T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 184 No. 6 909–921
Correspondence to Fang Lin: firstname.lastname@example.org; Heidi E. Hamm: heidi
.email@example.com; or Lilianna Solnica-Krezel: lilianna.solnica-krezel@
Abbreviations used in this paper: C & E, convergence and extension; cdh1 , cad-
herin1 ; CT, carboxy terminal; CyT, cytoplasmic terminus; dcm, deep cell margin;
df, dorsal forerunner; EVL, enveloping layer; GEF, guanine nucleotide exchange
factor; GPCR, G protein – coupled receptor; hab , half-baked ; LWR, length-to-
width ratio; MO, morpholino oligonucleotide; ntl , no tail ; WT, wild type; YCL,
yolk cytoplasmic layer; YSL, yolk syncytial layer; YSN, yolk syncytial nuclei.
During vertebrate gastrulation, an embryo of simple and sym-
metrical morphology is reshaped to reveal its fundamental
body plan. This process is accomplished by cooperation of
four morphogenetic movements — epiboly, internalization, and
convergence and extension (C & E) — that are largely conserved
among vertebrates ( Arendt and Nubler-Jung, 1999 ; Leptin,
2005 ; Solnica-Krezel, 2005 ). Epiboly starts at the late blastula
stage as the yolk cell pushes into the blastoderm, which thins
and expands vegetally until it encloses the entire yolk cell
( Warga and Kimmel, 1990 ; Solnica-Krezel, 2006 ; Rohde and
Heisenberg, 2007 ). At this stage, the embryo is composed of
four cell layers: the enveloping layer (EVL), deep cells, the
yolk syncytial layer (YSL), and the yolk cell. The EVL is a
superfi cial epithelial layer that covers a mass of deep cells,
which give rise to embryonic tissues. The YSL is a shallow and
superfi cial cytoplasmic layer within the yolk cell ( Solnica-
Krezel and Driever, 1994 ; Rohde and Heisenberg, 2007 ).
Proper epiboly involves coordinated movements of all of these
layers, and the underlying cellular and molecular mechanisms
remain to be fully defi ned ( Solnica-Krezel, 2006 ; Rohde and
Heisenberg, 2007 ).
Recent studies indicate that E-cadherin – mediated cell – cell
adhesion plays a critical role in zebrafish epiboly. In both
E-cadherin mutant embryos and embryos injected with E-cadherin
morpholino oligonucleotides (MOs) to block its translation, the
epibolic movement of the deep cells is delayed or arrested at
midgastrulation, although the YSL and EVL expand vegetally
in a relatively normal fashion ( Babb and Marrs, 2004 ; Kane et al.,
2005 ; McFarland et al., 2005 ; Shimizu et al., 2005 ). This epi-
bolic delay has been attributed to impaired radial intercalation
resulting from decreased adhesion among the deep cells and be-
tween the deep cells and the EVL ( Kane et al., 2005 ; Montero
et al., 2005 ; Shimizu et al., 2005 ). An additional cell – cell adhesion
Although recent studies have begun to elucidate the
processes that underlie these epibolic movements, the
cellular and molecular mechanisms involved remain to
be fully defi ned. Here, we show that gastrulae with al-
tered G ? 12/13 signaling display delayed epibolic move-
ment of the deep cells, abnormal movement of dorsal
forerunner cells, and dissociation of cells from the blasto-
derm, phenocopying e-cadherin mutants. Biochemical
piboly spreads and thins the blastoderm over the
yolk cell during zebrafi sh gastrulation, and in-
volves coordinated movements of several cell layers.
and genetic studies indicate that G ? 12/13 regulate epi-
b oly, in part by associating with the cytoplasmic termi-
nus of E-cadherin, and thereby inhibiting E-cadherin
activity and cell adhesion. Furthermore, we demonstrate
that G ? 12/13 modulate epibolic movements of the envel-
oping layer by regulating actin cytoskeleton organiza-
tion through a RhoGEF/Rho-dependent pathway. These
results provide the fi rst in vivo evidence that G ? 12/13 reg-
ulate epiboly through two distinct mechanisms: limiting
E-cadherin activity and modulating the organization of
the actin cytoskeleton.
G ? 12/13 regulate epiboly by inhibiting E-cadherin
activity and modulating the actin cytoskeleton
Fang Lin , 1,3 Songhai Chen , 2 Diane S. Sepich , 4 Jennifer Ray Panizzi , 4 Sherry G. Clendenon , 5 James A. Marrs , 5
Heidi E. Hamm , 1 and Lilianna Solnica-Krezel 4
1 Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232
2 Department of Pharmacology and 3 Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
4 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235
5 Department of Medicine, Indiana University Medical Center, Indianapolis, IN 46202
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JCB • VOLUME 184 • NUMBER 6 • 2009 910
bands and the EVL margin, moving the EVL toward the vege-
tal pole ( Koppen et al., 2006 ). Disruption of these actin struc-
tures, as a consequence of either cytochalasin B treatment
( Cheng et al., 2004 ), interference with myosin2 ( Koppen et al.,
2006 ), or the homeobox transcription factor Mtx2 ( Wilkins et al.,
2008 ), results in a delay or failure in epiboly. Similarly, an ab-
normal cytoskeleton contributes to the epibolic delay in Pou5fl -
defi cient embryos ( Lachnit et al., 2008 ).
We previously demonstrated that G ? 12/13 are required for
C & E gastrulation movements in zebrafi sh ( Lin et al., 2005 ).
Here, we fi nd that G ? 12/13 regulate epiboly in zebrafi sh, and pro-
vide evidence that G ? 12/13 interact with E-cadherin to negatively
modulate E-cadherin – mediated cell – cell adhesion. Moreover,
we show that G ? 12/13 also regulate epiboly by promoting actin
microfi lament assembly through a Rho guanine nucleotide ex-
change factor (GEF)-dependent signaling pathway. Our results
therefore identify a novel G ? 12/13 -dependent mechanism for
modulating epiboly during vertebrate gastrulation.
