A Trio-RhoA-Shroom3 pathway is required for apical constriction and epithelial invagination

Abstract

Epithelial invagination is a common feature of embryogenesis. An example of invagination morphogenesis occurs during development of the early eye when the lens placode forms the lens pit. This morphogenesis is accompanied by a columnar-to-conical cell shape change (apical constriction or AC) and is known to be dependent on the cytoskeletal protein Shroom3. Because Shroom3-induced AC can be Rock1/2 dependent, we hypothesized that during lens invagination, RhoA, Rock and a RhoA guanine nucleotide exchange factor (RhoA-GEF) would also be required. In this study, we show that Rock activity is required for lens pit invagination and that RhoA activity is required for Shroom3-induced AC. We demonstrate that RhoA, when activated and targeted apically, is sufficient to induce AC and that RhoA plays a key role in Shroom3 apical localization. Furthermore, we identify Trio as a RhoA-GEF required for Shroom3-dependent AC in MDCK cells and in the lens pit. Collectively, these data indicate that a Trio-RhoA-Shroom3 pathway is required for AC during lens pit invagination.

Fig. 1.

Rock1/2 activity is required for lens pit invagination and apical constriction. (A-F) In situ hybridization of cryosectioned chicken embryos cultured in the presence of DMSO (A,B,D,E), or 50 mM Y27632 (C,F) for the indicated time, using a probe specific for δ-crystallin, an early lens differentiation marker. Embryos were age matched according to somite number. (G-O) Cryosections of chicken embryos cultured for 16 hours were immunofluorescently labeled (green or red) with the indicated lens differentiation markers. Hoechst staining was performed simultaneously to visualize the nuclei (blue). (P-T) A local dose of Y27632 was administered to the left eye of stage 13 chick embryos and cultured for 3.5 hours. The embryos were immunofluorescently labeled for ZO1 to mark the apical junctions of the right control eye (Q,S) or the Y27632-treated eye (R,T). The average apical area of cells from each eye was quantified and is depicted in the graph (P). Error bars represent s.e.

Fig. 2.

RhoA activity is required for Shroom3 induced apical constriction. (A-D) MDCK cells were transfected with the indicated expression plasmid and subsequently treated with the specified chemical inhibitor. Immunofluorescent labeling of ZO1 (green) was used to visualize the apical junctions, and for the epitope tags (red), to visualize transfected RhoA (B) or Shroom3 (D). Apical views of cells are visualized in the xy plane and the apical-basal axis is visualized in the xz plane. The average apical area for cells was calculated for each condition and is displayed in the bar graphs (A,C). Error bars represent s.e.m. ^*P<0.05. (E) The amino acids 594-1060 of Rock1 fused to GST, constitutively active RhoA (RhoA^L63) and the Rho-kinase binding domain of Shroom3 (SD2) were bacterially expressed and used in a pull-down assay. Coomasie Blue staining of an SDS-PAGE gel reveals protein that was resolved from the pellet fraction (P), which indicates binding to GST-Rock1, or from the supernatant (S).

Fig. 3.

RhoA activity is necessary and sufficient for Shroom3 apical localization. (A,B) Immunofluorescent labeling of MDCK cells co-expressing exogenous Shroom3 (red) and regionally targeted versions of constitutively active RhoA (green). Nuclei are in blue. The xz plane allows visualization of the apical-basal axis and the xy plane a view of the apex or base. The xz view shows that VSVG-RhoA^L63 (Ap-RhoA^L63) is targeted apically (A) whereas Fcgr2b-RhoA^L63 (Bl-RhoA^L63) targets basolaterally (B). (C) Immunofluorescent labeling for ZO1 (green) of an MDCK cell expressing Bl-RhoA^L63 (red). Nuclei are in blue. (D) xz plane of a field of MDCK cells labeled for ZO1 (green) and nuclei (blue) in which one is expressing Bl-RhoA^L63 (red). In this cell, ZO1 labeling is adjacent to the adhesion substrate. (E,F) Immunofluorescent labeling of Shroom3 (E) or phospho-MRLC (F) in wild-type or mutant E10.5 lens pit cryosections. (G-I) The average pixel intensity following immunolabeling with Shroom3 (G) or phospho-MRLC (H-I) specific antibodies was quantified over apical-basal line intervals for control (gray) or mutant (red) genotypes. The means are calculated from ≥17 cells from at least four different lens pits. Statistically significant differences were evaluated by calculating P-values (see Materials and methods) for each pixel along the apical-basal axis. The blue line indicates the trend of statistical P-values subtracted from 1 and values greater than 0.95 (P<0.05) indicate regions in which a statistically significant difference in signal intensity is found.

