N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis

Abstract

Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development.

Keywords: Lens; Morphogenesis; N-cadherin; Tissue development.

Figure 1. Expression of cadherin junctional proteins and F-actin in the developing lens

Cryosections of E13.5 (A,D,G,J), E14.5 (B,E,H,K), and E16.5 (C,F,I,L) eyes were labeled for F-actin (A,B,C), β-catenin (D,E,F), E-cadherin (G,H,I) or N-cadherin (J,K,L). (A–C) F-actin localized to cell-cell borders and along the epithelial fiber interface (EFI) where epithelial and fiber cell apical tips interact (A, arrowhead). (D–F) β-catenin was localized to cell-cell borders of lens epithelial and fiber cells, and in a punctate pattern along the EFI that is shown as a higher magnification of the boxed areas in insets (arrowheads). (G,H,I) E-cadherin was expressed only in the lens epithelium, including distinct puncta just adjacent to the EFI, shown at a higher magnification of the boxed areas in the insets (arrowheads). (J,K,L) N-cadherin was localized along cell-cell borders of lens epithelial and fiber cells and in a punctate pattern along the EFI shown at a higher magnification of the boxed areas in the insets (arrowheads). (Mag bar=20μm; n=5)

Figure 2. Loss of N-cadherin expression in N-cadcKO lenses blocks migration/elongation of the apical tips of lens fiber cells along the EFI resulting in disrupted lens morphogenesis

Cryosections of E13.5 wildtype (A) and N-cadcKO (B) eyes labeled for N-cadherin show loss of N-cadherin in N-cadcKO lenses by E13.5. (C) Immunoblot for N-cadherin in E16.5 wildtype and N-cadcKO lenses confirms the loss of N-cadherin protein in the N-cadcKO lens, with GAPDH as a loading control. (D) Quantification of immunoblot analysis shows highly significant loss of N-cadherin in knockout lenses, data presented as a ratio to GAPDH. Cryosections of wildtype (E–H) and N-cadcKO (I–L) eyes were labeled for F-actin at E13.5 (E,I), E14.5 (F,J), E16.5 (G,K) and E18.5 (H,L). By E13.5 primary fiber cells of wildtype lenses have elongated coordinately in a straight path from the posterior of the lens to the anterior epithelium (E). A small number of secondary fiber cells have begun to form from the equatorial epithelium, and have migrated anteriorly along the EFI towards the anterior of the lens (E, arrowhead). In N-cadcKO lenses at E13.5 the primary fiber cells have elongated normally, their apical tips interacting with the overlying anterior epithelium (I, arrow), but the apical tips of secondary fibers have failed to migrate along the EFI and remain clustered adjacent to the lens fulcrum (I, arrowhead). By E14.5 there is increased clustering of secondary fiber cell apical tips at the fulcrum of N-cadcKO lenses (J, arrowhead) due to failure to migrate along the EFI. Primary fibers in these lenses begin to exhibit signs of disorganization and separation from the EFI (J, arrow). Later in development at E16.5, the migration failure of secondary fibers cells differentiating in the absence of N-cadherin causes major morphogenetic defects (K). Secondary fiber cells accumulate, contiguous with the equatorial epithelium but failing to elongate beyond the equatorial plane (K, closed arrowhead). Note that the elongation defective differentiating secondary fiber cells maintain lateral cohesion in the absence of N-cadherin (K, arrow). At this time of development, primary fiber cells become extensively disorganized (K, open arrowhead). The phenotype at E18.5 is similar to E16.5 with continued addition of new secondary fiber cells at the equatorial plane and increased disorganization of lens primary fibers (L). Measurements were made from confocal microscopy images acquired of F-actin-labeled cryosections from E13.5, E14.5, E16.5, and E18.5 wildtype (WT) and N-cadcKO eyes of lens width (M), height (N), and area (O). By E18.5 there was significant loss of height leading to decreased area in N-cadcKO lenses. (P) Shortened fiber cells that failed to elongate along the EFI in N-cadcKO lenses at E16.5 had a corresponding increase in width. (Mag bar=20μm; A–D n=12; E–L n=12; p<0.05*;p<0.01**; p<0.001***).

