Galpha12/13 regulate epiboly by inhibiting E-cadherin activity and modulating the actin cytoskeleton

Abstract

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Galpha(12/13) signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Galpha(12/13) regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Galpha(12/13) modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Galpha(12/13) regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

Figure 1.

Gα_12/13 regulate epibolic movements of the deep cells. (A and B) Nomarski images of control WT embryos (A) and embryos overexpressing Gα₁₃a (B) at 80% epiboly (A′ and B′ are schematic drawings of A and B), showing the dcm and YSL nuclei (YSLn; green arrows and dots), which move together in control embryos (A and A′) but are separated in embryos overexpressing Gα₁₃a (B and B′). (C and D) Nomarski images of yolk cell region at high magnification in a control WT embryo (C) and an embryo overexpressing Gα₁₃a (D), showing distortions in the YCL (white arrowheads). (A–D) Lateral view, with dorsal shown toward the right and vegetal toward the bottom. (E–H) Nomarski images of control WT embryos (E), embryos overexpressing either full-length Gα₁₃a (F), or the CT fragment of Gα₁₃a (Gα₁₃-CT; G), and embryos injected with 3MOs against gna13a, gna13b, and gna12 (4 ng each; H) at 95% epiboly. (E′–H′) Schematic drawings of E–H. Vegetal view is shown. df, df cells (red arrowheads). Note: in F–H versus E, the vegetal opening is much larger, and dfs are separated from the dcm; in F and H, the dfs are split. (I–L) Expression of the ntl mRNA at 90% epiboly. Images show ntl expression domains at dcm and df. Dorsal view, with the vegetal pole (VP; blue lines) toward the bottom. Yellow lines with double arrows, distance from dcm to VP. Bars, 100 µm. (M) The percentage of embryos with epibolic defects. Data are compiled from two to three different experiments. Error bars represent mean ± SEM.

Figure 2.

Overexpression of Gα₁₃a results in cell adhesion defects in embryonic tissues. (A–C′) Nomarski images of uninjected WT embryos, embryos overexpressing Gα₁₃a, and hab^vu44/vu44 mutant (E-cadherin–deficient) embryos. Higher magnification images of the boxed areas are shown in A′–C′. Red arrows indicate cells detaching from the blastoderm. Lateral view is shown, with dorsal (D) toward the right and the vegetal pole (VP) toward the bottom. (D and E) Nomarski images of notochord and somites in the WT embryos and embryos overexpressing Gα₁₃a at the 4–5 somite stage. Red arrowheads indicate gaps between the notochord and somites. Dorsal view is shown, with anterior to the left. Bars, 100 µm.

Figure 3.

Altered Gα_12/13 expression does not change the levels and distribution of E-cadherin and β-catenin. (A) Western blots showing the expression levels of E-cadherin, the G protein β subunit, and β-catenin in the uninjected WT, Gα₁₃a-overexpressing, and three MOs (3MO)-injected gastrulae. (B and C) Confocal images showing the cellular distribution of E-cadherin (red) in the anterior mesendoderm of embryos at 70% E (B; gsc-GFP labels the prechordal mesoderm), and of β-catenin in the lateral mesoderm in embryos at 80% E (C). Bars, 10 µm.

Figure 4.

Gα_12/13 signaling modulates the phenotype of hab^vu44 mutant embryos. (A–C) Different phenotypic classes of progeny of hab^vu44/+ parents revealed by ntl staining: normal pattern (A), type I (B), and type II (C). See text for details. (D) A representative image showing exacerbation of epibolic defects of hab^vu44 mutant embryos overexpressing Gα₁₃a (20 pg; see text for details). A dorsal view is shown. AP, animal pole; VP, vegetal pole. Bars, 100 µm. (E) Effects of altered Gα_12/13 signaling on distribution of the phenotypic classes of progeny from hab^vu44/+ parents. The data were generated from at least three separate experiments, with the total number of embryos indicated below the graph. Error bars represent mean ± SEM. *, P < 0.001; **, P < 0.05; †, P < 0.01; #, P < 0.001 versus control.

