Regulation of cochlear convergent extension by the vertebrate planar cell polarity pathway is dependent on p120-catenin

Abstract

The vertebrate planar cell polarity (PCP) pathway consists of conserved PCP and ciliary genes. During development, the PCP pathway regulates convergent extension (CE) and uniform orientation of sensory hair cells in the cochlea. It is not clear how these diverse morphogenetic processes are regulated by a common set of PCP genes. Here, we show that cellular contacts and geometry change drastically and that the dynamic expression of N-cadherin and E-cadherin demarcates sharp boundaries during cochlear extension. The conditional knockout of a component of the adherens junctions, p120-catenin, leads to the reduction of E-cadherin and N-cadherin and to characteristic cochlear CE defects but not misorientation of hair cells. The specific CE defects in p120-catenin mutants are in contrast to associated CE and hair cell misorientation defects observed in common PCP gene mutants. Moreover, the loss-of-function of a conserved PCP gene, Vangl2, alters the dynamic distribution of N-cadherin and E-cadherin in the cochlea and causes similar abnormalities in cellular morphology to those found in p120-catenin mutants. Conversely, we found that Pcdh15 interacts genetically with PCP genes to regulate the formation of polar hair bundles, but not CE defects in the cochlea. Together, these results indicate that the vertebrate PCP pathway regulates CE and hair cell polarity independently and that a p120-catenin-dependent mechanism regulates CE of the cochlea.

Fig. 1.

Remarkable cellular contact remodeling and cell geometry changes during cochlear extension. (A,B) Surface views of the mouse cochlear epithelia at E14.5 (A) and postnatal day (P0) (B). The E14.5 cochlea (A) was stained for F-actin (green), γ-tubulin (red) and acetylated α-tubulin (blue), whereas the P0 cochlea (B) was stained for F-actin (green). IHC, inner hair cells; OHC, outer hair cells; Iph, inner phallangeal cells; Ip, inner pillar cells; Op, outer pillar cells; Dc, Deiters’ cells; M and L, the center or medial side and the periphery or lateral side of the cochlear duct, respectively. Red lines and a red star outline a Dc (B). (C-G) Surface views of the cochlear duct at E14. Phalloidin (green) and Sox2 (blue) staining visualizes the outline of cells and the developing organ of Corti domain within the cochlear epithelium, respectively. (H-J) Surface views of the organ of Corti from E14.5 to E15.5. The samples were stained for F-actin. IHCs and the cellular rosettes are indicated by asterisks and circles, respectively. The distance between adjacent IHCs is indicated by white arrowheads. The white arrow marks the separation between IHCs and OHCs (J).

Fig. 2.

Dynamic expression of N-cadherin and E-cadherin demarcates sharp boundaries within the developing organ of Corti. (A-H) Surface views of mouse cochleae from E15 to E18.5. The samples were stained for F-actin (green) and E-cadherin (red). The white arrowheads mark the separation between IHCs and OHCs. The OHC and Hensen cell regions are indicated by brackets and an asterisk, respectively (H). (I-N) Surface views of the organ of Corti from E14 to E18.5. The samples were stained for F-actin (green) and N-cadherin (red). The arrowheads and asterisks mark the separation between IHCs and OHCs, and the nascent IHCs, respectively. The medial (M) to lateral (L) arrows indicate the direction of PCP in the cochlea.

Fig. 3.

Reduced levels of cadherins and normal hair cell polarity in p120^CKO/CKO mice. (A-F) Surface views of the organs of Corti from E18.5 (A-D) and E14.5 (E,F) embryos. Wild-type (A,C,E) and p120^CKO/CKO (B,D,F) samples were stained for F-actin (green) and p120-catenin (red) (A,B), F-actin (green) and E-cadherin (red) (C,D) or F-actin (green) and N-cadherin (red) (E,F). The boxes in A and B are the enlarged images of the organs of Corti from wild-type (A) and mutant (B) animals. The white arrowheads mark the inner pillar cell region that separates IHCs from OHCs. (G,H) Surface views of the organ of Corti from E18.5 wild-type (G) and p120^CKO/CKO (H) animals carrying the Vangl2-eGFP transgene (green). F-actin (red) outlines the cellar cortex. (I-K) Oriana graphs showing the distribution of hair cell orientation from wild-type (I) and mutant (J) animals. The orientation of hair cells was determined (K) by measuring the angle formed between the medial-to-lateral axis of the cochlea and the line bisecting the stereociliary bundle from the center of the hair cell to the vertex of the hair bundle.

Fig. 4.

p120-catenin conditional inactivation leads to the formation of a shorter and wider organ of Corti. (A-T) Cochleae from E18.5 (A-L) and E14.5 (M-T) control (A,D,G,J,M,O,Q,S), p120^CKO/CKO (B,E,H,K) or p120^CKO/CKO;Vangl2^Lp/+ (C,F,I,L,N,P,R,T) littermates were analyzed for their width by staining for F-actin (A-I, green) to visualize the rows of hair cells, and for their relative length (J-L,S,T) by measuring the total length of the cochlear ducts using ImageJ. Sox2 staining (M-R, blue) visualizes the width of the developing organ of Corti at E14.5. The arrowheads (A-I) and the asterisks (G-I) indicate the separation between IHCs and OHCs, and between the nascent neighboring IHCs, respectively.

Fig. 5.

The conditional inactivation of p120-catenin affects cellular morphology during CE. (A-J) The cellular outlines (A-D,F-I, white lines) in the developing organ of Corti in control (A-D) and p120^CKO/CKO (F-I) E14.5 animals were extracted from F-actin-stained cochlear whole-mount images, and the cellular long axes were calculated and plotted. The distribution of the orientation of the long axis in the control (E) and p120^CKO/CKO (J) is plotted using Oriana3 program. The colored lines indicate the angle that forms between the long axis of each cell and the longitudinal axis of the cochlear duct. (K) Quantification of tri-cellular and 4⁺-cellular vertices. A total of ∼3200 vertices were analyzed for each genotype. The percentage of cellular vertices formed by 3, 4 or 4⁺ cells were counted in control (blue), p120^CKO/CKO (red) and Vangl2^Lp/Lp (green) animals at E14.5. The brackets and the matching asterisks indicate that the Welch’s t-test P-value is less than 0.05 between the two conditions. Error bars represent s.d.

Fig. 6.

Mutations in PCP gene Vangl2 alter the expression of N-cadherin and E-cadherin in the cochlea. (A-D) Surface views of the cochleae from wild-type (A,B) and Vangl2^Lp/Lp (C,D) animals at E14.5. Samples were stained for F-actin (green) and N-cadherin (red). Arrowheads mark the separation between IHCs and OHCs. The developing organ of Corti is identified by cellular morphology and cortical actin enrichment in the nascent hair cells, and is outlined between two dotted lines. (E-H) Surface views of the organs of Corti from wild-type (E,F) and Vangl2^Lp/Lp (G,H) animals at E18.5. The samples were stained for F-actin (green) and E-cadherin (red). Arrowheads and ‘H’ mark the separation between IHCs and OHCs, and the Hensen cell region, respectively. (I,J) Western blot analysis (I) of E-cadherin proteins in the Vangl2^Lp/Lp mutant and control cochleae, which is quantified and plotted (J). The densities of the Vangl2 protein bands were normalized with housekeeping proteins HSP-90 or α-tubulin (not shown) (J). Error bars represent s.d.