p53 regulates metanephric development

Abstract

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, p53(-/-) 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(-/-) 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(-/-) 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.

Figure 1.

p53 gene expression is shown during metanephric development. p53 mRNA is expressed in the WD, the mesonephric tubules (Mes), and the surrounding mesenchyme (whole-mount ISH [A; section ISH [B]). (A through E, G, and H) Expression is higher in the duct and UB derivatives (black arrows) than in the mesenchyme. p53 mRNA is expressed in nephron precursors (red arrows) from the time they appear at E13.5 (E), at E16.5 (G) and PN1 (H). (F) p53 expression is developmentally regulated in the kidney. Q-PCR from metanephroi of various embryonic ages shows approximately four-fold decline in p53 mRNA expression from E15.5 to adulthood. p53 expression normalized to β-actin expression.

Figure 2.

Duplex kidney phenotype in conventional p53-null mice and delayed primary branch formation in p53^−/− metanephroi are shown. (A through F′) Growth and branching patterns of p53-null metanephroi ex vivo are examined. Metanephroi from p53^+/+ and p53^−/− littermates were cultured on transwell filters for 88 h, then stained with anti-cytokeratin antibody. At 0 h, the p53^+/+ metanephroi (A and A′) show the T-shaped UB characteristic at E11.5. The UB in p53^−/− metanephroi has not yet branched (D and D′) at E11.5, resulting in fewer UB tips at 88h (C and C′ versus F and F′). (D) White arrows point to two UBs emerging at distinct points from the WD and invading the MM (black arrow). At 40 h (E), the two points of UB emergence seem to have merged into one, thus giving the appearance of a bifurcated ureter (E and F). Larger condensates in p53^−/− kidneys (black arrowheads, E and E′) than in p53^+/+ littermates (B and B′). Blind ectopic UBs (E and F, yellow arrowheads) are clearly visible at 40 and 88 h. (G through G″) Regression of an ectopic ureter over time (0 versus 88 h, G and G′) results in a single ureter with an abnormal lobulated kidney. (H) Periodic acid-Schiff–stained p53^−/− PN1 kidney section is shown. Red arrows point to the duplex lobes that are separated by a deep nephrogenic zone (boxed [H]; high magnification [H′]). (I) Section from a heterozygous kidney shows WT morphology.

Figure 3.

GDNF, c-Ret, and Spry1 mRNA levels and domains are unchanged in p53^−/− embryos. E10.5 litters from p53^+/− pairings were dissected and used for in situ hybridization. AP, anterior-posterior; h, hind-limb bud.

Figure 4.

Hypoplasia in p53^−/− kidneys is shown. (A) In situ hybridization for Spry1 was done on E12.5 metanephroi. Branch and tip formation are lagging in the p53^−/− metanephros in comparison with the age-appropriate branching observed in a p53^+/+ littermate. There was no change in Spry1 mRNA levels. (B) Decreased proliferation in p53-null kidneys was visualized by phospho-H3 antibody staining compared with kidney section from WT littermate. (C) UB tip numbers were counted in four litters from p53^+/− pairings. (D) The number of tips was consistently lower in p53^−/− (yellow bar) versus p53^+/+ metanephroi (purple bar), with the null metanephroi showing 30% fewer tips than the WT.

Figure 5.

WD/UB^Δp53 mice have kidneys with double papillae. (A, B, and D through H) p53 was conditionally deleted in the WD/UB by crossing Ksp-cadherin–driven (A) or Hoxb7-driven (B) Cre transgenic mice to p53^flox mice or by expressing p53DN driven by Hoxb7 promoter (D through H). (C) Hoxb7-p53DN construct. p53 cDNA encoding a miniprotein (amino acids 1 through 14 and 302 through 390) was inserted into a Hoxb7-IRES-EGFP construct to drive expression of the mutant protein (p53DN) in WD lineage. (D and D′) Immunofluorescence staining with anti–p53-P-ser392 is shown. Section from a transgenic mouse kidney expressing p53DN shows intense staining in UB trunks and branches (D periphery, and inset) and deeper collecting ducts. Section from a nontransgenic mouse kidney shows much lower intensity of staining, representing detection of endogenous levels of p53-P-ser392 (D′, magnified inset). (E) Transgenic mice expressing this miniprotein exhibit duplex ureters. (E′) Periodic acid-Schiff staining of a section reveals an internalized nephrogenic zone (arrow).)H and H′) The double papilla (H, black arrows) stains positively for AQP2 (H′). (F and G) Hoxb7-p53DN gain-of-function phenotype: Extrarenal nodules (F) and pitted surface (G).

Figure 6.

p53-deficient nephric ducts exhibit increased sensitivity to GDNF. (A through C) WT nephric ducts grow ectopic c-Ret⁺ UBs in response to exposure to high dosage (>50 ng/ml) GDNF (B and C). Exposure to subthreshold dosage (10 ng/ml) does not elicit bud growth (A). (D through E′) Subthreshold dosage exposure with GDNF of p53-deficient ducts induces multiple ectopic bud formation in mutant (D′ and E′) ducts but not in ducts from WT littermates (D and E, respectively). (F and F′) WT ducts treated with p53-inhibitor Pifithrin also respond to 10-ng/ml dose of GDNF with ectopic buds (F′); the contralateral DMSO-treated duct does not (F). (G through I) p53 stabilization by treatment with 5μM (H) or 10 μM (I) Nutlin3a renders the duct refractory to 50 ng/ml GDNF, whereas DMSO-treated duct responds appropriately (G).

Figure 7.

(A) p53 knockdown by shRNA potentiates PI3K activation by GDNF in UB cells. UB cells were transfected with GFP, scrambled, or p53 shRNA plasmids for 72 h. Transfected cells were treated with GDNF+GFRα1 20 h before harvesting for Western blot analyses. Decrease in p53 levels is accompanied by increase in P-Akt, without an increase in total Akt. PTEN levels did not decrease with p53 knockdown. Fold differences in levels are normalized for total Akt. (B) Model of p53 function in WD is shown. p53 opposes pathways activated by growth stimuli, such as GDNF→cRet→PI3K. Antagonism may occur at more that one site along the pathway; p53 regulates expression of genes such as PTEN that antagonize PI3K function, or p21/Cip1 and FAK, inhibitors of proliferation and cell migration, respectively.^, p53 negatively regulates expression of PI3KCA, the catalytic subunit of PI3K.