Histone deacetylases (HDACs) regulate fundamental biological processes such as cellular proliferation, differentiation, and survival via genomic and nongenomic effects. This study examined the importance of HDAC activity in the regulation of gene expression and differentiation of the developing mouse kidney. Class I HDAC1-3 and class II HDAC4, -7, and -9 genes are developmentally regulated. Moreover, HDAC1-3 are highly expressed in nephron precursors. Short term treatment of cultured mouse embryonic kidneys with HDAC inhibitors (HDACi) induced global histone H3 and H4 hyperacetylation and H3K4 hypermethylation. However, genome-wide profiling revealed that the HDAC-regulated transcriptome is restricted and encompasses regulators of the cell cycle, Wnt/␤-catenin, TGF-␤/Smad, and PI3K-AKT pathways. Further analysis demonstrated that base-line expression of key developmental renal regulators, including Osr1, Eya1, Pax2/8, WT1, Gdnf, Wnt9b, Sfrp1/2, and Emx2, is dependent on intact HDAC activity. Treatment of cultured embryonic kidney cells with HDACi recapitulated these gene expression changes, and chromatin immunoprecipitation assays revealed that HDACi is associated with histone hyperacetylation of Pax2/Pax8, Gdnf, Sfrp1, and p21. Gene knockdown studies demonstrated that HDAC1 and HDAC2 play a redundant role in regulation of Pax2/8 and Sfrp1 but not Gdnf. Long term treatment of embryonic kidneys with HDACi impairs the ureteric bud branching morphogenesis program and provokes growth arrest and apoptosis. We conclude that HDAC activity is critical for normal embryonic kidney homeostasis, and we implicate class I HDACs in the regulation of early nephron gene expression, differentiation, and survival.Kidney development depends on reciprocal inductive interactions between the metanephric mesenchyme (MM), 4 a specified region in the caudal intermediate mesoderm, and the ureteric bud (UB), an epithelial outgrowth from the Wolffian (nephric) duct (1-3). Recent years have witnessed significant progress in our understanding of the gene regulatory networks of early kidney development (3-6). For example, the Osr1/ Eya1/Pax2/Six/Sall/WT1/Hoxd11 gene regulatory network specifies the MM and is absolutely required for expression of glia-derived neurotrophic factor (Gdnf) (7,8). Gdnf, in turn, is essential for UB outgrowth and subsequent branching (9 -11).Gdnf acts via activation of a c-Ret/PI3K/ERK-dependent gene network (Wnt11, Spry1, Etv4, Etv5, Cxcr4, Myb, Met, and Mmp14) in UB tip cells to control the branching morphogenesis program (12-14). Various growth factor/receptor signaling pathways, including FGFs, bone morphogenic proteins, VEGF, semaphorins, hepatocyte growth factor, EGF, among others, share signaling components with the c-Ret pathway and are required for optimal metanephric growth and patterning (15)(16)(17)(18)(19)(20)(21)(22). Following induction of the MM, activation of the Wnt/␤-catenin signaling pathway plays a key role in nephrogenesis. Release of Wnt9b from the UB branches triggers a ␤-catenindependent mo...

p53 is best known as a tumor suppressor that regulates cell-cycle, differentiation, and apoptosis pathways, but its potential role in embryonic development and organogenesis remains controversial. Here, p53Ϫ/Ϫ embryos bred on C57Bl6 background exhibited a spectrum of congenital abnormalities of the kidney and urinary tract, including ureteric bud (UB) ectopia, double ureters/collecting systems, delayed primary branching of the UB, and hypoplastic metanephroi. We observed ectopic UB outgrowth from the Wolffian duct (WD) in one third of p53 Ϫ/Ϫ embryos. The prevalence of duplex was higher in embryos than in neonates, and ex vivo organ culture suggested that ectopic ureters can regress over time, leaving behind a dysplastic pole ("segmental dysgenesis"). Transgenic expression of dominant negative p53 or conditional inactivation of p53 in the UB but not in the metanephric mesenchyme lineage recapitulated the duplex phenotype. Mechanistically, p53 inactivation in the WD associated with enhanced sensitivity to glial cell line-derived neurotrophic factor (GDNF)-induced ectopic budding and potentiated phosphatidylinositol-3 kinase activation by GDNF in UB cells. Unlike several other models of UB ectopia, hypersensitivity of p53 Ϫ/Ϫ WD to GDNF is not accompanied by reduced Sprouty-1 or anterior expansion of the GDNF domain. In summary, our data lend support for a restrictive role for p53 activity in UB outgrowth from the WD. Organogenesis is dependent on a multitude of growth factors, signaling molecules, and transcription factors that temporally and spatially define a developmental program. Metanephric development is dependent on formation of the Wolffian duct (WD) and the adjacent metanephric mesenchyme (MM) from the intermediate mesoderm. In mice, the ureteric bud (UB) develops at embryonic day 10.5 (E10.5) from the caudal end of the WD. Inductive interactions between the UB and the MM ensure cell survival and subsequent onset of metanephrogenesis. Emergence of the UB from a specific site in the WD adjacent to the MM is controlled by the glial cell line-derived neurotrophic factor (GDNF)-cRet signaling pathway. 1-6 GDNF is a growth factor that is secreted by the MM and binds to its receptor c-Ret and co-receptor GFR␣1 that are expressed along the WD and UB tips. Stimulation of the GDNF-cRet pathway results in cell proliferation and chemotaxis. 7,8 After the initial broad distribution of the GDNF field along the posterior half of the WD, the GDNF expression domain is progressively restricted to the caudal end of the WD in the immediate vicinity of the site of UB outgrowth 9 -11 ; however, the potential for bud emergence remains along the length of the WD, which continues to express the c-Ret/GFR␣1 receptor/co-receptor. Evidence from mutant mouse models and metanephric organ culture implicates impaired restriction of the GDNF field and the GDNF/c-Ret signaling pathway in ureter duplica-

