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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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Altered Gα12/13 expression does not change thelevels and distribution of E-cadherin and β-catenin.(A) Western blots showing the expression levels of E-cadherin, the Gprotein β subunit, and β-catenin in the uninjected WT,Gα13a-overexpressing, and three MOs(3MO)-injected gastrulae. (B and C) Confocal images showing the cellulardistribution of E-cadherin (red) in the anterior mesendoderm of embryosat 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.
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fig3: Altered Gα12/13 expression does not change thelevels and distribution of E-cadherin and β-catenin.(A) Western blots showing the expression levels of E-cadherin, the Gprotein β subunit, and β-catenin in the uninjected WT,Gα13a-overexpressing, and three MOs(3MO)-injected gastrulae. (B and C) Confocal images showing the cellulardistribution of E-cadherin (red) in the anterior mesendoderm of embryosat 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.

Mentions: To test the hypothesis that Gα12/13 regulate epiboly bymodulating the function of E-cadherin, we first determined ifGα12/13 affect the expression of E-cadherin in embryoswith reduced or excess signaling. We performed Western blot analyses using ananti–E-cadherin antibody with protein extracts prepared either fromgastrulae injected with gna13a RNA or 3MO, or from uninjectedcontrol siblings. As shown in Fig. 3 A,two prominent bands of E-cadherin, which may correspond to two glycosylationforms of E-cadherin, were detected, as described previously (Babb and Marrs, 2004). There was no cleardifference in the expression level of E-cadherin protein between the controlembryos and embryos with excess or reduced Gα12/13signaling (Fig. 3 A). We then performedwhole-mount immunostaining to determine if Gα12/13 regulatethe cellular distribution of E-cadherin. It has been shown that E-cadherin isexpressed at a higher level at the anterior region of the hypoblast duringgastrulation (Babb and Marrs, 2004). Toidentify this region, we used embryos obtained from transgenicTG:[gsc-GFP] fish, in which GFP isexpressed in the dorsal midline (Doitsidou etal., 2002; Inbal et al.,2006). As shown in Fig. 3 B, incontrol embryos at 70% E, E-cadherin was expressed in all blastomeres,predominantly on the cell membranes, but also in a punctate pattern in thecytosol, as described previously (Babb andMarrs, 2004; Montero et al.,2005). Our analyses revealed that neither Gα13aoverexpression nor Gα12/13 down-regulation (3MO-mediated)affected the expression level or the cellular distribution of E-cadherin (Fig. 3 B).


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)

Altered Gα12/13 expression does not change thelevels and distribution of E-cadherin and β-catenin.(A) Western blots showing the expression levels of E-cadherin, the Gprotein β subunit, and β-catenin in the uninjected WT,Gα13a-overexpressing, and three MOs(3MO)-injected gastrulae. (B and C) Confocal images showing the cellulardistribution of E-cadherin (red) in the anterior mesendoderm of embryosat 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2664974&req=5

fig3: Altered Gα12/13 expression does not change thelevels and distribution of E-cadherin and β-catenin.(A) Western blots showing the expression levels of E-cadherin, the Gprotein β subunit, and β-catenin in the uninjected WT,Gα13a-overexpressing, and three MOs(3MO)-injected gastrulae. (B and C) Confocal images showing the cellulardistribution of E-cadherin (red) in the anterior mesendoderm of embryosat 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.
Mentions: To test the hypothesis that Gα12/13 regulate epiboly bymodulating the function of E-cadherin, we first determined ifGα12/13 affect the expression of E-cadherin in embryoswith reduced or excess signaling. We performed Western blot analyses using ananti–E-cadherin antibody with protein extracts prepared either fromgastrulae injected with gna13a RNA or 3MO, or from uninjectedcontrol siblings. As shown in Fig. 3 A,two prominent bands of E-cadherin, which may correspond to two glycosylationforms of E-cadherin, were detected, as described previously (Babb and Marrs, 2004). There was no cleardifference in the expression level of E-cadherin protein between the controlembryos and embryos with excess or reduced Gα12/13signaling (Fig. 3 A). We then performedwhole-mount immunostaining to determine if Gα12/13 regulatethe cellular distribution of E-cadherin. It has been shown that E-cadherin isexpressed at a higher level at the anterior region of the hypoblast duringgastrulation (Babb and Marrs, 2004). Toidentify this region, we used embryos obtained from transgenicTG:[gsc-GFP] fish, in which GFP isexpressed in the dorsal midline (Doitsidou etal., 2002; Inbal et al.,2006). As shown in Fig. 3 B, incontrol embryos at 70% E, E-cadherin was expressed in all blastomeres,predominantly on the cell membranes, but also in a punctate pattern in thecytosol, as described previously (Babb andMarrs, 2004; Montero et al.,2005). Our analyses revealed that neither Gα13aoverexpression nor Gα12/13 down-regulation (3MO-mediated)affected the expression level or the cellular distribution of E-cadherin (Fig. 3 B).

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