Fig. 7.

Fig. 7.

frizzled9b-dependent apical constriction guides orthogonal interconnection of tubules and functional fluid filtration. (A) New nephron lumens are narrow and composed of apically constricted cells with small apical surfaces (arrowhead). DT, distal tubule (dotted outline). (B) fzd9b mutants show distended lumens but do not show apical constriction and they fail to make orthogonal interconnections (arrowhead). (C) Apical constriction is disrupted in wnt9b^−/− mutants, phenocopying fzd9b mutants. (D) Apical constriction requires Rho kinase signaling. ROCK inhibitor Y-27632 treatment phenocopies the fzd9b mutant phenotype. (E) Quantification of apical surface area in fzd9b and wnt9b mutants: ROCK/Y-27632 and Rac1/EHT1826 inhibited new nephrons. Data are mean±s.d. (control: 0.5±0.3, n=105; fzd9b−/−: 3.32±1.45, n=37; wnt9b−/−: 3.03±1.26, n=58; Y-27632: 1.80±1.77, n=104; EHT1826: 0.56±0.32, n=38). (F,G) fzd9b mutant new nephron cells fail to undergo cell shape change from apical to basal constriction (see Fig. 1) and instead maintain a primarily rectangular shape with roughly equivalent apical (a) and basal (b) surface areas. (H,I) Compared to wild-type tubule interconnections (H, arrowhead), fzd9b mutant interconnections (I) are delayed and narrow (arrowhead in I). (J) In wild-type kidneys at 14 dpi, intraperitoneally injected 10 K fluorescent dextran (blue) marks both proliferating EdU⁺ (magenta) new nephrons (asterisk) as well as non-proliferating pre-existing nephrons (hashtag). (K) In fzd9b mutants, pre-existing nephrons take up injected dextran (hashtag); however, EdU⁺ new nephrons show reduced frequency of dextran uptake (asterisk). White fluorescence indicates Hoechst-stained nuclei. Scale bars: 5 µm. Arrows indicate the direction of fusion with the distal tubule (DT). ****P<0.0001; ns, not significant (ordinary one-way ANOVA).