p120ctn and Arvcf are broadly expressed during metanephric development. (A) Western blot of E17.5 kidney lysates in control (Ctl; ctnnd1^flox/flox) and mutant (Mut; ctnnd1^flox/flox; pax3-cre^tg/0) mice with anti-p120ctn mAb pp120 (2B12; anti-p120ctn C-terminus) shows two isoforms. Western blot with anti-p120ctn isoform 1 (6H11, N-terminus mAb) identifies the higher molecular weight band as isoform 1. The lower molecular weight band co-migrates with p120ctn from MDCK cells, which express only isoform 3 (Ohkubo and Ozawa, 2004). (B) Schematic of renal development. The ureteric bud (UB) is surrounded by condensed (CM) and uncondensed (UM) mesenchyme. CM differentiates to form pre-tubular aggregates (PA), which epithelialize to form renal vesicles (RV). These lengthen into comma-shaped bodies (CB) and subsequently s-shaped bodies (SB). The connecting tubule (CT) of the UB fuses with the SB to form a continuous lumen. (C-H) Localization of p120ctn in E17.5 control shows that p120ctn (green) is present in UM, CM and UB, and the CM derivative RV (C-E). Mutant kidneys lack p120ctn in UM and CM (F-H). NCAM (red) identifies CM, RV, CB and SB. (I,J) Expression of p120ctn in the CM derivatives CB (I) and SB (J). (K-M) Downregulation of p120ctn isoform 1 during mesenchymal-to-epithelial differentiation. (K,L) Localization of p120ctn isoform 1 (mAb 6H11) (green) and cytokeratin (red), a UB marker, at E17.5. Low isoform 1 levels are present in UB compared with CM. Note high isoform 1 in connecting tubules (CT). (M) There is minimal isoform 1 expression in proximal tubules (PT), identified with lotus tetragonolobus lectin (LTL, not shown). (N-P) Arvcf expression at E17.5. (N-O) Arvcf (green) is expressed in UB, RV (not shown), CB, SB and a developing glomerulus (asterisk, O). (P) Localization of Arvcf (green) and LTL (red) shows little Arvcf is present in mature PT. Tubules with low LTL (arrows) express Arvcf and might be immature PT or descending loops of Henle. (Q,R) Arvcf is present in a short loop of Henle, as identified by cadherin 6 (red) and Tamm Horsfall protein (THP, blue). (S,T) Arvcf is present in a distal tubule (DT), identified by the sodium chloride co-transporter (NCC), but not an adjacent PT (LTL, blue). White and red dotted lines delineate structures. Scale bars: 10 μm in I-M,Q-T; 20 μm in C-H,N-P.

p120ctn absence results in hypoplastic cystic kidneys. (A-C) Gross histology of P0 control (ctnnd1^flox/flox; A) and mutant (ctnnd1^flox/flox; pax3-cre^tg/0; B,C) mouse kidneys. (D,E) Quantification of differences between control and mutant in (D) renal size [kidney area (mm²) per mouse weight (g)] P<0.003 and (E) P0 animal weight P<0.10. Data are mean ± s.d. Statistical analysis by the Mann-Whitney U-test. (F,G) Hematoxylin and Eosin (H&E)-stained sections from P0 control (F) and mutant (G) kidneys. Panels in G show sections from a single kidney with a cyst indicated (arrow). (H-K) Higher magnification images of H&E-stained cortex in control (H) and mutant (I-K) kidneys. I shows a cystic tubule involving Bowman's capsule (arrow) and proximal tubule (PT). J and K illustrate the most common morphologies observed: cuboidal epithelium with luminal cells and areas of multilayering (arrowheads). Arrows in H,J highlight the nuclear crowding in J compared with H. Scale bars: 1 mm in A-C; 250 μm in F,G; 25 μm in H-K.

p120ctn absence leads to proximal tubule cysts. (A-D) Visualization with a proximal tubule marker, LTL, in control (A,C) and mutant (B,D) mouse kidneys shows cysts and widened tubules. (E,F) Absence of cysts in ureteric bud and collecting ducts, identified by calbindin. (G-J) Mild luminal widening in mutant distal tubules, identified using anti-NaCl cotransporter (NCC; G,H) and absence of dilation in mutant thick ascending limb tubules, identified using anti-Tamm Horsfall Protein (I,J, dashed lines). Scale bars: 500 μm in A,B,E,F; 200 μm in C,D; 10 μm in G-J.