Disruption of G ? 12/13 function results in
We have previously identifi ed one G ? 12 and two G ? 13 (G ? 13 a
and G ? 13 b isoforms) in zebrafi sh and demonstrated that proper
G ? 12/13 signaling is essential for C & E movements, as well as for
epiboly during zebrafi sh gastrulation ( Lin et al., 2005 ). To de-
fi ne further the mechanisms whereby G ? 12 and G ? 13 regulate
epiboly, we used gain- and loss-of-function approaches. To en-
hance G ? 13 function, we injected embryos with a synthetic
RNA encoding G ? 13 a. To inhibit G ? 12/13 function, we injected
embryos with a mixture of antisense MOs (3MO) that interfere
with translation of the three G ? 12/13 transcripts ( gna13a , gna13b ,
and gna12 ; 4 ng each). Alternatively, we injected a synthetic
RNA encoding the carboxy-terminal (CT) peptides of G ? 12/13 ,
which have been shown to disrupt the coupling of G ? 12/13 to
their cognate receptors ( Akhter et al., 1998 ; Gilchrist et al.,
1999 ; Arai et al., 2003 ; Lin et al., 2005 ). In zebrafi sh, epiboly
initiates at the sphere stage and is complete when the blasto-
derm encloses the yolk cell ( Warga and Kimmel, 1990 ). Embryos
with either an excess or defi ciency of G ? 12/13 expression initi-
ated epiboly and progressed through early stages at rates com-
parable to those of their uninjected siblings. Furthermore, they
underwent internalization normally and formed embryonic
shields of normal morphology (unpublished data). However,
when the blastoderm covered 70% of the yolk cell in control
embryos (70% E), embryos with altered G ? 12/13 activity lagged
in epibolic movements behind their siblings by 10 – 20%. By
80% E, only a very small fraction of uninjected embryos (1.1 ±
1.9%; 136 embryos) showed epiboly defects, yet a majority of
embryos overexpressing G ? 13 a (98.5 ± 2.6%, 258 embryos), in-
jected with the G ? 13 -CT RNA (86.5 ± 6.5%, 106 embryos) or
injected with 3MO (84.4 ± 5.7%, 143 embryos), exhibited epib-
oly defects ( Fig. 1 M ).
The YSL consists of an internal YSL and an external YSL
that is populated with YSN ( Solnica-Krezel and Driever, 1994 ).
The EVL is tightly linked to the YSL margin. Therefore, as
defect was observed in E-cadherin – defi cient embryos, with cells
bulging and detaching from the embryonic surface ( Babb and
Marrs, 2004 ; Kane et al., 2005 ; McFarland et al., 2005 ; Shimizu
et al., 2005 ).
E-cadherin is a plasma membrane glycoprotein that is
indirectly linked to the actin cytoskeleton through ? -catenin
( Barth et al., 1997 ). The involvement of E-cadherin in morpho-
genesis and differentiation during the early development has
been also demonstrated in many species including mouse, chick,
and frog ( Halbleib and Nelson, 2006 ). In addition, E-cadherin is
essential for cell migration and polarity, as well as neuronal
synapse function. E-cadherin expression is regulated at various
levels including gene expression, protein stability, and intra-
cellular protein distribution ( Halbleib and Nelson, 2006 ). Down-
regulation of E-cadherin is regarded as the hallmark of the
epithelial – mesenchymal transition, and is often observed in in-
vasive tumor cells ( Behrens, 1999 ).
In comparison to our fairly detailed knowledge about the
regulation of E-cadherin expression, we know very little about
regulation of its activity. However, recent studies in cell culture
indicate that heterotrimeric G proteins of the G ? 12 family (G ? 12
and G ? 13 ) can modulate E-cadherin function: G ? 12/13 can bind
E-cadherin at its cytoplasmic domain to block the ? -catenin –
binding site, resulting in inhibition of cell – cell adhesion ( Kaplan
et al., 2001 ; Meigs et al., 2001 ; Meigs et al., 2002 ). Nevertheless,
the signifi cance of the G ? 12/13 and E-cadherin interaction during
morphogenesis remains to be tested.
During epiboly, the yolk cell may serve as a towing motor
to drive the movements of epiboly. Nuclei of the YSL move
vegetally even after removal of the blastoderm ( Trinkaus, 1951 ),
which indicates that the YSL can undergo epiboly autono-
mously. Because the EVL and the YSL are tightly attached
( Betchaku and Trinkaus, 1986 ), EVL epiboly is believed to de-
pend on the YSL expansion. In addition, endocytosis in the YSL
near the blastoderm margin results in removal of the yolk cyto-
plasmic membrane and could play a role in epiboly by drawing
the blastoderm to the vegetal pole ( Trinkaus, 1993 ; Solnica-
Krezel and Driever, 1994 ).
The cytoskeleton plays many important roles during
epiboly. Extensive microtubule networks in the yolk cell may
facilitate the epibolic movements, as microtubule disruption
completely inhibits the movement of yolk syncytial nuclei
(YSN) and impairs the epibolic movements of the deep cells
and the EVL ( Strahle and Jesuthasan, 1993 ; Solnica-Krezel
and Driever, 1994 ). A decrease in the amount of polymerized
microtubules in the yolk cell also leads to epiboly delay ( Hsu
et al., 2006 ). Actin microfi laments throughout the embryo
contribute to epiboly as well ( Zalik et al., 1999 ; Cheng et al.,
2004 ; Koppen et al., 2006 ). Three distinct actin structures are
elaborated during late epiboly stages: two rings at the margin
of the deep cells and the EVL, and a punctate band of actin ac-
cumulation in the external YSL adjacent to the EVL margin
( Cheng et al., 2004 ). It has been proposed that the actin rings
act as a “ purse string ” to pull the EVL vegetally, thereby ad-
vancing the epiboly process ( Cheng et al., 2004 ), whereas the
punctate band of contractile elements including actin and
myosin 2 in the YSL contributes to the shortening of the actin
12/13 REGULATE ZEBRAFISH EPIBOLY • Lin et al.
Figure 1. G ? 12/13 regulate epibolic movements of the deep cells. (A and B) Nomarski images of control WT embryos (A) and embryos overexpress-
ing G ? 13 a (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 G ? 13 a (B and B ? ). (C and D) Nomarski images of yolk cell
region at high magnifi cation in a control WT embryo (C) and an embryo overexpressing G ? 13 a (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 over-
expressing either full-length G ? 13 a (F), or the CT fragment of G ? 13 a (G ? 13 -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 μ m. (M) The percentage of embryos with epibolic defects. Data are compiled from two to three different experiments. Error bars
represent mean ± SEM.