Fig. 4.

Inhibition of the Trio RhoA-GEF domain prevents Shroom3 induced AC. (A,B) RT-PCR performed on RNA isolated from MDCK cells (A) or chicken embryo eyes from the indicated stages (B) reveals the presence of both Trio and kalirin mRNA. (C) Lens pit cells from the eye region of E10.5 transgenic mouse embryos expressing GFP (the Le-cre line) were isolated by flow cytometry. Scatter plot depicts the cell populations isolated. qPCR for lens and retina marker genes was performed on RNA from the GFP+ and GFP– cell populations. The fold expression difference is listed in the table. Both Trio and kalirin are expressed in the GFP+ and GFP– cell populations. –RT, without reverse transcriptase. (D-G) MDCK cells were transfected with the indicated plasmid(s). Where indicated, they were treated with Y27632 or G04. Immunofluorescent labeling of Shroom3-Flag (red, E) or Trio-GFP (green, G) was used to identify transfected cells. ZO1 labeling (green in E, red in G) allowed visualization and quantification of the apical area. Error bars represent s.e.m. and the asterisks identify experimental groups significantly different from the control (P<0.05). The number of cells analyzed for each experimental group is listed at the base of each bar in the histogram.

Fig. 5.

Trio activation of RhoA is required for apical constriction during lens pit invagination. (A) Stage 11 chicken embryos were electroporated in ovo with the indicated plasmid(s) and allowed to develop until the lens pits began to invaginate. Transgenic embryos were cryosectioned and the outline of GFP-positive cells was determined (dashed lines). (B) The average apical width along the apical basal axis of transgenic cells from each experimental group is depicted. Error bars represent s.e.m. and the asterisk identifies significant differences from the control (P<0.05). (C) The average cell shape from each experimental group is depicted. The number value above or below each shape is the fold difference between the average basal width of control cells and the apical or basal widths, respectively, of the transgenic cells. The number of cells analyzed for each group is indicated in the middle of each outline. (D) Electroporated chicken lens pit cells labeled for GFP (green), nuclei (blue) and epitope tagged Ap-RhoA^L63 (red). The arrowheads indicate the apical localization of Ap-RhoA^L63.

Fig. 6.

Trio regulates apical constriction during mouse lens pit invagination. (A,B) Wild-type (A) or Trio-deficient (B) cryosectioned E10.5 mouse embryos were labeled for, phospho-myosin (red) and nuclei (blue). (C) Lines describing the apical surface curvature of lens pits from embryos of the indicated genotypes. The lines represent average curvature of lens pits from five embryos, compiled using coordinate geometry. (D) Examples of phospho-MRLC labeling of cryosectioned lens pits from control and Trio-deficient mouse embryos. The yellow lines depict the axes of immunofluorescent quantification. (E) The average pixel intensity along apical-basal line intervals (depicted in D) in lens pit cells from control (gray line) or Trio-deficient (red line) embryos immunolabeled for phospho-MRLC. The means are calculated from ≥17 cells from at least four different lens pits. Statistical significant differences were evaluated by calculating P-values (see Materials and methods) for each pixel along the apical-basal axis. The blue line indicates the trend of statistical P-values subtracted from 1 and values greater than 0.95 (P<0.05) indicate regions in which a statistically significant difference in signal intensity is found. (F) The average cell shape for control and Trio-deficient lens pit cells was quantified. The numerical values above and below each shape indicate the fold difference compared with the average basal width of control cells.

Fig. 7.

A Trio-RhoA-Shroom3 pathway is required for apical constriction during lens pit invagination. Model depicting the proposed molecular pathway by which lens pit cell apical constriction drives invagination. Pax6 and RhoA regulate Shroom3 expression and apical localization, respectively. The Shroom3-dependent apical constriction machinery requires Trio activation of RhoA. This leads to activation of the contractile, actin-myosin network and a reduced apical circumference of cells. The coordinated action of a localized group of cells that are apically constricting causes inward bending and invagination of the epithelial placode forming the lens pit.