Cadherins and catenins are downregulated dramatically in p120ctn mutant. (A) Western blots of cadherins and catenins from E17.5 control (Ctl) and mutant (Mut) mouse kidneys with GAPDH as a loading control. Active β-catenin is dephosphorylated Ser37/Thr41 (mAb 8E7) (van Noort et al., 2002). (B) Densitometry using ImageJ of cadherin and catenin levels normalized to GAPDH. n=5 for control (ctnnd1^flox/flox) and n=7 for mutant mice. Data are mean ± s.d. All decreased levels are statistically significant (Mann-Whitney U-test, P<0.005). (C-L) Localization of E-cadherin (red) and cadherin 6 (green) illustrates that cadherin 6 is present in control and mutant renal vesicles (D,H, blue dotted lines and arrows indicate renal vesicles). E-cadherin is in the distal s-shaped body, whereas cadherin 6 is in the mid-s-shaped body in control and mutant (C,H, white dotted lines delineate s-shaped bodies). Panel I shows two abnormal comma and s-shaped structures (arrowheads) in the mutant. Boxed area in D is shown at higher magnification in E-G. Boxed area in I is shown at higher magnification in J-L. (M,R) Localization of cadherin 11 (green), NCAM (blue) and cytokeratin (red, UB marker) shows loss of cadherin 11 from mesenchyme. (N-Q,S-V) Localization of E-cadherin (green, N,O,S,T), β-catenin (green, P,Q,U,V) and LTL (red, proximal tubule marker, O,T,Q,V) show reduced levels of E-cadherin and β-catenin in mutant proximal tubules. Scale bars: 25 μm in C,D,H,I; 10 μm in E-G,J-L,N-Q,S-V; 30 μm in M,R.

p120ctn absence leads to defects in glomerulogenesis. (A-H) The podocyte marker nephrin (green) in P0 control (A,B) and mutant (E,F) mouse kidneys shows smaller, less compact glomeruli in the mutant. Anti-PECAM (red, C,D,G,H) identifies endothelial cells. (I-N) R-cadherin (green) and nephrin (red) in maturing glomeruli. R-cadherin is reduced in mutant presumptive podocytes. (O-Q,S-U) Localization of slit-diaphragm marker podocin (green), PECAM (red) and nephrin (blue). (R,V,W) Localization of podocyte marker podocalyxin (Pcx, green), endothelial marker IB4 (blue) and laminin (red) at the capillary loop stage at E17.5. (X) Quantification of glomerular number and glomerular density with ImageJ. n=4 embryos from three separate litters for each genotype. Data are mean ± s.d. There is a significant reduction in glomerular count (P<0.03) but not density (P>0.50). Mann-Whitney U-test. Scale bars: 500 μm in A,E; 100 μm in B,F; 10 μm C,D,G,H,I-W.