JCB • VOLUME 184 • NUMBER 6 • 2009 912
affect the expression of E-cadherin in embryos with reduced
or excess signaling. We performed Western blot analyses using
an anti – E-cadherin antibody with protein extracts prepared
either from gastrulae injected with gna13a RNA or 3MO, or
from uninjected control siblings. As shown in Fig. 3 A , two
prominent bands of E-cadherin, which may correspond to two
glycosylation forms of E-cadherin, were detected, as described
previously ( Babb and Marrs, 2004 ). There was no clear differ-
ence in the expression level of E-cadherin protein between the
control embryos and embryos with excess or reduced G ? 12/13
signaling ( Fig. 3 A ). We then performed whole-mount immuno-
staining to determine if G ? 12/13 regulate the cellular distribu-
tion of E-cadherin. It has been shown that E-cadherin is
expressed at a higher level at the anterior region of the hypo-
blast during gastrulation ( Babb and Marrs, 2004 ). To identify
this region, we used embryos obtained from transgenic
TG :[ gsc-GFP ] fi sh, in which GFP is expressed in the dorsal
midline ( Doitsidou et al., 2002 ; Inbal et al., 2006 ). As shown
in Fig. 3 B , in control embryos at 70% E, E-cadherin was ex-
pressed in all blastomeres, predominantly on the cell mem-
branes, but also in a punctate pattern in the cytosol, as described
previously ( Babb and Marrs, 2004 ; Montero et al., 2005 ). Our
analyses revealed that neither G ? 13 a overexpression nor
G ? 12/13 down-regulation (3MO-mediated) affected the expres-
sion level or the cellular distribution of E-cadherin ( Fig. 3 B ).
seen in Fig. 1 (A and A ? ) , during the course of normal epiboly,
the YSN and the deep cell margin (dcm) stay together (the
EVL is invisible, as it is not in the focal plane; Trinkaus, 1984 ;
Solnica-Krezel and Driever, 1994 ). However, in embryos over-
expressing G ? 13 a, a sizable gap was formed between the YSN
and the dcm ( Fig. 1, B and B ? ), which indicates that epibolic
movement of the deep cells lags behind the movement of the
YSL. Thus, the distance between the dcm and the vegetal pole
is signifi cantly greater in embryos overexpressing G ? 13 a than
in the uninjected control ( Fig. 1, A and B ). In addition, we
noted that in contrast to the uniform and smooth appearance of
the yolk cytoplasmic layer (YCL; a thin anuclear cytoplasmic
layer covering the yolk mass) in control embryos ( Fig. 1 C ),
this structure was frequently distorted in G ? 13 a-expressing
embryos, exhibiting an uneven thickness ( Fig. 1 D , arrowheads).
As epiboly progressed to 95% E, the dcm of wild-type (WT)
embryos moved closer to the vegetal pole ( Fig. 1, E and E ? ),
but those of embryos overexpressing G ? 13 a, the dominant-
negative G ? 13 -CT peptide, or injected with 3MO had a much
larger vegetal opening ( Fig. 1, E – H ? ). Moreover, the dorsal
forerunners (dfs), a small dorsal cell population that normally
moves toward the vegetal pole as a single cluster in close asso-
ciation with the dcm ( Fig. 1 E ; Cooper and D ’ Amico, 1996 ),
were well separated from the dcm and far ahead of the remain-
ing deep cells in embryos with reduced or excess G ? 12/13 func-
tion. Interestingly, in these embryos, the df cells split and
formed several smaller clusters ( Fig. 1, F – H ). These observa-
tions in live embryos were confi rmed by analyzing the expres-
sion of the no tail ( ntl ) gene, which marks the mesodermal
precursors at the dcm and the df cells ( Schulte-Merker et al.,
1994 ). As seen in Fig. 1 (I – L) , the distance between the dcm
and the vegetal pole was signifi cantly greater in embryos with
either reduced or excess G ? 12/13 function than that in control
embryos, and dfs were separated from the dcm and divided
into several smaller clusters. The observed delay in epiboly
and abnormal behavior of the dfs resemble aspects of the pheno-
types that have been described for the half-baked ( hab )
mutants, which harbor mutations in the cadherin1 ( cdh1 ;
E-cadherin) gene ( Kane et al., 1996 ; Kane and Warga, 2004 ),
and in embryos injected with an MO that targets cdh1 ( Babb
and Marrs, 2004 ). This observation suggested a possible link
between G ? 12/13 function and E-cadherin activity.
In embryos overexpressing G ? 13 a, but not those injected
with G ? 13 -CT RNA or 3MO (not depicted), cells frequently dis-
sociated from the embryonic surface ( Fig. 2, A – C ? ), and gaps
formed between the paraxial and axial mesoderm during seg-
mentation ( Fig. 2 E ). These phenotypic changes have also been
observed in hab mutant embryos and have been attributed to
defects in cell – cell adhesion ( Kane et al., 2005 ; McFarland
et al., 2005 ). Together, these observations suggest that G ? 12/13
signaling may negatively regulate E-cadherin – mediated cell –
cell adhesion during zebrafi sh gastrulation.
G ? 12/13 do not infl uence E-cadherin
expression or intracellular distribution
To test the hypothesis that G ? 12/13 regulate epiboly by modu-
lating the function of E-cadherin, we fi rst determined if G ? 12/13
Figure 2. Overexpression of G ? 13 a results in cell adhesion defects in
embryonic tissues. (A – C ? ) Nomarski images of uninjected WT embryos, em-
bryos overexpressing G ? 13 a, and hab vu44/vu44 mutant (E-cadherin – defi cient)
embryos. Higher magnifi cation 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 G ? 13 a 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 μ m.
913 G ?
12/13 REGULATE ZEBRAFISH EPIBOLY • Lin et al.
G ? 12/13 signaling should suppress the phenotypic changes
caused by E-cadherin defi ciency. Among the uninjected prog-
eny from hab vu44/+ parents, 63 ± 11% embryos showed a nor-
mal pattern of ntl expression ( Fig. 4 A ); 16 ± 9% exhibited
mild defects in epiboly, in which their df cells were divided
into smaller clusters in spite of being tightly associated with
the margin (type I defect; Fig. 4 B ); and 20 ± 3.3% showed a
strong epiboly delay in the deep cells and obvious separation
of the df cells from the dcm (type II defect; Fig. 4 C ). This
phenotypic distribution is consistent with a partial penetrance
of both the dominant df defect and the recessive epiboly pheno-
type of hab vu44 mutation ( Kane et al., 2005 ). A reduction in the
expression of either G ? 12 or G ? 13 in the progeny of hab vu44/+
heterozygotes partially suppressed the mutant epibolic de-
fects, as indicated by a signifi cant increase in the proportion of
embryos showing normal ntl expression in the blastoderm
margin and df cells, and a decrease in the percentage of em-
bryos with severe epibolic defects (type II; Fig. 4 E ). Con-
versely, a slight increase in G ? 13 activity exacerbated these
defects ( Fig. 4, D – E ). These results support the notion that
G ? 12/13 regulate epiboly through E-cadherin by acting as nega-
tive regulators of E-cadherin activity.
G ? 12/13 regulate epiboly by inhibiting
Next, we aimed to determine whether G ? 12/13 modulate
E-cadherin function in vivo by testing their genetic interactions.
