) Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6. Development 127: 4325–4334.
[15]
Torres M, Gómez-Pardo E, Dressler GR, Gruss P (1995) Pax-2 controls multiple steps of urogenital development. Development 121: 4057–4065.
[16]
Sanyanusin P, Schimmenti LA, McNoe LA, Ward TA, Pierpont ME, et al. (1995) Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nat Genet 9: 358–364.
[17]
Porteous S, Torban E, Cho NP, Cunliffe H, Chua L, et al. (2000) Primary renal hypoplasia in humans and mice with PAX2 mutations: evidence of increased apoptosis in fetal kidneys of Pax2(1Neu) +/？ mutant mice. Hum Mol Genet 9: 1–11.
[18]
Gong K-Q, Yallowitz AR, Sun H, Dressler GR, Wellik DM (2007) A Hox-Eya-Pax Complex Regulates Early Kidney Developmental Gene Expression. Mol Cell Biol 27: 7661–7668.
[19]
Brodbeck S, Englert C (2004) Genetic Determination of Nephrogenesis: the Pax/Eya/Six Gene Network. Pediatr Nephrol 19: 249–255.
[20]
Brophy PD, Ostrom L, Lang KM, Dressler GR (2001) Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene. Development 128: 4747–4756.
[21]
Torban E, Dziarmaga A, Iglesias D, Chu LL, Vassilieva T, et al. (2006) PAX2 activates WNT4 expression during mammalian kidney development. J Biol Chem 281: 12705–12712.
[22]
Stiewe T (2007) The p53 family in differentiation and tumorigenesis. Nat Rev Cancer 7: 165–168.
[23]
Zhao T, Yu X (2010) p53 and Stem cells: New Developments and New Concerns. Trends in Cell Biology 20: 170–175.
[24]
Horn HF, Vousden KH (2007) Coping with stress: multiple ways to activate p53. Oncogene 26: 1306–1316.
[25]
Lin T, Chao C, Saito S, Mazur SJ, Murphy ME, et al. (2005) p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat Cell Biol 7: 165–171.
[26]
Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, et al. (2009) The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell 138: 1083–1095.
[27]
Zhao Z, Zuber J, Diaz-Flores E, Lintault L, Kogan SC, et al. (2010) p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal. Genes Dev 24: 1389–1402.
[28]
Armata HL, Golebiowski D, Jung DY, Ko HJ, Kim JK, et al. (2010) Requirement of the ATM/p53 Tumor Suppressor Pathway for Glucose Homeostasis. Mol Cell Biol 30: 5787–5794.
[29]
Zhang L, Yu D, Hu M, Xiong S, Lang A, et al. (2000) Wildtype p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res 60: 3655–3661.
[30]
Teodoro JG, Parker AE, Zhu X, Green MR (2006) p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase. Science 313: 968–971.
[31]
Schmid P, Lorenz A, Hameister H, Montenarh M (1991) Expression of p53 during mouse embryogenesis. Development 113: 857–865.
[32]
Choi J, Donehower LA (1999) p53 in embryonic development: maintaining a fine balance. Cell Mol Life Sci 55: 38–47.
[33]
Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, et al. (1994) Tumor spectrum analysis in p53-mutant mice. Curr Biol 4: 1–7.
[34]
Sah VP, Attardi LD, Mulligan GJ, Williams BO, Bronson RT, et al. (1995) A subset of p53-deficient embryos exhibit exencephaly. Nat Genet 10: 175–180.
[35]
Baatout S, Jacquet P, Michaux A, Buset J, Vankerkom J, et al. (2002) Developmental abnormalities induced by X-irradiation in p53 deficient mice. In Vivo 16: 215–221.
[36]
Ikeda S, Hawes NL, Chang B, Avery CS, Smith RS, et al. (1999) Severe ocular abnormalities in C57BL/6 but not in 129/Sv p53-deficient mice. Invest Ophthalmol Vis Sci 40: 1874–1878.
[37]
Rotter V, Schwartz D, Almon E, Goldfinger N, Kapon A, et al. (1993) Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome. Proc Natl Acad Sci U S A 90: 9075–9079.
