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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 signaling modulates the phenotype ofhabvu44 mutant embryos.(A–C) Different phenotypic classes of progeny ofhabvu44/+ parents revealedby ntl staining: normal pattern (A), type I (B), andtype II (C). See text for details. (D) A representative image showingexacerbation of epibolic defects of habvu44mutant embryos overexpressing Gα13a (20 pg; seetext for details). A dorsal view is shown. AP, animal pole; VP, vegetalpole. Bars, 100 µm. (E) Effects of alteredGα12/13 signaling on distribution of thephenotypic classes of progeny fromhabvu44/+ parents. The data weregenerated from at least three separate experiments, with the totalnumber 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.
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fig4: Gα12/13 signaling modulates the phenotype ofhabvu44 mutant embryos.(A–C) Different phenotypic classes of progeny ofhabvu44/+ parents revealedby ntl staining: normal pattern (A), type I (B), andtype II (C). See text for details. (D) A representative image showingexacerbation of epibolic defects of habvu44mutant embryos overexpressing Gα13a (20 pg; seetext for details). A dorsal view is shown. AP, animal pole; VP, vegetalpole. Bars, 100 µm. (E) Effects of alteredGα12/13 signaling on distribution of thephenotypic classes of progeny fromhabvu44/+ parents. The data weregenerated from at least three separate experiments, with the totalnumber 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.

Mentions: Next, we aimed to determine whether Gα12/13 modulateE-cadherin function in vivo by testing their genetic interactions. We tookadvantage of a zebrafish mutant, habvu44, harboringa premature stop codon at amino acid residue L553 within the EC4 domain of theextracellular portion of the cdh1 gene (Kane et al., 2005).habvu44/vu44 embryos display an epiboly delay/arrestafter midgastrulation, probably due to a moderating effect of the maternalcontribution of E-cadherin, which has been shown to cooperate with thezygotically expressed E-cadherin to regulate epiboly (Shimizu et al., 2005). We injected embryos derived fromcrosses among habvu44 heterozygous fish with eithera small dose of synthetic RNA encoding Gα13a (10 pg) or asingle MO against Gα13a or Gα12 (4 ng)to elevate or reduce the function of Gα13 orGα12, respectively. Such treatments alone had no effecton the epiboly in WT embryos (unpublished data). We then assessed whether thismanipulation of Gα12/13 function can modulate thephenotypic changes caused by E-cadherin deficiency by analyzing thentl expression profile. We reasoned that ifGα12/13 negatively regulate the E-cadherin activity,then excess Gα12/13 function exacerbates it, and decreasedGα12/13 signaling should suppress the phenotypicchanges caused by E-cadherin deficiency. Among the uninjected progeny fromhabvu44/+ parents, 63± 11% embryos showed a normal pattern of ntlexpression (Fig. 4 A); 16 ± 9%exhibited mild defects in epiboly, in which their df cells were divided intosmaller clusters in spite of being tightly associated with the margin (type Idefect; Fig. 4 B); and 20 ±3.3% showed a strong epiboly delay in the deep cells and obvious separation ofthe df cells from the dcm (type II defect; Fig.4 C). This phenotypic distribution is consistent with a partialpenetrance of both the dominant df defect and the recessive epiboly phenotype ofhabvu44 mutation (Kane et al., 2005). A reduction in the expression ofeither Gα12 or Gα13 in the progeny ofhabvu44/+heterozygotes partially suppressed the mutant epibolic defects, as indicated bya significant increase in the proportion of embryos showing normalntl expression in the blastoderm margin and df cells, and adecrease in the percentage of embryos with severe epibolic defects (type II;Fig. 4 E). Conversely, a slightincrease in Gα13 activity exacerbated these defects (Fig. 4, D–E). These resultssupport the notion that Gα12/13 regulate epiboly throughE-cadherin by acting as negative regulators of E-cadherin activity.


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 signaling modulates the phenotype ofhabvu44 mutant embryos.(A–C) Different phenotypic classes of progeny ofhabvu44/+ parents revealedby ntl staining: normal pattern (A), type I (B), andtype II (C). See text for details. (D) A representative image showingexacerbation of epibolic defects of habvu44mutant embryos overexpressing Gα13a (20 pg; seetext for details). A dorsal view is shown. AP, animal pole; VP, vegetalpole. Bars, 100 µm. (E) Effects of alteredGα12/13 signaling on distribution of thephenotypic classes of progeny fromhabvu44/+ parents. The data weregenerated from at least three separate experiments, with the totalnumber 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Gα12/13 signaling modulates the phenotype ofhabvu44 mutant embryos.(A–C) Different phenotypic classes of progeny ofhabvu44/+ parents revealedby ntl staining: normal pattern (A), type I (B), andtype II (C). See text for details. (D) A representative image showingexacerbation of epibolic defects of habvu44mutant embryos overexpressing Gα13a (20 pg; seetext for details). A dorsal view is shown. AP, animal pole; VP, vegetalpole. Bars, 100 µm. (E) Effects of alteredGα12/13 signaling on distribution of thephenotypic classes of progeny fromhabvu44/+ parents. The data weregenerated from at least three separate experiments, with the totalnumber 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.
Mentions: Next, we aimed to determine whether Gα12/13 modulateE-cadherin function in vivo by testing their genetic interactions. We tookadvantage of a zebrafish mutant, habvu44, harboringa premature stop codon at amino acid residue L553 within the EC4 domain of theextracellular portion of the cdh1 gene (Kane et al., 2005).habvu44/vu44 embryos display an epiboly delay/arrestafter midgastrulation, probably due to a moderating effect of the maternalcontribution of E-cadherin, which has been shown to cooperate with thezygotically expressed E-cadherin to regulate epiboly (Shimizu et al., 2005). We injected embryos derived fromcrosses among habvu44 heterozygous fish with eithera small dose of synthetic RNA encoding Gα13a (10 pg) or asingle MO against Gα13a or Gα12 (4 ng)to elevate or reduce the function of Gα13 orGα12, respectively. Such treatments alone had no effecton the epiboly in WT embryos (unpublished data). We then assessed whether thismanipulation of Gα12/13 function can modulate thephenotypic changes caused by E-cadherin deficiency by analyzing thentl expression profile. We reasoned that ifGα12/13 negatively regulate the E-cadherin activity,then excess Gα12/13 function exacerbates it, and decreasedGα12/13 signaling should suppress the phenotypicchanges caused by E-cadherin deficiency. Among the uninjected progeny fromhabvu44/+ parents, 63± 11% embryos showed a normal pattern of ntlexpression (Fig. 4 A); 16 ± 9%exhibited mild defects in epiboly, in which their df cells were divided intosmaller clusters in spite of being tightly associated with the margin (type Idefect; Fig. 4 B); and 20 ±3.3% showed a strong epiboly delay in the deep cells and obvious separation ofthe df cells from the dcm (type II defect; Fig.4 C). This phenotypic distribution is consistent with a partialpenetrance of both the dominant df defect and the recessive epiboly phenotype ofhabvu44 mutation (Kane et al., 2005). A reduction in the expression ofeither Gα12 or Gα13 in the progeny ofhabvu44/+heterozygotes partially suppressed the mutant epibolic defects, as indicated bya significant increase in the proportion of embryos showing normalntl expression in the blastoderm margin and df cells, and adecrease in the percentage of embryos with severe epibolic defects (type II;Fig. 4 E). Conversely, a slightincrease in Gα13 activity exacerbated these defects (Fig. 4, D–E). These resultssupport the notion that Gα12/13 regulate epiboly throughE-cadherin by acting as negative regulators of E-cadherin activity.

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