We took advantage of a zebrafi sh mutant, hab vu44 , harboring a pre-
mature stop codon at amino acid residue L553 within the EC4
domain of the extracellular portion of the cdh1 gene ( Kane
et al., 2005 ). hab vu44/vu44 embryos display an epiboly delay/arrest
after midgastrulation, probably due to a moderating effect of
the maternal contribution of E-cadherin, which has been
shown to cooperate with the zygotically expressed E-cadherin
to regulate epiboly ( Shimizu et al., 2005 ). We injected embryos
derived from crosses among hab vu44 heterozygous fi sh with
either a small dose of synthetic RNA encoding G ? 13 a (10 pg) or
a single MO against G ? 13 a or G ? 12 (4 ng) to elevate or reduce
the function of G ? 13 or G ? 12 , respectively. Such treatments
alone had no effect on the epiboly in WT embryos (unpublished
data). We then assessed whether this manipulation of G ? 12/13
function can modulate the phenotypic changes caused by
E-cadherin defi ciency by analyzing the ntl expression profi le. We
reasoned that if G ? 12/13 negatively regulate the E-cadherin activ-
ity, then excess G ? 12/13 function exacerbates it, and decreased
Figure 3. Altered G ? 12/13 expression does
not change the levels and distribution of
E-cadherin and ? -catenin. (A) Western blots
showing the expression levels of E-cadherin,
the G protein ? subunit, and ? -catenin in the un-
injected WT, G ? 13 a-overexpressing, and three
MOs (3MO)-injected gastrulae. (B and C) Con-
focal images showing the cellular distribution
of E-cadherin (red) in the anterior mesendo-
derm of embryos at 70% E (B; gsc -GFP labels
the prechordal mesoderm), and of ? -catenin in
the lateral mesoderm in embryos at 80% E (C).
Bars, 10 μ m.
JCB • VOLUME 184 • NUMBER 6 • 2009 914
E-cad–CyT but not by GST alone, which suggests a specifi c
association between zebrafi sh G ? 13 a and the E-cad–CyT. In addi-
tion, we demonstrated that ? -catenin can compete with G ? 13 a for
the binding to E-cadherin in a dose-dependent manner ( Fig. 5 B ),
confirming that the ? -catenin – and G ? 13 a-binding sites on
E-cadherin are close to one another ( Kaplan et al., 2001 ). How-
ever, we did not observe any obvious change in the expression
level and intracellular distribution of ? -catenin in embryos with
altered G ? 12/13 expression ( Figs. 3 C and S1 ).
E-cadherin is known to regulate cell – cell adhesion in zebra-
fi sh ( Meigs et al., 2002 ; Montero et al., 2005 ) and many other
animals ( Halbleib and Nelson, 2006 ). Moreover, the binding of
G ? 13 to E-cadherin interferes with its cell adhesive function in
mammalian cultured cells ( Meigs et al., 2002 ). To determine if
G ? 12/13 can infl uence cell adhesion in zebrafi sh, we performed a
cell tracing experiment in embryos ( Warga and Kane, 2003 ). In
this assay, zygotes were fi rst injected with gna13a RNA to en-
hance G ? 13 function. At the 256-cell stage, a single cell at the
animal pole of an uninjected or gna13a -RNA – injected blastula
was then injected with fl uorescein dextran, then the distribution
of the progeny of the labeled cells at several time points up to
50%E was analyzed. During embryonic development, blasto-
meres at the animal pole become separated from each other by
intercalating radially from deeper layers to the more superfi cial
layers without signifi cant directional migration ( Warga and
Kimmel, 1990 ). This phenomenon is thought to be mediated by
E-cadherin – dependent cell – cell adhesion interactions, because
in hab mutants (E-cadherin defi cient), cells intercalate from the
deeper to the more superfi cial layers but fail to maintain this
position and often fall back into the deeper layer ( Warga and
Kane, 2003 ; Kane et al., 2005 ). We found that progeny of the
labeled cells gradually dispersed over time in control embryos
( Fig. 5 C ) and in embryos overexpressing G ? 13 a ( Fig. 5 D ). To
quantify the scattering, we marked the outside edge of the re-
gions containing the labeled cells, and calculated the areas. We
then determined a scattering factor by comparing the areas at
different time points to the initial area for each embryo. 1 h after
injection, the scattering factor for the G ? 13 a-expressing em-
bryos was similar to that of control embryos. However, by the
second and third hour, the ratio in embryos overexpressing
G ? 13 a was signifi cantly greater than that in control embryos
( Fig. 5, C – E ). These results indicate that overexpression of
G ? 13 a enhanced dispersion in the blastoderm during epiboly,
which suggests that G ? 13 a-expressing cells have a reduced ten-
dency to adhere to one another. These fi ndings provide further
support for the notion that signaling via G ? 13 negatively regu-
lates E-cadherin activity.
G ? 12/13 regulate actin cytoskeleton
assembly during epiboly via a
Although embryos with enhanced or decreased G ? 12/13 showed
similar epibolic defects in deep cells as E-cadherin mutant em-
bryos, we noted that G ? 13 a-overexpressing embryos exhibited
additional defects such as a distorted YCL ( Fig. 1 D ), which
suggests that G ? 13 a signaling may contribute to the regulation
of epiboly via additional mechanisms that are independent of
G ? 12/13 interact with E-cadherin and inhibit
To better understand the mechanisms by which G ? 12/13 regulate
E-cadherin activity, we set out to test these two proteins for
physical interactions in vivo. Because previous studies in cul-
tured cells had shown that mammalian G ? 12 or G ? 13 can bind the
cytoplasmic domain of E-cadherin ( Kaplan et al., 2001 ; Meigs
et al., 2001 ), we performed the following procedures. First, we
cotransfected HEK 293 cells with zebrafi sh G ? 13 a and a GST-
tagged construct encoding the E-cadherin cytoplasmic terminus
(E-cad–CyT) or GST only, and performed a GST pull-down
assay. As shown in Fig. 5 A , G ? 13 a was pulled down by GST –
Figure 4. G ? 12/13 signaling modulates the phenotype of hab vu44 mutant
embryos. (A – C) Different phenotypic classes of progeny of hab vu44/+ par-
ents 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 G ? 13 a (20 pg;
see text for details). A dorsal view is shown. AP, animal pole; VP, vegetal
pole. Bars, 100 μ m. (E) Effects of altered G ? 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.
915 G ?
12/13 REGULATE ZEBRAFISH EPIBOLY • Lin et al.
EVL and the deep cells move together toward the vegetal pole
during epiboly. Consistent with previous papers on studies per-
formed in hab vu44/vu44 mutant embryos, the deep cells exhibited
impaired epiboly and lagged behind the EVL margin ( Fig. 6 D );
whereas the EVL underwent epiboly at a relatively normal rate,
as revealed by the observation that the distance between the
EVL margin and the vegetal pole ( Fig. 6 , yellow lines with
arrows) in the mutant was comparable to that in WT embryos
( Fig. 6, A and D ; Kane et al., 2005 ; Koppen et al., 2006 ). As ex-
pected, embryos with reduced or excess G ? 12/13 function dis-
played similar epibolic defects of the deep cells (separation
from EVL margin), although the defects were more minor than
those in hab vu44/vu44 mutant embryos ( Fig. 6, B – D ). However,
embryos with altered G ? 12/13 function exhibited an epibolic de-
lay of the EVL, as the distance between the EVL margin and
vegetal pole ( Fig. 6 , yellow lines with arrows) was signifi cantly
increased relative to that in the age-matched uninjected WT em-
bryos ( Fig. 6, A – C ).
