Notch2 activation in the embryonic kidney depletes nephron progenitors

Abstract

Successive activation of Wnt4 and Notch2 generates nephrons from the metanephric mesenchyme. Mesenchymal-to-epithelial transition requires Wnt4, and normal development of the proximal nephron (epithelia of glomeruli and proximal tubules) requires Notch2. It is unknown, however, whether Notch2 dictates the fate of the proximal nephron directly. Here, we generated a mutant strain of mice with activated Notch2 in Six2-containing nephron progenitor cells of the metanephric mesenchyme. Notch2 activation did not skew the cell fate toward the proximal nephron but resulted in severe kidney dysgenesis and depletion of Six2-positive progenitors. We observed ectopic expression of Wnt4 and premature tubule formation, similar to the phenotype of Six2-deficient mice. Activation of Notch2 in the progenitor cells suppressed Pax2, an upstream regulator of Six2, possibly through Hesr genes. Taken together, these data suggest that a positive feedback loop exists between Notch2 and Wnt4, and that Notch2 stabilizes, rather than dictates, nephron fate by shutting down the maintenance of undifferentiated progenitor cells, thereby depleting this population.

Figure 1.

R26Notch2 mice are generated. (A) Targeting strategy of Flag-Notch2 in the Rosa26 locus. A Flag-Notch2 fragment is inserted into the pBigT vector containing an adenovirus splice acceptor sequence followed by a PGK-neo cassette and a tpA stop sequence flanked by two loxP sites. The resultant plasmid is inserted into pRosa26pl239pA (a modified pRosa26-1 vector) and subsequently integrated into the Rosa26 locus in ES cells by homologous recombination. (B) Targeted ES clones are confirmed by Southern blot analysis. Genomic DNA is digested with EcoRI and hybridized with probe A. (C) PCR amplification of mouse tail DNA.

Figure 2.

Overexpression of Notch2 in embryonic nephron progenitors leads to severe kidney dysgenesis. (A) Kidneys in a newborn wild-type mouse (P0). k, kidney; t, testis; b, bladder; a, adrenal gland. (B) Kidney size is reduced in a newborn Six2Cre/R26Notch2 mouse (P0). (C and D) HE staining of newborn kidneys. Severe dysgenesis is observed in the Six2Cre/R26Notch2 kidney. Arrowheads show glomerular cysts. (E and F) Immunostaining for Lotus tetragonobulus lectin (LTL; green; proximal tubule marker) and cytokeratin 8 (CK8; red; ureteric bud marker). The upper right panels show higher-magnification images of the dotted square regions. They are ×2 of the original panels. Proximal tubules are not overproduced under Notch2 activation. Arrows, dilated proximal tubules. Scale bar = 100 μm.

Figure 3.

Notch2 overexpression causes nephron progenitor depletion. (A and B) HE staining at E14.5 during gestation. The mutant kidney is reduced in size and shows a thin cortical nephrogenic zone and poor nephron development, compared with the control kidney (R26Notch2). (C and D) Immunostaining for Pax2 (green) and cytokeratin 8 (CK8; red). (E and F) Higher-magnification images of the dotted square regions in panels C and D. They are ×6 of the panels C and D. Pax2-positive/cytokeratin-negative cells (arrowhead) that surround the ureteric bud-derived epithelia (arrow) are missing in the mutant cortex. Ureteric branching is also impaired. (G and H) Six2 immunostaining is significantly reduced in the mutant cortex. (I and J) Immunostaining of Sall1. Mesenchymal expression of Sall1 is reduced in the mutant kidney, whereas its stromal expression is retained (arrowhead). (K and L) In situ hybridization of Foxd1, a stromal marker. Foxd1 continues to be expressed in the mutant kidney (arrowhead). Scale bar = 100 μm.

Figure 4.

Notch2 overexpression leads to ectopic Wnt4 expression and premature tubule formation. (A) Immunostaining of Six2. Six2 is expressed dorsally to the ureteric buds in the control kidney (R26Notch2) at E12.5. (B) Six2 immunostaining is reduced in the Six2Cre/R26Notch2 kidney. (C) In situ hybridization of Wnt4. Wnt4 is expressed ventrally against the ureteric buds in the control kidney at E12.5. (D) Wnt4 expression is not confined to the ventral side of the ureteric buds in the Six2Cre/R26Notch2 kidney. (E) Immunostaining for laminin (Lam; green; epithelial marker) and cytokeratin 8 (CK8; red; ureteric bud marker). Mesenchyme-derived tubules (arrowhead) are formed ventrally to the cortical ureteric bud-derived epithelia (arrow) in the control kidney at E14.5. (F) Ectopic tubules (arrowhead) are observed dorsal to the poorly branched ureteric bud-derived epithelia (arrow). Scale bar = 100 μm. (G and H) Higher-magnification images of the dotted square regions in panels E and F. They are ×3 of the panels E and F (I and J) In situ hybridization of the intracellular domain of Notch2. Note that all of the mesenchyme-derived epithelia in the mutants express Notch2 (J). The upper right panel in J shows a higher-magnification image of glomerular podocytes. I and J are taken at ×100, and the inset is taken at ×400.

Figure 5.

Expression of Pax2 is impaired under Notch2 activation. (A) HE staining of a control kidney (R26Notch2) at E11.5. The ureteric bud invades into the mesenchyme. (B) HE staining of a Six2Cre/R26Notch2 kidney at E11.5. The mutant kidney is indistinguishable from the control kidney. (C and D) Six2 staining is reduced in the mutant mesenchyme. (E and F) In situ hybridization of Wnt4. The Wnt4 expression domain is expanded dorsally in the mutant kidney. (G and H) Pax2 staining in the mesenchyme is reduced in the mutant kidney, whereas its expression in the ureteric bud is unaffected. (I and J) In situ hybridization of Hoxd11. Hoxd11 expression is unaffected in the mutant kidney. (K and L) In situ hybridization of Gdnf. Gdnf expression is reduced in the mutant kidney. (M and N) Sall1 staining is minimally affected in the mutant kidney. (O and P) Wt1 staining is minimally affected in the mutant kidney. Scale bar = 100 μm. (Q) Quantitative RT-PCR of the E11.5 kidney. The columns represent means ± SD (n = 3). *P < 0.05, R26Notch2 mice versus Six2Cre/R26Notch2 mice.

Figure 6.

A Notch2-Wnt4-positive feedback loop stabilizes the differentiated state. (A and B) In situ hybridization of Hesr1. Hesr1 expression is significantly increased in the Six2Cre/R26Notch2 kidney at E12.5. (C and D) In situ hybridization of Hesr3. Hesr3 expression is significantly increased in the Six2Cre/R26Notch2 kidney at E12.5. Scale bar = 100 μm. (E) Quantitative RT-PCR of Hesr1 and Hesr3 in the E11.5 kidney. The columns represent means ± SD (n = 3). *P < 0.05, R26Notch2 mice versus Six2Cre/R26Notch2 mice. (F) RT-PCR of the exogenous intracellular domain of Notch2 in E11.5 kidneys from wild-type mice (n = 2) and Six2Cre/R26Notch2 mice (n = 2). (G) Proposed functions of Notch2 for the nephron progenitors. See the Discussion section for details.