p73 is a member of the p53 gene family, which also includes p53 and p63. These proteins share sequence similarity and target genes but also have divergent roles in cancer and development. Unlike p53, transcription of the p73 gene yields multiple full-length (transactivation (TA) domain) and amino terminus-truncated (⌬N) isoforms. ⌬Np73 acts in a dominant negative fashion to inhibit the actions of TAp73 and p53 on their target genes, promoting cell survival and proliferation and suppressing apoptosis. The balance between TAp73 and its negative regulator, ⌬Np73, may therefore represent an important determinant of developmental cell fate. There is little if anything known regarding the developmental regulation of the p73 gene. In this study, we showed that TAp73 and ⌬Np73 exhibit reciprocal spatiotemporal expression and functions during nephrogenesis. TAp73 was predominantly expressed in the differentiation domain of the renal cortex in an overlapping manner with the vasopressin-sensitive water channel aquaporin-2 (AQP-2). Chromatin immunoprecipitation assays demonstrated that the endogenous AQP-2 promoter was occupied by TAp73 in a developmentally regulated manner. Furthermore TAp73 stimulated AQP-2 promoter-driven reporter expression. TAp73 also activated the bradykinin B2 receptor (B2R) promoter, a developmentally regulated gene involved in regulation of sodium excretion. The transcriptional effects of TAp73 on AQP-2 and B2R were independent of p53. In marked contrast to TAp73, ⌬Np73 isoforms were induced early in development and were preferentially expressed in proliferating nephron precursors. Moreover ⌬Np73 was a potent repressor of B2R gene transcription. We conclude that the p73 gene is developmentally regulated during kidney organogenesis. The spatiotemporal switch from ⌬Np73 to TAp73 may play an important role in the terminal differentiation program of the developing nephron.

In response to gestational high salt intake, BdkrB2-/- embryos acquire an aberrant renal phenotype mimicking renal dysplasia in humans. Genetic analysis identified p53 as a mediator of the renal dysplasia in salt-stressed BdkrB2-/- mice, acting partly via repression of terminal epithelial differentiation genes. The present study tested the hypothesis that inactivation of BdkrB2 predisposes the salt-stressed embryo to p53-mediated metanephric apoptosis. Newborn BdkrB2-/- pups exhibited hyperphosphorylation of metanephric p53 on serine 20 (mouse serine 23), a modification known to increase p53 stability and apoptotic activity. As a result, there was widespread, ectopic expression of p53 in the BdkrB2-/- kidney. However, no differences were found in the apoptosis index or gene expression in BdkrB2-/- and +/+ kidneys, indicating that p53 stabilization as a result of BdkrB2 inactivation is not sufficient to induce metanephric apoptosis. On gestational salt stress, fulminant metanephric apoptosis and enhanced Bax gene expression occurred in BdkrB2-/- but not their +/- or +/+ littermates. Germline deletion of p53 from BdkrB2-/- mice prevented Bax activation and normalized the apoptosis index. Rescue of metanephric apoptosis in BdkrB2-/- mice was similarly achieved by Bax gene deletion. Aberrant apoptosis in salt-stressed BdkrB2-/- mice was triggered on embryonic day E15.5 and involved both ureteric bud (UB) and metanephric mesenchyme-derived nephron elements. Cultured E12.5 salt-stressed BdkrB2-/- metanephroi manifested stunted UB branching compared with +/- and +/+ littermates; the abnormal UB branching was corrected by p53 deletion. Our results suggest a model whereby a seemingly silent genetic mutation of BdkrB2 predisposes mice to renal dysplasia by creating a "preapoptotic" state through p53 activation.

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