E-cadherin. Such distortions of the YCL have also been ob-
served in embryos with cytoskeleton abnormalities; e.g., in em-
bryos treated with taxol to stabilize microtubules ( Solnica-Krezel
and Driever, 1994 ) or in Pou5fl mutants ( Lachnit et al., 2008 ).
We have previously shown that, like their mammalian counter-
parts, zebrafi sh G ? 12/13 can promote actin stress fi ber formation
in cultured cells ( Lin et al., 2005 ). Based on these observations,
we tested whether G ? 12/13 can also regulate cytoskeletal func-
tion in zebrafi sh gastrulae.
To assess the organization of actin cytoskeleton during
gastrulation, we visualized actin by whole-mount immunostain-
ing with phalloidin. As shown in Fig. 6 (A – D) , the confocal im-
ages revealed the periphery of the superfi cial EVL cells and the
deep cells beneath, as well as two actin rings at the margins of
the deep cells and the EVL ( Fig. 6, A – D , red and green arrow-
heads, respectively), as reported previously ( Cheng et al., 2004 ).
In WT embryos, the actin rings adjacent to the deep cells and
the EVL are closely associated ( Fig. 6 A ), which indicates that
Figure 5. G ? 13 a interacts with E-cadherin and inhibits cell adhesion. (A) G ? 13 a interacts with the cytoplasmic domain of E-cadherin. The GST pull-down
assay was performed on cell extracts from HEK 293 cells cotransfected with G ? 13 a and either GST or a GST-tagged cytoplasmic domain of E-cadherin
(GST – E-cad–CyT). The precipitates were immunoblotted with anti-G ? 13 and anti-GST antibodies. The level of G ? 13 a expression in the lysates is shown at
the bottom of the panel. (B) ? -catenin competes with E-cadherin for binding to G ? 13 a in a dose-dependent manner. HEK 293 cells were transfected with
G ? 13 a and GST – E-cad–CyT with or without ? -catenin at various doses, and the GST pull-down assay was performed. The expression levels of ? -catenin
and G ? 13 a in the lysates are shown. (C and D) Overexpression of G ? 13 a enhances cell scattering in the blastoderm. Shown are representative images of
labeled cells in the blastoderm of control WT embryos and embryos overexpressing G ? 13 a 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 μ m. (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
JCB • VOLUME 184 • NUMBER 6 • 2009 916
EVL cells in hab vu44/vu44 mutant embryos were elongated and
aligned properly (LWR = 1.79 ± 0.54; angle = 64 ± 16 ° C; 171
cells, 3 embryos; P > 0.05 vs. WT). In contrast, in embryos
with reduced or excess G ? 12/13 function, the EVL cells were
signifi cant rounder (smaller LWR; 3MO: LWR = 1.46 ± 0.32,
379 cells, 8 embryos; G ? 13 a: LWR = 1.55 ± 0.44, 342 cells,
8 embryos; P < 0.001 vs. control; Fig. 6, E – H ). In addition,
these EVL cells were more disorganized and failed to align
their cell bodies along the direction of epibolic movement,
with orientations of 52 ± 26 ° or 54 ± 25 ° (P < 0.001 vs. con-
trol) in G ? 12/13 -depleted or G ? 13 a-overexpressing embryos, re-
spectively. Moreover, only 41 – 49% of the cells from these
embryos exhibited an angle within the range of 60 – 120 ° ,
which suggests that most EVL cells in these embryos were
oriented in random directions ( Fig. 6, E – G and I – K ). Interest-
ingly, the punctate actin accumulation adjacent to the EVL
margin was markedly reduced in embryos overexpressing
G ? 13 a ( Fig. 6 G ; compare the yellow arrows Fig. 6, E and G ).
Moreover, these embryos exhibited abnormal formation of ac-
tin bundles in the YCL, although these are rarely observed in
the yolk cells of WT embryos. This is possibly due to the ag-
gregation or contraction of F-actin, which was absent in some
areas of the cortical cytoplasmic layer ( Fig. 6, C and G ).
Work in mammalian systems has established that G ? 12/13
regulate actin cytoskeleton dynamics to modulate cell shape
During epiboly, the constriction of the marginal EVL
cells leads to dramatic cell-shape changes in the EVL cells, and
to the elongation of the EVL cells along the animal – vegetal
axis. Failure of such cell-shape changes has been implicated in
epibolic defects ( Koppen et al., 2006 ). To further evaluate the
morphology of the EVL cells in embryos with altered G ? 12/13
function, we took confocal images of phalloidin-stained em-
bryos at higher magnifi cation, and analyzed cell shape (length-
to-width ratio [LWR]) and orientation (the angle of the long
axis of the EVL cells relative to a line parallel to the EVL
margin) of the EVL cells near the margin. As shown in Fig. 6
(E – H) , there was no signifi cant difference in the intensity of
F-actin staining in the EVL cells between the uninjected WT
embryos and embryos injected with 3MO or the G ? 13 a RNA.
However, both the shape and orientation of the EVL cells in
embryos with reduced or excess G ? 12/13 function were signifi -
cantly altered with respect to those in the control embryos
( Fig. 6 E-G ). In the uninjected control embryos, the EVL cells
were elongated, with a mean LWR of 1.73 ± 0.3 (220 cells,
6 embryos), and were orientated at an angle of 67 ± 20 ° . Of
220 cells counted, 73% aligned their cell bodies at an angle in
the range of 60 – 120 ° with respect to the EVL margin; this in-
dicates that most of these cells elongate vegetally along a line
roughly perpendicular to the EVL margin, which is consistent
with the direction of the epibolic movement. Similarly, the
Figure 6. G ? 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
magnifi cation. The cell boundaries of a few EVL cells of each group are highlighted. Note: the EVL cells in embryos injected with 3MO and embryos over-
expressing G ? 13 a are rounder and not correctly aligned. Yellow arrows indicate an actin ring in the vegetal margin of the EVL. Bars, 100 μ m. (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.
917 G ?
12/13 REGULATE ZEBRAFISH EPIBOLY • Lin et al.