[38]
Hoever M, Clement JH, Wedlich D, Montenarh M, Knochel W (1994) Overexpression of wild-type p53 interferes with normal development in Xenopus laevis embryos. Oncogene 9: 109–120.
[39]
Wallingford JB, Seufert DW, Virta VC, Vize PD (1997) p53 activity is essential for normal development in Xenopus. Curr Biol 7: 747–757.
[40]
Cordenonsi M, Dupont S, Maretto S, Insinga A, Imbriano C, et al. (2003) Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads. Cell 113: 301–314.
[41]
Saifudeen Z, Dipp S, Stefkova J, Yao X, Lookabaugh S, et al. (2009) p53 regulates metanephric development. J Am Soc Nephrol 20: 2328–2337.
[42]
Saifudeen Z, Dipp S, El-Dahr SS (2002) A role for p53 in terminal epithelial cell differentiation. J Clin Invest 109: 1021–1030.
[43]
Molchadsky A, Shats I, Goldfinger N, Pevsner-Fischer M, Olson M, et al. (2008) p53 plays a role in mesenchymal differentiation programs, in a cell fate dependent manner. PLoS One 3: e3707.
[44]
Almog N, Rotter V (1997) Involvement of p53 in cell differentiation and development. Biochim Biophys Acta 1333: F1–27.
[45]
Qin Q, Baudry M, Liao G, Noniyev A, Galeano J, et al. (2009) A Novel Function for p53: Regulation of Growth Cone Motility through Interaction with Rho Kinase. 29: 5183–5192.
[46]
Montes de Oca Luna R, Wagner DS, Lozano G (1995) Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378: 203–206.
[47]
Hilliard S, Aboudehen K, Yao X, El-Dahr SS (2011) Tight regulation of p53 activity by Mdm2 is required for ureteric bud growth and branching. Dev Biol 353: 354–366.
[48]
Aboudehen K, Hilliard S, Saifudeen Z, El-Dahr SS (2012) Mechanisms of p53 activation and physiological relevance in the developing kidney. American Journal of Physiology - Renal Physiology 302: F928–F940.
[49]
Dermitzakis ET, Reymond A, Antonarakis SE (2005) Conserved non-genic sequences – an unexpected feature of mammalian genomes. Nat Rev Genetics 6: 151–157.
[50]
Valerius MT, Patterson LT, Witte DP, Potter SS (2002) Microarray analysis of novel cell lines representing two stages of metanephric mesenchyme differentiation. Mech Dev 112: 219–232.
[51]
Patel SR, Dressler GR (2004) Expression of Pax2 in the intermediate mesoderm is regulated by YY1. Dev Biol 267: 505–516.
[52]
el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B (1992) Definition of a consensus binding site for p53. Nat Genet 1: 45–49.
[53]
Stuart ET, Haffner R, Oren M, Gruss P (1995) Loss of p53 function through PAX-mediated transcriptional repression. EMBO J 14: 5638–5645.
[54]
Pfeffer PL, Payer B, Reim G, di Magliano MP, Busslinger M (2002) The activation and maintenance of Pax2 expression at the mid-hindbrain boundary is controlled by separate enhancers. Development 129: 307–318.
[55]
Torres M, Gomez-Pardo E, Gruss P (1996) Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122: 3381–3391.
[56]
Sasai N, Yakura R, Kamiya D, Nakazawa Y, Sasai Y (2008) Ectodermal factor restricts mesoderm differentiation by inhibiting p53. Cell 133: 878–890.
[57]
Takebayashi-Suzuki K, Funami J, Tokumori D, Saito A, Watabe T, et al. (2003) Interplay between the tumor suppressor p53 and TGF beta signaling shapes embryonic body axes in Xenopus. Development 130: 3929–3939.
[58]
Grieshammer U, Cebrian C, Ilagan R, Meyers E, Herzlinger D, et al. (2005) FGF8 is required for cell survival at distinct stages of nephrogenesis and for regulation of gene expression in nascent nephrons. Development 132: 3847–3857.
[59]
Perantoni AO, Timofeeva O, Naillat F, Richman C, Pajni-Underwood S, et al. (2005) Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development. Development 132: 3859–3871.
