Balanced Rac1 and RhoA activities regulate cell shape and drive invagination morphogenesis in epithelia

Abstract

Epithelial bending is a central feature of morphogenesis in animals. Here we show that mutual antagonism by the small Rho GTPases Rac1 and RhoA determines cell shape, tissue curvature, and invagination activity in the model epithelium of the developing mouse lens. The epithelial cells of the invaginating lens placode normally elongate and change from a cylindrical to an apically constricted, conical shape. RhoA mutant lens placode cells are both longer and less apically constricted than control cells, thereby reducing epithelial curvature and invagination. By contrast, Rac1 mutant lens placode cells are shorter and more apically restricted than controls, resulting in increased epithelial curvature and precocious lens vesicle closure. Quantification of RhoA- and Rac1-dependent pathway markers over the apical-basal axis of lens pit cells showed that in RhoA mutant epithelial cells there was a Rac1 pathway gain of function and vice versa. These findings suggest that mutual antagonism produces balanced activities of RhoA-generated apical constriction and Rac1-dependent cell elongation that controls cell shape and thus curvature of the invaginating epithelium. The ubiquity of the Rho family GTPases suggests that these mechanisms are likely to apply generally where epithelial morphogenesis occurs.

Fig. 1.

Rac1 and RhoA mutations have opposite effects on epithelial curvature. (A–C) The appearance of control (A), Le-cre; RhoA^fl/fl (B), and Le-cre; Rac1^fl/fl (C) lens pits at E10.5. (D) Curves representing the average shape of the apical surface of E10.5 lens pits from control (gray), Le-cre; RhoA^fl/fl (red), and Le-cre; Rac1^fl/fl (blue) and Le-cre; Rac1^fl/fl; RhoA^fl/fl (green). Although individual examples of any genotype show some variability in lens pit shape, coordinate geometry averaging shows that the RhoA mutant pit has less curvature, whereas that for Rac1 has more. Quantification in E10.5 lens pits (n = 10) of cell numbers.

Fig. 2.

Rac1 and RhoA mutations have opposite effects on cell shape. (A–D) Cell profiles from the lens pits of control and somatic mutants were generated from β-catenin–labeled cryosections. The profiles were exported, measured for width and height, and combined to produce the average profiles in E. (E) Apical and basal cell dimensions are indicated (above and below the profile) relative to the control basal dimension. Similarly, the absolute dimensions in micrometers are indicated for the average control cell. Significance values for apical dimension: control (2.08 ± 0.14) to RhoA (2.74 ± 0.18), P < 0.001; control to Rac1 (1.72 ± 0.12), P = 0.043; RhoA to Rac1, P < 0.001. Significance values for cell length: control (26.1 ± 0.46) to RhoA (32.6 ± 0.37), P < 0.001; control to Rac1 (23.8 ± 0.44), P = 0.008; RhoA to Rac1, P < 0.001. Basal dimensions are not significantly different. This analysis shows that Le-cre; RhoA^fl/fl cells (red) were longer and less apically constricted and that Le-cre; Rac1^fl/fl cells (blue) were shorter and more apically constricted. Control cells occupied an angle of 6.0°, whereas Le-cre; RhoA^fl/fl and Le-cre; Rac1^fl/fl cells occupied angles of 4.0° and 7.4°, respectively.

Fig. 3.

Mutual antagonism of RhoA and Rac1 in regulating the cytoskeletal machinery during lens pit invagination. (A, C, E, G, I, and K) Labeling for F-actin (A), myosin IIB (C), phospho-MRLC (E), Arpc2 (G), cortactin (I), and Rac1 (K) in control and Le-cre; RhoA^fl/fl and Le-cre; Rac1^fl/fl (except Rac1) lens pit cells at E10.5. (B, D, F, H, J, and L) Quantification of the labeling shown in (A, C, E, G, I, and K) for lens pits over an apical–basal line interval (n = 5 eyes, n = 85 line intervals). The gray line shows quantification of the indicated marker in control cells and the red and blue lines, the quantification in Le-cre; RhoA^fl/fl and Le-cre; Rac1^fl/fl lens pit cells, respectively. The red and blue arrowheads in A, C, E, G, I, and K (only red) indicate the apical and basal marker labeling, and where numbered, cross-reference a peak on the quantification profiles.

Fig. 4.

Schematic describing the role of RhoA–Rac1 mutual antagonism in epithelial bending in the lens pit. From the current analysis, we can infer that both Rac1 and RhoA have dual functions. Rac1 has a role in elongating cells through Arpc2 and cortactin but also suppresses the production of phospho-MRLC and thus the generation of contractile actin. By contrast, RhoA is required for apical constriction through the production of phospho-MRLC and contractile actin, but also suppresses the basal Arpc2 and cortactin complexes and thus inhibits cell elongation. In this way, a balance between the activities of RhoA and Rac1 controls the apical width and cell length. In turn, the ratio of these two dimensions controls the angle formed by the cells and, in aggregate, the curvature of the epithelium.