To determine if zebrafi sh G ? 12/13 modulate epiboly via a
RhoGEF-dependent signaling pathway, we fi rst examined the
effect of Arhgef11 overexpression on epiboly. The overexpres-
sion of Arhgef11 resulted in similar epiboly defects and dis-
tortions in the YCL, similar to those observed for G ? 12/13
overexpression ( Fig. 7 D and not depicted). Actin staining re-
vealed that embryos overexpressing Arhgef11 also exhibited
delayed epiboly of the deep cells and the EVL as well as the
formation of thick actin bundles in the YCL ( Fig. 7 H ). Similar
defects were observed in embryos overexpressing a constitu-
tively activated zebrafi sh RhoA (data not shown). To test
whether RhoGEF acts downstream of G ? 12/13 in the regulation
of the actin cytoskeleton, we coexpressed G ? 13 a together with
a dominant-negative form of Arhgef11 lacking the DH and PH
and migration via a RhoGEF/Rho-dependent signaling path-
way ( Buhl et al., 1995 ; Gohla et al., 1998 ; Hart et al., 1998 ;
Kozasa et al., 1998 ). We have shown previously that, like zebra-
fish G ? 12/13 , one of the zebrafish RhoGEFs, PDZRhoGEF
(Arhgef11), can induce stress fi ber formation in HEK 293 cells
( Lin et al., 2005 ; Panizzi et al., 2007 ), which suggests that zebra-
fi sh G ? 12/13 also function through RhoGEF to regulate actin or-
ganization. Furthermore, we showed that, when coexpressed in
HEK 293 cells, G ? 13 a specifi cally coprecipitated with myc-
tagged full-length Arhgef11, and this interaction was not ob-
served when an Arhgef11 mutant lacking the RGS domain,
known to be required for target binding, was coexpressed. This
indicates that G ? 12/13 physically interact with PDZRhoGEF via
the RGS domain ( Fig. 7 J ).
Figure 7. G ? 13 a promotes actin assembly via a PDZ RhoGEF-dependent pathway. (A – D) Nomarski images of live WT embryos (A), embryos over-
expressing G ? 13 a alone (B), embryos overexpressing G ? 13 a and a dominant-negative mutant zebrafi sh Arhgef11, ? DHPH (C), or embryos overexpressing
Arhgef11 (D) at 80% epiboly. Bar, 250 μ m. (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 G ? 13 a and a ? DHPH-encoding RNA. VP, vegetal pole. Bars, 100 μ m. (I) The percentage of embryos with actin bundles in the
embryos expressing G ? 13 a alone or both G ? 13 a and ? DHPH. *, P < 0.05 versus G ? 13 a. (J) G ? 13 a interacts with zebrafi sh Arhgef11. Coimmunoprecipita-
tion was performed on cell extracts from HEK 293 cells transfected with G ? 13 a or Arhfef11 alone, or with both G ? 13 a and myc-tagged Arhgef11 forms
(WT, dominant-negative mutants lacking the RGS domain [ ? RGS], or lacking the DH and PH domains [ ? DHPH]). Immunoblotting was performed with the
indicated antibodies. Error bars represent mean ± SEM.
JCB • VOLUME 184 • NUMBER 6 • 2009 918
coinjection of G ? 13 a-RNA exacerbated these phenotypes (un-
published data). The region of E-cadherin that binds G ? 12/13 was
found to be located near the binding site for ? -catenin ( Kaplan
et al., 2001 ). This is supported by our result showing that ? -catenin
can compete with G ? 13 for E-cadherin binding in HEK cells
( Fig. 5 B ). Furthermore, it has been hypothesized that the binding
of G ? 12/13 to E-cadherin interferes with the ability of E-cadherin
to form a complex with ? -catenin. In fact, G ? 12/13 overexpres-
sion can cause ? -catenin to dissociate from E-cadherin and to
translocate from the membrane to the cytosol in cultured cells
( Meigs et al., 2001 ). We speculate that the competition of G ? 12/13
with ? -catenin for binding to E-cadherin may be one of the un-
derlying mechanisms in embryos. However, we did not observe
any overt change in expression level or intracellular distribution
of ? -catenin in embryos overexpressing G ? 13 a ( Fig. 3 ). The rea-
son for this discrepancy between studies in cell culture and our
studies in zebrafi sh embryos is unclear. However, one possibil-
ity is that the levels of G ? 12/13 we used were suffi cient to alter
E-cadherin activity but not to produce detectable changes in
? -catenin distribution.
Studies from hab mutant embryos indicate that E-cadherin
regulates epiboly in part by impinging on cell – cell adhesion
( Warga and Kimmel, 1990 ; Montero et al., 2005 ; Shimizu et al.,
2005 ). Considering these fi ndings in the light of our biochemical
and genetic data showing that G ? 12/13 functionally interact with
E-cadherin in zebrafi sh, we propose that G ? 12/13 modulate epi-
bolic movement by inhibiting E-cadherin – mediated cell – cell
adhesion. Accordingly, cells in embryos overexpressing G ? 13 a
during early epiboly scattered across a larger area ( Fig. 5 ). In ad-
dition, we also demonstrated that a larger scattering area in em-
bryos overexpressing G ? 13 a is not due to an increase in cell
number ( Fig. S2 ). However, we cannot rule out the possibility that
other functions of G ? 12/13 could contribute to reduced cohesion or
abnormal cell movements.
In addition to the impaired epiboly of the deep cells, al-
tered G ? 12/13 signaling resulted in epibolic defects of the EVL
( Fig. 6 ). This is in contrast to hab vu44 and maternal-zygotic cdh1 rk3
mutant embryos, in which the EVL appears to undergo normal
epiboly in spite of the fact that the deep cells exhibit severe epi-
bolic defects ( Fig. 6 D ; Shimizu et al., 2005 ). These results sug-
gest that G ? 12/13 may impinge on pathways other than the
E-cadherin pathway to regulate epiboly in EVL cells. Recent
evidence indicates that proper organization of the F-actin – based
cytoskeleton plays critical roles in the normal epiboly of zebra-
fi sh embryos ( Zalik et al., 1999 ; Cheng et al., 2004 ; Koppen
et al., 2006 ). The actin contractile elements in the YSL are nec-
essary for facilitating the proper EVL cell shape changes during
late gastrulation ( Koppen et al., 2006 ). Notably, in E-cadherin –
defi cient embryos, actin organization appeared to be normal,
and EVL cells were elongated and orientated properly ( Fig. 6, D
and H ; Shimizu et al., 2005 ), which indicates that E-cadherin
does not play a signifi cant role in actin organization and EVL
epiboly in zebrafi sh.