[60]
Poladia DP, Kish K, Kutay B, Hains D, Kegg H, et al. (2006) Role of fibroblast growth factor receptors 1 and 2 in the metanephric mesenchyme. Developmental Biology 291: 325–339.
[61]
Sims-Lucas S, Cusack B, Baust J, Eswarakumar VP, Masatoshi H, et al. (2011) Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development. Dev Dyn 240: 240–249.
[62]
Naim E, Bernstein A, Bertram JF, Caruana G (2005) Mutagenesis of the epithelial polarity gene, discs large 1, perturbs nephrogenesis in the developing mouse kidney. Kidney Int 68: 955–965.
[63]
Mah SP, Saueressig H, Goulding M, Kintner C, Dressler GR (2000) Kidney Development in Cadherin-6 Mutants: Delayed Mesenchyme-to-Epithelial Conversion and Loss of Nephrons. Developmental Biology 223: 38–53.
[64]
Sorenson CM (2004) Interaction of bcl-2 with Paxillin through Its BH4 Domain Is Important during Ureteric Bud Branching. Journal of Biological Chemistry 279: 11368–11374.
[65]
Hollander MC, Alamo I, Jackman J, Wang MG, McBride OW, et al. (1993) Analysis of the mammalian gadd45 gene and its response to DNA damage. Journal of Biological Chemistry 268: 24385–24393.
[66]
Morris GF, Bischoff JR, Mathews MB (1996) Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53. Proceedings of the National Academy of Sciences 93: 895–899.
[67]
Thornborrow EC, Manfredi JJ (1999) One Mechanism for Cell Type-specific Regulation of thebax Promoter by the Tumor Suppressor p53 Is Dictated by the p53 Response Element. Journal of Biological Chemistry 274: 33747–33756.
[68]
Zauberman A, Flusberg D, Haupt Y, Barak Y, Oren M (1995) A functional p53-responsive intronic promoter is contained within the human mdm2 gene. Nucleic Acids Research 23: 2584–2592.
[69]
Bouchard M, Grote D, Craven SE, Sun Q, Steinlein P, et al. (2005) Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region. Development 132: 2633–2643.
[70]
El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, et al. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817–825.
[71]
Park D, Jia H, Rajakumar V, Chamberlin HM (2006) Pax2/5/8 proteins promote cell survival in C. elegans. Development 133: 4193–4202.
[72]
Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B (2001) PUMA Induces the Rapid Apoptosis of Colorectal Cancer Cells. Molecular Cell 7: 673–682.
[73]
Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, et al. (2000) Noxa, a BH3-Only Member of the Bcl-2 Family and Candidate Mediator of p53-Induced Apoptosis. Science 288: 1053–1058.
[74]
Clark P, Dziarmaga A, Eccles M, Goodyer P (2004) Rescue of Defective Branching Nephrogenesis in Renal-Coloboma Syndrome by the Caspase Inhibitor, Z-VAD-fmk. Journal of the American Society of Nephrology 15: 299–305.
[75]
Dressler GR, Douglass EC (1992) Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proceedings of the National Academy of Sciences 89: 1179–1183.
[76]
Rothenpieler UW, Dressler GR (1993) Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development. Development 119: 711–720.
[77]
Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, et al. (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9: R137.
[78]
Saifudeen Z, Diavolitsis V, Stefkova J, Dipp S, Fan H, et al. (2005) Spatiotemporal switch from DeltaNp73 to TAp73 isoforms during nephrogenesis: impact on differentiation gene expression. J Biol Chem 280: 23094–23102.
[79]
Chen S, Bellew C, Yao X, Stefkova J, Dipp S, et al. (2011) Histone Deacetylase (HDAC) Activity Is Critical for Embryonic Kidney Gene Expression, Growth, and Differentiation. J Biol Chem 286: 32775–32789.
[80]
Saifudeen Z, Dipp S, Fan H, El-Dahr SS (2005) Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation. Am J Physiol Renal Physiol 288: F899–909.
[81]
Saifudeen Z, Du H, Dipp S, El-Dahr SS (2000) The bradykinin type 2 receptor is a target for p53-mediated transcriptional activation. J Biol Chem 275: 15557–15562.
