Disruption of Core Planar Cell Polarity Signaling Regulates Renal Tubule Morphogenesis but Is Not Cystogenic

Abstract

Oriented cell division (OCD) and convergent extension (CE) shape developing renal tubules, and their disruption has been associated with polycystic kidney disease (PKD) genes, the majority of which encode proteins that localize to primary cilia. Core planar cell polarity (PCP) signaling controls OCD and CE in other contexts, leading to the hypothesis that disruption of PCP signaling interferes with CE and/or OCD to produce PKD. Nonetheless, the contribution of PCP to tubulogenesis and cystogenesis is uncertain, and two major questions remain unanswered. Specifically, the inference that mutation of PKD genes interferes with PCP signaling is untested, and the importance of PCP signaling for cystogenic PKD phenotypes has not been examined. We show that, during proliferative stages, PCP signaling polarizes renal tubules to control OCD. However, we find that, contrary to the prevailing model, PKD mutations do not disrupt PCP signaling but instead act independently and in parallel with PCP signaling to affect OCD. Indeed, PCP signaling that is normally downregulated once development is completed is retained in cystic adult kidneys. Disrupting PCP signaling results in inaccurate control of tubule diameter, a tightly regulated parameter with important physiological ramifications. However, we show that disruption of PCP signaling is not cystogenic. Our results suggest that regulating tubule diameter is a key function of PCP signaling but that loss of this control does not induce cysts.

Keywords: oriented cell division; planar cell polarity; polycystic kidney disease; renal tubule diameter.

Figure 1. Phenotypes of WT, Vangl2^Lp/Lp and Fz3^−/−; Fz6^−/− mutant kidneys at E18.5

(A–B) Control and Vangl2^Lp/Lp kidneys. (C,C′,D,D′) H&E histology of control and Vangl2^Lp/Lp kidneys. (E) Fz3^−/−; Fz6^−/− kidneys. (E–E′) H&E histology of Fz3^−/−; Fz6^−/− kidneys. (F–G) Immunofluorescence images of control and Vangl2^Lp/Lp kidneys stained for DAPI (blue; nuclei), LTL (green; proximal tubule) and AQP2 (red; collecting duct). (H–I) Median tubule diameters for WT control, Vangl2^Lp/Lp and Fz3^−/−; Fz6^−/− collecting ducts (H) and proximal tubules (I). Images illustrate measurement method (see Experimental Methods). Asterisks indicate significant differences: (H) 22.7 μm and 20.1 μm respectively versus 16.6 for WT control, * = p<0.0001 and (I) 28.0 and 25.4 respectively versus 22.4 for WT control, * = p<0.0001 by Mann-Whitney U test). (J–K) Distribution plots of data from H and I. Scale bars: A,B,E, 1 mm; C,C′,D,D′,E′,E″ 100 μm; F,G, 200 μm; H,I 25 μm. See also Figure S1.

Figure 2. Core PCP proteins localize asymmetrically in proximal tubule and collecting duct

(A) Control kidney stained for phalloidin (blue; actin), LTL (green; proximal tubule) and DBA (red; collecting duct). (B,B′,B″) Collecting duct stained for Vangl1 (green) and E-Cadherin (blue) showing asymmetric proximal localization of Vangl1 relative to E-Cadherin (arrowheads). (C–C″) Collecting duct stained for Vangl1 (green) and Fz6 (red) showing asymmetric proximal localization of Vangl1 and distal localization of Fz6 (arrowheads). (D–D″) Collecting duct showing distal localization of Fz3 (red) relative to β-catenin (blue). (E–E″) Proximal tubule (LTL; red) showing proximal localization of Vangl1 (green) relative to E-Cadherin (blue) (arrowheads). Asymmetry was quantified as an intensity ratio (see STAR methods) and indicated in the white box in associated figure panels and graphically in Figure S3C. Scale bars: A, 500 μm; B, 25 μm; B″,C″,D″,E″, 5 μm. See also Figure S2.

Figure 3. Asymmetric localization of core PCP protein localization is lost in PCP mutants

(A–C) E18.5 Vangl2^Lp/Lp collecting ducts (DBA, red) stained for (A) Vangl1 (green) and E-Cadherin (blue), (B) Fz6 (red) and E-Cadherin (blue) or (C) Fz3 (red) and E-Cadherin (blue) showing similar proximal and distal localization of Vangl1, Fz6 and Fz3 (arrowheads). (D) E18.5 Fz3^−/−; Fz6^−/− collecting duct stained for Vangl1 (green) and E-Cadherin (arrowheads). (E) P1 Vangl1,2DKO, Ksp-Cre collecting duct stained for Fz6 (red) and E-Cadherin (blue). Scale bars: 5 μm. See also Figure S3.