In mammalian cultured cells, G ? 12/13 are known to be in-
volved in the regulation of actin polymerization and the mainte-
nance of proper cell morphology, which suggests that G ? 12/13 may
affect EVL epiboly by regulating actin cytoskeleton organization
domains ( ? DHPH), which are needed for interacting with
downstream proteins ( Panizzi et al., 2007 ). We found that
Arhgef11 ? DHPH bound to G ? 13 ( Fig. 7 J ) and suppressed both
the formation of actin bundles in the YCL and the epiboly de-
fects associated with G ? 13 a overexpression ( Figs. 1 M and 7 ).
Although actin bundles were found in 86 ± 5% of the embryos
overexpressing G ? 13 a, only 33 ± 3% of the embryos coex-
pressing G ? 13 a and Arhgef11 ? DHPH showed this phenotype
( Fig. 7 I ). However, we observed that coexpression of Arhgef11
? DHPH did not fully rescue the epibolic delay in the deep
cells ( Fig. 7 G ), which suggests that cytoskeletal assembly
regulated by Arhgef11 only partially accounts for the function
of G ? 12/13 in epiboly. Collectively, these results indicate that
G ? 12/13 can regulate epiboly through a PDZ RhoGEF/RhoA-
dependent signaling pathway to modulate the function of the
In this paper, we demonstrate that G ? 12/13 signaling can regulate
different aspects of epiboly movements by two distinct mecha-
nisms: inhibiting E-cadherin activity and modulating actin cyto-
Excess or reduced G ? 12/13 signaling during gastrulation
resulted in delayed epiboly of the deep cells and in the splitting
of the df cell cluster ( Fig. 1 ). Moreover, excess G ? 12/13 activity
led to the detachment of cells from embryonic tissues, which
suggests that cell adhesion is defective under these circum-
stances ( Fig. 2 ). All of these phenotypic characteristics resem-
ble those observed in hab ( cdh1 ) mutant embryos ( Kane et al.,
1996 ; Kane and Warga, 2004 ), which suggests a possible link
between G ? 12/13 signaling and E-cadherin. Indeed, although al-
tered G ? 12/13 expression did not change the expression level and
cellular distribution of E-cadherin ( Fig. 3 ), our in vivo genetic
experiments demonstrated that G ? 12/13 can inhibit the function
of E-cadherin. In particular, we found that a reduction in the
expression of either G ? 12 or G ? 13 function by MO injection
partially suppressed, whereas an increase in G ? 13 activity exac-
erbated the epibolic defects in hab mutant mutants ( Fig. 4 ).
Interestingly, decreased G ? 12/13 function reduced the fraction of
embryos with a weak epibolic defect, as well as the fraction
with a strong epibolic delay ( Fig. 4 ). This suggests that reduced
G ? 12/13 function may suppress not only the epibolic defects in
heterozygous embryos, but also those in homozygous mutants.
We speculate that such an effect might be caused by the reduced
inhibition of the maternal E-cadherin protein by G ? 12/13 in homo-
zygous mutants ( Babb and Marrs, 2004 ; Kane et al., 2005 ).
Our biochemical studies support the notion that G ? 12/13
can interact with E-cadherin. We showed that zebrafi sh G ? 13 a
was pulled down with the CT fragment of zebrafi sh E-cadherin
(E-cad–CyT) in HEK cells ( Fig. 4 ), which is consistent with the
physical interaction between mammalian G ? 12/13 and E-cadherin
shown previously ( Kaplan et al., 2001 ; Meigs et al., 2001 ). The
E-cad–CyT has been shown to act as a dominant-negative pro-
tein ( Sadot et al., 1998 ). Accordingly, we observed that embryos
expressing this fragment exhibited cleavage defects, and the de-
tachment of blastodermal cells during early development and
12/13 REGULATE ZEBRAFISH EPIBOLY • Lin et al.
epiboly and C & E defects but gave rise to defects associated
with ciliated epithelia ( Panizzi et al., 2007 ). We speculate that
Arhgef11 and G ? 12/13 may act in both overlapping and different
signaling pathways during gastrulation. These results further
support the idea that G ? 12/13 regulate actin organization and cell
adhesion via distinct mechanisms.
In summary, our studies establish G ? 12/13 as novel regula-
tors of epiboly in zebrafi sh. Our data indicate that G ? 12/13 may
regulate different aspects of epibolic movements by two distinct
pathways. In the deep cells, G ? 12/13 bind the intracellular do-
main of E-cadherin and inhibit its activity to modulate epiboly;
in the EVL and the yolk cell, G ? 12/13 promote actin cytoskeleton
assembly through RhoGEF/RhoA in order to regulate epibolic
movement of the EVL. It has been shown that G ? 12/13 can trans-
mit signals from different GPCRs and suggested that different
GPCRs may activate distinct signaling pathways through G ? 12/13
( Riobo and Manning, 2005 ). It will be interesting to determine
if zebrafi sh epiboly involves different extracellular signals acting
through distinct GPCRs via G ? 12/13 to specify distinct cell behav-
iors in different cell types. G ? 12/13 are oncogenes with trans forming
potential and growth-promoting activity ( Chan et al., 1993 ;
Voyno-Yasenetskaya et al., 1994 ; Radhika and Dhanasekaran,
2001 ). Furthermore, the down-regulation of E-cadherin is associ-
ated with tumor metastasis and cancer progression ( Behrens,
1999 ). Thus, our fi ndings on the in vivo role of G ? 12/13 in epiboly
may have signifi cant implications for the mechanisms whereby
G ? 12/13 function during tumorigenesis and metastasis, as well as
during other morphogenetic processes in multicellular systems.
Materials and methods
Zebrafi sh strain and maintenance
WT, transgenic Tg [ gsc:GFP ] ( Doitsidou et al., 2002 ), and hab vu44 mutant
strains of zebrafi sh were maintained as described previously ( Solnica-
Krezel et al., 1994 ). Embryos were obtained by natural mating and staged
according to morphology as described previously ( Kimmel et al., 1995 ).
Generation of a GST-tagged cytoplasmic fragment of E-cadherin
The cytoplasmic domain of zebrafi sh E-cadherin (708-864AA) was cloned
by PCR using the cdh1 cDNA as a template ( Babb et al., 2001 ). The GST
sequence was inserted in front of the 5 ? end of the fragment, and the con-
struct was verifi ed by sequencing and by its expression (as ascertained by
immunostaining with anti-GST antibody).
mRNA and antisense MO injections, in situ hybridization
Capped sense mRNAs were synthesized using the SP6 mMessage machine
(Applied Biosystems). The injection of synthetic mRNAs encoding G ? 13 a
(60 pg), G ? 13 -CT (800 pg), myc-tagged PDZ RhoGEF (Arhgef11, 2pg), the
dominant-negative mutant Arhgef11 ( ? DHPH, 600 pg), constitutively activated
RhoA (10 pg), and antisense MOs targeting zebrafi sh gna12 , gna13a , and
gna13b transcripts (4 ng each) has been described previously ( Lin et al.,
2005 ). Whole-mount in situ hybridization using an antisense ntl RNA probe
was performed as described previously ( Thisse and Thisse, 1998 ), except
that BM Purple (Roche) was used for the chromogenic reaction.