Figure 3. Absence of N-cadherin alters lens fiber cell morphology and membrane distribution

(A–F) Cryosections at E16.5 from wildtype (A–C) and N-cadcKO (D–F) eyes were labeled with F-actin (B,C,E,F) and WGA (A,C,D,F) to examine lens morphology. N-cadcKO lenses showed a differential labeling of WGA between primary fiber cells and secondary fiber cells (D,F), highlighting the distinction between these compartments in the N-cadcKO lens and the dysmorphogenesis of differentiating secondary fiber cells seen also with F-actin labeling (E,F). (Mag bar=20μm; n=3)

Figure 4. Absence of N-cadherin alters lens fiber cell nuclear distribution but does not result in cell death

Cryosections at E16.5 (A,B,E,F) and E18.5 (C,D,G,H) from wildtype (A,C,E,G) and N-cadcKO (B,D,F,H) eyes were labeled by TUNEL assay (red) and counterstained for nuclei (blue). Boxes in A–D represent areas shown in E–H, respectively. Neither wildtype nor N-cadcKO lenses demonstrated TUNEL positivity at E16.5 (A,B,E,F). By E18.5 wildtype lenses (C,G) had formed a large Organelle Free Zone (OFZ) in the center of the lens, as is typical of this stage of development. N-cadcKO lenses (D,H) had formed a smaller OFZ at E18.5, which was flanked on either side by a few layers of fiber cells with TUNEL-positive nuclei, likely representing the ongoing expansion of the OFZ in the mutant lenses. Cells in the primary fiber zone began to exhibit TUNEL-positive labeling at E18.5 (D) (mag bar in low power images= 200μm; in high power images=20μm; n=8)

Figure 5. βB–crystallin is expressed in N-cadcKO lenses, including newly differentiating fiber cells that aberrantly express E-cadherin

Cryosections of E16.5 wild-type (A,B,C,I,J) and N-cadcKO (D,E,F,K,L) eyes were labeled for (A,C,D,F,I–L) βB–crystallin, (I–L) E-cadherin, and (B,C,E,F,I–L) F-actin. βB–crystallin expression was maintained in lens fiber cells in the absence of N-cadcKO (D,F). In the N-cadcKO lens E-cadherin expression fails to be turned off in new secondary fiber cells located near the lens fulcrum whose differentiation is confirmed by their expression of βB–crystallin (K,L). βB–crystallin expression was also examined by immunoblot analysis of proteins from E16.5 wild-type and N-cadcKO lenses, which confirmed that there was no significant change in βB–crystallin protein level (G,H). (mag bar=20μm; n=3; p<0.05*;p<0.01**; p<0.001***)

Figure 6. E-cadherin/β-catenin junctions maintained but apical ZO-1 junctions more extensive, linking apical domains of migration-defective fiber cells in the N-cadcKO lens

Cryosections of wild-type (A–C,H–J, O–Q) or N-cadcKO (D–G,K–N,R–U) eyes were labeled for E-cadherin (A,C,D,F,G), β-catenin(H,J,K,M,N), ZO-1(O,Q,R,T,U), F-actin (B,C,E–G,I,J,L–N,P,Q,S–U), and nuclei (C,F,Q,T) at E16.5. E-cadherin is restricted to the epithelium of normal lenses (A,C). In N-cadcKO lenses E-cadherin is maintained in lens epithelial cells (arrow) and expressed aberrantly in the apical tips of migration-defective, differentiating cortical fiber cells close to the lens equator (D,G, arrowhead; G is a high magnification of the boxed area in F). β-catenin, which normally localizes to all cell-cell borders (H,J), is retained only by cells expressing E-cadherin junctions in N-cadcKO lenses (K,M), including the apical tips of the migration-defective cortical fiber cells near the lens fulcrum (K,N, closed arrowhead; N is a high magnification of the boxed area in M. No β-catenin labeling was detected in the remainder of the lens fiber cell mass (K, open arrowhead). ZO-1 is most highly expressed at the EFI of wildtype lenses (O,Q, arrows). In the N-cadcKO lens, ZO-1 junctions were maintained along the apical domains of lens epithelial cells and extended across the apical domains of the secondary fiber cell population that fails to elongate and migrate along the EFI (R,T,U, denoted by arrows in R and U; U is a high magnification image of the area boxed in T), co-localized with F-actin (T,U). Loss of ZO-1 correlates with discontinuity of the fiber cells’ apical domains (R,S,T, arrowhead). High magnification images of E-cadherin (G, arrowhead) and ZO-1 (U, arrow) co-labeled for F-actin show ZO-1 is localized apical to E-cadherin and β-catenin and coincident with apical F-actin (denoted by arrows in G, N and U). (Mag bar=20μm; n=5)