p120 knockdown in 3D cyst culture results in enlarged cysts with reduced E-cadherin. (A,C) p120ctn and E-cadherin in p120ctn knockdown cysts. Panels show staining of phalloidin, ZO1 and gm130 as indicated. Merged images with Hoescht 33342 (blue, nuclei) show that p120 KD cysts contain intraluminal cells. (B) Western blot of p120ctn. Lanes 1 and 2: E17.5 kidney lysates from control and mutant. Lanes 3-5: MDCK cyst lysates from scramble and two p120 shRNA (KD1 and KD2). (D) Western blots of E-cadherin and cadherin 6 in control and p120ctn knockdown cysts. Percentages are E-cadherin and cadherin 6 levels normalized to GAPDH. (E) Quantification of percentage of cysts containing ≥1 luminal cell(s). Results are mean ± s.d. from three independent experiments with >300 cysts each (P<0.001 for scramble versus p120 KD1, and scramble versus p120 KD2). (F) Quantification of cyst area using DIC images analyzed in ImageJ. Results are mean ± s.d. of the upper quartile of cyst areas measured from three independent experiments done in triplicate with >300 cysts each (P<0.01 for scramble versus p120 KD1). (G) Localization of podocalyxin (Pcx, an apical membrane marker), β-catenin and Hoescht shows enlarged cysts and multilayered epithelium (arrow). (H,I) Densitometry of western blots of cyst lysates with phosphohistone-3 and active caspase-3 normalized to GAPDH. For phosphohistone-3, P<0.04 for scramble versus p120 KD1. For caspase-3, P<0.001 for scramble versus p120 KD2; P<0.09 scramble versus p120 KD1. (J) Cellular aggregation in scramble- and p120ctn-knockdown MDCK cells. Cells dissociated with EDTA were added to wells containing 1 mM CaCl₂ or 1 mM EGTA, and cellular aggregation assessed at times 0, 30 and 60 minutes. Percentage aggregation=(Na/N)×100 where Na=number of cells in aggregates ≥3 cells and N=total number of cells. Graph shows one of two similar experiments with three replicates for each time. Aggregation (mean ± s.d.) in p120ctn knockdown was reduced compared with control at 30 minutes (P<0.003) and 60 minutes (P<0.02). All statistical analysis was carried out using unpaired, two-tailed Student's t-test.

E-cadherin knockdown but not RhoA inhibition phenocopies loss of p120ctn in MDCK cysts. (A) Rho activity assays. Quantification of GTP-Rho pull-downs using GST Rhotekin-RBD. GTP-Rho levels are normalized to total Rho. (B) Quantification of the effect of ROCK inhibition (Y-27632, 10 μM) on cyst area using DIC images analyzed in ImageJ. Results are mean ± s.d. of the upper quartile of cyst areas. Cysts treated with Y-27632 are smaller than control (P<0.03). (C) Quantification of the effect of ROCK inhibitor (Y-27632, 10 μM) on the percentage of cysts with luminal cells (P<0.4 for scramble versus scramble +Y-27632). (D) Western blot of E-cadherin from MDCK lysates with scramble or two E-cadherin shRNA (KD1 and KD2). (E) Quantification of cyst area performed as described for panel B (P<0.001 for scramble versus E-cadherin KD1 or KD2). (F) Visualization of E-cadherin reduction in E-cadherin KD cysts. Panels show β-catenin and phalloidin as indicated. Merged images with Hoescht 33342 (blue, nuclei) show intraluminal cells in E-cadherin KD cysts. (G) Quantification of percentage of cysts containing >1 luminal cell (P<0.001 for scramble versus each KD). (H) Cellular aggregation in scramble, E-cadherin KD and p120 KD cells. Cells dissociated with EDTA were added to wells containing 1 mM CaCl₂ and cellular aggregation assessed at 0 and 30 minutes. Percentage aggregation was determined as described for Fig. 6J. Graph shows one of two similar experiments with four replicates for each time (P<0.15 for scramble versus E-cadherin KD1; P<0.03 for scramble versus E-cadherin KD2; P<0.001 for scramble versus p120 KD1, all at 30 minutes). All graphical results are mean ± s.d. and were analyzed with an unpaired two-tailed Student's t-test. Panels B,C,E and G are from three independent experiments done in triplicate with >300 cysts each.

Proposed model of p120ctn function in renal tubulogenesis. p120ctn maintains cadherin levels for appropriate migration and/or sorting of cells in renal vesicles into the distinct domains of comma- and s-shaped bodies. In the absence of p120ctn, cadherin levels are reduced, with a fraction of the vesicles failing to form normal s-shaped bodies. As the s-shaped body elongates into tubules, p120 regulates proliferation and migration.