Figure 5.

Gα₁₃a interacts with E-cadherin and inhibits cell adhesion. (A) Gα₁₃a interacts with the cytoplasmic domain of E-cadherin. The GST pull-down assay was performed on cell extracts from HEK 293 cells cotransfected with Gα₁₃a and either GST or a GST-tagged cytoplasmic domain of E-cadherin (GST–E-cad–CyT). The precipitates were immunoblotted with anti-Gα₁₃ and anti-GST antibodies. The level of Gα₁₃a expression in the lysates is shown at the bottom of the panel. (B) β-catenin competes with E-cadherin for binding to Gα₁₃a in a dose-dependent manner. HEK 293 cells were transfected with Gα₁₃a and GST–E-cad–CyT with or without β-catenin at various doses, and the GST pull-down assay was performed. The expression levels of β-catenin and Gα₁₃a in the lysates are shown. (C and D) Overexpression of Gα₁₃a enhances cell scattering in the blastoderm. Shown are representative images of labeled cells in the blastoderm of control WT embryos and embryos overexpressing Gα₁₃a scattering over time. The area of cell scattering is indicated by the yellow broken lines, which mark the cells at the outer edge. Bars, 100 µm. (E) Quantitative data from four separate experiments (eight embryos in each group), showing the ratio of the area of cell scattering relative to the starting point, at different time points. Error bars represent mean ± SEM. *, P < 0.05 versus control.

Figure 6.

Gα_12/13 regulate cytoskeleton organization during epiboly. (A–D) Confocal images show phalloidin staining of F-actin in gastrulae. Red and green arrowheads indicate the margin of the deep cells and the EVL, respectively; yellow lines with arrows indicate the distance between the EVL margin and the vegetal pole (VP; white lines). Pink asterisks indicate the actin bundles in the yolk. (E–G) Representative images of the EVL cells indicated at high magnification. The cell boundaries of a few EVL cells of each group are highlighted. Note: the EVL cells in embryos injected with 3MO and embryos overexpressing Gα₁₃a are rounder and not correctly aligned. Yellow arrows indicate an actin ring in the vegetal margin of the EVL. Bars, 100 µm. (H) Quantitative data showing the LWRs of the EVL cells close to the margin. Error bars represent mean ± SEM. *, P < 0.05 versus WT. #, P > 0.05 versus control. (I–K) The half-Rose diagrams show the numbers of EVL cells for which the angle of the long axis relative to a line parallel to the EVL margin falls within each sector.

Figure 7.

Gα₁₃a promotes actin assembly via a PDZ RhoGEF-dependent pathway. (A–D) Nomarski images of live WT embryos (A), embryos overexpressing Gα₁₃a alone (B), embryos overexpressing Gα₁₃a and a dominant-negative mutant zebrafish Arhgef11, ΔDHPH (C), or embryos overexpressing Arhgef11 (D) at 80% epiboly. Bar, 250 µm. (E–H) Confocal z-projection images show phalloidin staining of F-actin. Red and green arrowheads indicate the dcm and the EVL, respectively; pink asterisks show the actin bundles in the yolk. Note the gap between dcm and the EVL, and the lack of actin bundles in embryos coinjected with Gα₁₃a and a ΔDHPH-encoding RNA. VP, vegetal pole. Bars, 100 µm. (I) The percentage of embryos with actin bundles in the embryos expressing Gα₁₃a alone or both Gα₁₃a and ΔDHPH. *, P < 0.05 versus Gα₁₃a. (J) Gα₁₃a interacts with zebrafish Arhgef11. Coimmunoprecipitation was performed on cell extracts from HEK 293 cells transfected with Gα₁₃a or Arhfef11 alone, or with both Gα₁₃a and myc-tagged Arhgef11 forms (WT, dominant-negative mutants lacking the RGS domain [ΔRGS], or lacking the DH and PH domains [ΔDHPH]). Immunoblotting was performed with the indicated antibodies. Error bars represent mean ± SEM.