Figure 7. Connexin-50 and Aquaporin-0 are affected by loss of N-cadherin

Cryosections of E16.5 wild-type (A–C,I–K) and N-cadcKO (D–F, L–N) eyes were labeled for Connexin-50 (Cx50) (A,C,D,F), Aquaporin-0 (Aqp0), (I,K,L,N),F-actin (B,C,E,F,J,K,M,N), nuclei(C,F,K,N). Immunolocalization for Cx50 in N-cadcKO lenses showed that it was elevated at the apical tips of the newly differentiating secondary fiber cells adjacent to the lens fulcrum (D, arrowhead, and F), and had an stippled appearance along these cells lateral domains (D, arrow). Immunoblot analysis (G,H) at E16.5 showed decrease expression of Cx50 protein in the N-cadcKO lens. Immunolocalization of Aqp0 is highly up-regulated at fiber cell interfaces in N-cadcKO lenses (L,N) compared to wildtype lenses at the same settings (I,K). A higher intensity image of Aquaporin-0 in wildtype E16.5 lenses is depicted to demonstrate distinct localization of Aqp0 at cell-cell interfaces of normal fiber cells (I, inset). However, immunoblots for Aqp0 in E16.5 lenses show no significant change in Aqp0 protein expression in the absence of N-cadherin, GAPDH included as control (O,P). (Mag bar=20μm; n=6; p<0.05*;p<0.01**; p<0.001***)

Figure 8. Loss of N-cadherin induces EphA2

Cryosections of E16.5 wild-type (A–C) or N-cadcKO (D–F) eyes were labeled for EphA2 (A,C,D,F), F-actin (B,C,E,F), and nuclei (C,F), denoted as N. There is considerable up-regulation of EphA2 at cell-cell borders throughout the N-cadcKO lens (D,F). E16.5 wildtype and N-cadcKO lenses were also immunoblotted for EphA2, with GAPDH as loading control (G,H) demonstrating significantly higher protein levels of EphA2 in N-cadcKO lenses (H). (Mag bar=20μm; n=4; p<0.05*;p<0.01**; p<0.001***)

Figure 9. Increased expression of myosin light chain kinase (MLCK) and activation of myosin II in N-cadcKO lenses

Cryosections of wild-type (A–D,J–K) or N-cadcKO (E–H,L–M) eyes at E16.5 (A–H) and E18.5 (J–M) were labeled for myosin-II (A,E), phospho-myosin (B,F,J,L), myosin light chain kinase (MLCK) (D,H), F-actin (C,G,K,M). E16.5 lenses were immunoblotted for myosin-II and dual phospho-myosin, with GAPDH as control (O), and quantified (P). Myosin II protein levels were increased in N-cadcKO lenses (O,P), but myosin II localization was unchanged (E). In contrast, increased activation of myosin in N-cadcKO lens fiber cells was shown at both E16.5 and E18.5 by immunofluorescence (F,L, arrows) and immunoblotting (O,P) and is correlated with induction of MLCK (H). Histogram analysis of immunofluorescence intensity in epithelium and fiber cells at E16.5 (I) and E18.5 (N) demonstrated that the increased intensity of phospho-myosin immunolocalization was fiber-cell specific and significant. (Mag bar=20μm; n=5 for immunolocalization; n=4 for immunoblot analysis; p<0.05*;p<0.01**; p<0.001***)

Figure 10. Rac 1 activity is lost in newly differentiating secondary fiber cells in the absence of N-cadherin

Cryosections of E16.5 wild-type (A,C) or N-cadcKO (B,D) eyes were labeled for total Rac1 (A,B) or active Rac1 (Rac-GTP; C,D). Rac1 localizes throughout the wild-type embryonic lens, highest in newly differentiating secondary fiber cells (A, arrow), and along the cells’ basal surfaces (A, arrowhead). In the N-cadcKO lens Rac1 protein is still expressed only in lens epithelial cells and newly differentiating secondary fiber cells (B, arrowhead). Rac is activated throughout the normal lens (C), and is highest along the cells’ basal surfaces (C, arrowhead). Rac activity is suppressed throughout the N-cadcKO lens (D), including the elongation-defective secondary fiber cells just past the lens fulcrum that retain expression of Rac1, but maintained along the cells’ basal surfaces that are rich in integrin junctions (D, arrow). These results were confirmed by immunoblotting for both total and active Rac1 levels (E), which demonstrated that both total and active Rac1 are significantly decreased in N-cadcKO lenses (F). (Mag bar=20μm; n=5; p<0.05*;p<0.01**; p<0.001***)