wnt9b and wnt4 are required for new nephron formation and invasive cell behavior. (A) Wild-type adult kidney shows a robust induction of lhx1a-expressing new nephrons in response to gentamicin acute injury 7 days post-injection. (B,C) Homozygous mutation in wnt4 (B) or wnt9b (C) significantly reduces new nephron formation after acute gentamicin injury. (D,E) Lumen formation and basal cell protrusions (arrowhead in D; DT, distal tubule, dotted outline) seen in wild-type lhx1a+ new nephrons are not observed in wnt4 mutant new nephrons (E, arrowhead). (F) New nephrons in wnt9b mutants lack extensive lhx1a:eGFP fluorescence and do not show basal protrusions (arrowhead). Blue fluorescence indicates Hoechst-stained nuclei. (G,H) Quantification of lhx1a+ new nephron aggregates (by in situ hybridization) in wnt4 (G) and wnt9b (H) heterozygous and homozygous mutants reveals a significant reduction in nephrogenesis after injury. G: *P=0.0242, **P=0.0033; H: *P=0.027, **P=0.0061 (ordinary one-way ANOVA). Data are mean±s.d. (wnt4 +/+: 5.86±4.44, n=13, wnt4 +/−: 6.96±4.64, n=17; wnt4 −/−: 2.34±3.31, n=15; wnt9b +/+: 13.22±8.12, n=7; wnt9b +/−: 7.5±3.83, n=7; wnt9b −/−: 3.38±2.61, n=8). Scale bars: 0.2 mm in A-C; 10 µm in D; 5 µm in E,F.
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