Figure 4. Phenotype of Vangl1,2 DKO kidneys at P1 and 16 weeks

(A–B) Longitudinal sections of WT control and Vangl1,2 DKO collecting ducts stained for AQP2 (marking lumens) showing more irregular diameter of the Vangl1,2 DKO ducts along their lengths. Samples are 30 μm thickness. See also Movies S1 and S2. (C–C″) Gross, and H&E sections of WT control and (D–D″) Vangl1,2 DKO mutant kidneys showing absence of cysts. (E–F) Distribution plots of collecting duct diameters of WT and Vangl1,2 DKO at P1 and 16 weeks, measured with standard fixation, showing broader distribution in Vangl1,2 DKO compared to WT. (G–H) Distribution plots of collecting duct diameters showing reduction in overall diameters from E18.5 to 16 weeks in both WT and mutant ducts. Scale bars: A,B,C″,C‴,D″,D‴, 100 μm; C,D, 5 mm; C′,D′, 1 mm. See also Figure S4 and Table S1.

Figure 5. CE and OCD in WT and Vangl1,2 DKO collecting ducts

(A–B) Cell number ± SEM per tubule cross-section at the indicated stages in WT control, Vangl2^Lp/Lp and Fz3^−/−; Fz6^−/−collecting ducts and proximal tubules, showing a slowed decrease in mutants compared to WT. Box and whisker format shows the minimum, lower quartile, median, upper quartile and maximum values; + indicates mean. * = p<0.0001 by Mann-Whitney U test. (C–D) Orientation of cell divisions in WT control and in Vl1,2DKO collecting ducts assayed using the histone H3 immunolabeling method[10], quantified at P1. Difference between WT control and Vl1,2DKO is significant at p<0.0001 by Mann-Whitney U test. Representative images showing cell divisions labeled for AQP2 (green; collecting duct), histone H3 (red; dividing nuclei) and DAPI (blue; all nuclei). See also Movies S1, S2 and Figure S5. Scale bars: 10 μm.

Figure 6. PCP in cystic kidneys

(A) Median collecting duct diameters at E18.5 for WT control, Vangl2^Lp/Lp, Fz3^−/−; Fz6^−/− and KspCre;Kif3a^cko/cko. Data for WT control, Vangl2^Lp/Lp, Fz3^−/−; Fz6^−/− are from figure 1H. For KspCre;Kif3a^cko/cko, median = 20.1 μM and * = p<0.0001 compared to wildtype by Mann-Whitney U test. (B) Distribution plots of the measurements from (A). (C) Circumferential cell number for E18.5 collecting ducts for WT control, Vangl2^Lp/Lp, Fz3^−/−; Fz6^−/− and KspCre;Kif3a^cko/cko. Displayed in box and whisker format showing the minimum, lower quartile, median, upper quartile and maximum values; + indicates mean. * = p<0.0001 by Mann-Whitney U test. Data for WT control, Vangl2^Lp/Lp, Fz3^−/−; Fz6^−/− are from figure 5A. (D–D″) E18.5 collecting duct from KspCre;Kif3a^cko/cko precystic kidneys showing asymmetrically localized Vangl1 (green); E-Cadherin (blue). (E–E″) P1 Pkhd1^del4/del4 collecting duct showing asymmetrically localized Vangl1 (green) and Fz6 (red). Scale bars: 5 μm. See also Figure S6.

Figure 7. PCP is active in Kif3a mutant cystic kidneys

(A–A′) H&E stained P30 control (NoCre; Kif3a^cko/cko) and (B–B′) KspCre; Kif3a^cko/cko kidneys showing a strongly cystic phenotype. (C–C‴) Control P30 (NoCre; Kif3a^cko/cko) collecting duct stained for Vangl1 (green), E-Cadherin (blue) and acetylated-α-tubulin (red; cilia). (D–D‴) KspCre; Kif3a^cko/cko cysts stained for Vangl1 (green), E-Cadherin (blue) and cilia acetylated-α-tubulin; red). Vangl1 signal is strong in cysts (no cilia) but not in the unaffected tubule (cilia present). (E–E″) High magnification images from 3D reconstruction (see Movie S3) showing asymmetric localization of Vangl1 (green) relative to E-Cadherin (blue) in cyst lining cells. Scale bars: A,A′,B,B′, 200 μm; C‴,D″,E″, 5 μm. See also Figure S7.