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

Lin F, Chen S, Sepich DS, Panizzi JR, Clendenon SG, Marrs JA, Hamm HE, Solnica-Krezel L - J. Cell Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA. fang-lin@uiowa.edu

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.

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Gα12/13 regulate cytoskeleton organizationduring epiboly. (A–D) Confocal images showphalloidin staining of F-actin in gastrulae. Red and green arrowheadsindicate the margin of the deep cells and the EVL, respectively; yellowlines with arrows indicate the distance between the EVL margin and thevegetal pole (VP; white lines). Pink asterisks indicate the actinbundles in the yolk. (E–G) Representative images of the EVLcells indicated at high magnification. The cell boundaries of a few EVLcells of each group are highlighted. Note: the EVL cells in embryosinjected with 3MO and embryos overexpressing Gα13aare rounder and not correctly aligned. Yellow arrows indicate an actinring 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 < 0.05versus WT. #, P > 0.05 versus control. (I–K)The half-Rose diagrams show the numbers of EVL cells for which the angleof the long axis relative to a line parallel to the EVL margin fallswithin each sector.
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fig6: Gα12/13 regulate cytoskeleton organizationduring epiboly. (A–D) Confocal images showphalloidin staining of F-actin in gastrulae. Red and green arrowheadsindicate the margin of the deep cells and the EVL, respectively; yellowlines with arrows indicate the distance between the EVL margin and thevegetal pole (VP; white lines). Pink asterisks indicate the actinbundles in the yolk. (E–G) Representative images of the EVLcells indicated at high magnification. The cell boundaries of a few EVLcells of each group are highlighted. Note: the EVL cells in embryosinjected with 3MO and embryos overexpressing Gα13aare rounder and not correctly aligned. Yellow arrows indicate an actinring 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 < 0.05versus WT. #, P > 0.05 versus control. (I–K)The half-Rose diagrams show the numbers of EVL cells for which the angleof the long axis relative to a line parallel to the EVL margin fallswithin each sector.

Mentions: To assess the organization of actin cytoskeleton during gastrulation, wevisualized actin by whole-mount immunostaining with phalloidin. As shown inFig. 6 (A–D), the confocalimages revealed the periphery of the superficial EVL cells and the deep cellsbeneath, as well as two actin rings at the margins of the deep cells and the EVL(Fig. 6, A–D, red and greenarrowheads, respectively), as reported previously (Cheng et al., 2004). In WT embryos, the actin ringsadjacent to the deep cells and the EVL are closely associated (Fig. 6 A), which indicates that EVL and thedeep cells move together toward the vegetal pole during epiboly. Consistent withprevious papers on studies performed in habvu44/vu44mutant embryos, the deep cells exhibited impaired epiboly and lagged behind theEVL margin (Fig. 6 D); whereas the EVLunderwent epiboly at a relatively normal rate, as revealed by the observationthat the distance between the EVL margin and the vegetal pole (Fig. 6, yellow lines with arrows) in themutant was comparable to that in WT embryos (Fig. 6, A and D; Kane et al.,2005; Koppen et al., 2006). Asexpected, embryos with reduced or excess Gα12/13 functiondisplayed similar epibolic defects of the deep cells (separation from EVLmargin), although the defects were more minor than those inhabvu44/vu44 mutant embryos (Fig. 6, B–D). However, embryoswith altered Gα12/13 function exhibited an epibolic delayof the EVL, as the distance between the EVL margin and vegetal pole (Fig. 6, yellow lines with arrows) wassignificantly increased relative to that in the age-matched uninjected WTembryos (Fig. 6, A–C).


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

Lin F, Chen S, Sepich DS, Panizzi JR, Clendenon SG, Marrs JA, Hamm HE, Solnica-Krezel L - J. Cell Biol. (2009)

Gα12/13 regulate cytoskeleton organizationduring epiboly. (A–D) Confocal images showphalloidin staining of F-actin in gastrulae. Red and green arrowheadsindicate the margin of the deep cells and the EVL, respectively; yellowlines with arrows indicate the distance between the EVL margin and thevegetal pole (VP; white lines). Pink asterisks indicate the actinbundles in the yolk. (E–G) Representative images of the EVLcells indicated at high magnification. The cell boundaries of a few EVLcells of each group are highlighted. Note: the EVL cells in embryosinjected with 3MO and embryos overexpressing Gα13aare rounder and not correctly aligned. Yellow arrows indicate an actinring 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 < 0.05versus WT. #, P > 0.05 versus control. (I–K)The half-Rose diagrams show the numbers of EVL cells for which the angleof the long axis relative to a line parallel to the EVL margin fallswithin each sector.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664974&req=5

fig6: Gα12/13 regulate cytoskeleton organizationduring epiboly. (A–D) Confocal images showphalloidin staining of F-actin in gastrulae. Red and green arrowheadsindicate the margin of the deep cells and the EVL, respectively; yellowlines with arrows indicate the distance between the EVL margin and thevegetal pole (VP; white lines). Pink asterisks indicate the actinbundles in the yolk. (E–G) Representative images of the EVLcells indicated at high magnification. The cell boundaries of a few EVLcells of each group are highlighted. Note: the EVL cells in embryosinjected with 3MO and embryos overexpressing Gα13aare rounder and not correctly aligned. Yellow arrows indicate an actinring 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 < 0.05versus WT. #, P > 0.05 versus control. (I–K)The half-Rose diagrams show the numbers of EVL cells for which the angleof the long axis relative to a line parallel to the EVL margin fallswithin each sector.
Mentions: To assess the organization of actin cytoskeleton during gastrulation, wevisualized actin by whole-mount immunostaining with phalloidin. As shown inFig. 6 (A–D), the confocalimages revealed the periphery of the superficial EVL cells and the deep cellsbeneath, as well as two actin rings at the margins of the deep cells and the EVL(Fig. 6, A–D, red and greenarrowheads, respectively), as reported previously (Cheng et al., 2004). In WT embryos, the actin ringsadjacent to the deep cells and the EVL are closely associated (Fig. 6 A), which indicates that EVL and thedeep cells move together toward the vegetal pole during epiboly. Consistent withprevious papers on studies performed in habvu44/vu44mutant embryos, the deep cells exhibited impaired epiboly and lagged behind theEVL margin (Fig. 6 D); whereas the EVLunderwent epiboly at a relatively normal rate, as revealed by the observationthat the distance between the EVL margin and the vegetal pole (Fig. 6, yellow lines with arrows) in themutant was comparable to that in WT embryos (Fig. 6, A and D; Kane et al.,2005; Koppen et al., 2006). Asexpected, embryos with reduced or excess Gα12/13 functiondisplayed similar epibolic defects of the deep cells (separation from EVLmargin), although the defects were more minor than those inhabvu44/vu44 mutant embryos (Fig. 6, B–D). However, embryoswith altered Gα12/13 function exhibited an epibolic delayof the EVL, as the distance between the EVL margin and vegetal pole (Fig. 6, yellow lines with arrows) wassignificantly increased relative to that in the age-matched uninjected WTembryos (Fig. 6, A–C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA. fang-lin@uiowa.edu

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.

Show MeSH
Related in: MedlinePlus