Embryos at 80% epiboly stage were manually deyolked and homogenized
in lysis buffer ( Chen et al., 2004 ) to prepare embryo extracts. Equal
amounts of protein were used for Western blot analysis. The following pri-
mary antibodies were used: anti – E-cadherin antibody (1:10,000; Babb
and Marrs, 2004 ), anti-G ? antibody (1:5,000; Santa Cruz Biotechnology,
Inc.), and anti – ? -catenin antibody (1:250; Sigma-Aldrich).
GST pull-down and coimmunoprecipitation assays
HEK 293 cells were transiently cotransfected with cDNAs encoding G ? 13 a
and GST or the GST-tagged CT fragment of E-cadherin; or zebrafi sh G ? 13 a
and/or function. Although there is no signifi cant change in the
organization of fi lamentous actin of EVL cells in embryos with
altered G ? 12/13 signaling, these cells displayed defects in cell
shape and orientation ( Fig. 6 ), which may contribute to the epi-
bolic defects of the EVL. In addition, in embryos with excess
G ? 12/13 signaling, the punctate F-actin ring adjacent to the EVL
was signifi cantly reduced ( Fig. 6 G ), and abnormal thick actin
bundles, separated by F-actin – free regions, were frequently
found in the yolk ( Fig. 6, C and G ). We speculate that the thick
actin bundles may cause abnormal “ contractile ” forces that dis-
rupt the YCL; alternatively, these forces may create resistance to
vegetal pulling of the EVL. Altogether, the changes in actin ar-
chitecture resulting from altered G ? 12/13 activities could prevent
the EVL from undergoing active cell rearrangement and shape
changes, ultimately affecting normal EVL epiboly. In E-cadherin
mutants, in contrast, the abnormal actin fi bers are not observed,
and thus they are unlikely to be caused by a decrease in E-cadherin
function. Interestingly, although the deep cells in embryos with
altered G ? 12/13 signaling displayed severe defects in epiboly,
they did not exhibit corresponding changes in F-actin organiza-
tion and cell shape (unpublished data). This further underscores
the notion that epiboly of the deep cells might involve distinct
mechanisms. However, we cannot exclude the possibility that in
the deep cells, G ? 12/13 also infl uence epiboly by modulating the
actin cytoskeleton. Indeed, in differentiated leukocyte-HL60,
G ? 12/13 were shown to infl uence the actomyosin network during
retraction of the trailing edge ( Xu et al., 2003 ). It will be inter-
esting in the future to investigate how cell migration contributes
to epiboly in zebrafi sh. Furthermore, in Xenopus laevis , it has
been shown that two G protein – coupled receptors (GPCRs; the
phospholipid lysophosphatidic acid [LPA] receptor and Xfl op)
that couple to G ? 12/13 in some cell types ( Ishii et al., 2004 ) can
regulate expression of a calcium-dependent EP-cadherin and
modulate the assembly of cortical actin ( Lloyd et al., 2005 ; Tao
et al., 2005 , 2007 ). Therefore, it will be important in the future
to investigate if LPA functions in a similar manner in zebrafi sh.
In addition, we found that embryos overexpressing G ? 13
exhibited microtubule organization defects similar to those ob-
served for F-actin, showing thick bundles of microtubules sur-
rounded by areas devoid of microtubules ( Fig. S3 ). However,
embryos with reduced G ? 12/13 do not show signifi cant defects in
microtubule organization (unpublished data), which suggests
that at their normal expression levels, G ? 12/13 do not play an es-
sential role in microtubule stabilization in zebrafi sh.
G ? 12/13 are known to regulate cytoskeletal function via a
Rho-dependent signaling cascade. Several observations indicate
that G ? 12/13 appear to operate through the same signaling path-
way to regulate actin organization during EVL epiboly. First,
coexpression of a dominant-negative zebrafi sh PDZ RhoGEF,
Arhgef11, with G ? 13 signifi cantly reduced the formation of ac-
tin bundles, and suppressed the epiboly defects in the EVL
( Figs. 1 M and 7 ). Conversely, overexpression of Arhgef11 or a
constitutively active RhoA resulted in similarly abnormal actin
organization in the yolk, and impaired epiboly ( Fig. 7, D and H ;
and unpublished data). Notably, embryos expressing Arhgef11
did not exhibit the detachment of cells from the embryo surface
(unpublished data), and Arhgef11 LOF did not result in obvious
JCB • VOLUME 184 • NUMBER 6 • 2009 920
EY07135-13 and NIH grant 1 K99 RR024119-01. This work was supported
in part by the following NIH grants: GM77770 (to L. Solnica-Krezel),
EY10291 (to H.E. Hamm), and HL60678 (to H.E. Hamm).
Submitted: 23 May 2008
Accepted: 24 February 2009
Akhter , S.A. , L.M. Luttrell , H.A. Rockman , G. Iaccarino , R.J. Lefkowitz , and
W.J. Koch . 1998 . Targeting the receptor-Gq interface to inhibit in vivo
pressure overload myocardial hypertrophy. Science . 280 : 574 – 577 .
Arai , K. , Y. Maruyama , M. Nishida , S. Tanabe , S. Takagahara , T. Kozasa , Y. Mori , T.
Nagao , and H. Kurose . 2003 . Differential requirement of G ? 12 , G ? 13 , G ? q ,
and G ? ? for endothelin-1-induced c-Jun NH2-terminal kinase and extra-
cellular signal-regulated kinase activation. Mol. Pharmacol. 63 : 478 – 488 .
Arendt , D. , and K. Nubler-Jung . 1999 . Rearranging gastrulation in the name
of yolk: evolution of gastrulation in yolk-rich amniote eggs. Mech. Dev.
81 : 3 – 22 .
Babb , S.G. , and J.A. Marrs . 2004 . E-cadherin regulates cell movements and tis-
sue formation in early zebrafi sh embryos. Dev. Dyn. 230 : 263 – 277 .
Babb , S.G. , J. Barnett , A.L. Doedens , N. Cobb , Q. Liu , B.C. Sorkin , P.C. Yelick ,
P.A. Raymond , and J.A. Marrs . 2001 . Zebrafi sh E-cadherin: expression
during early embryogenesis and regulation during brain development.
Dev. Dyn. 221 : 231 – 237 .
Barth , A.I. , I.S. Nathke , and W.J. Nelson . 1997 . Cadherins, catenins and APC
protein: interplay between cytoskeletal complexes and signaling path-
ways. Curr. Opin. Cell Biol. 9 : 683 – 690 .
Behrens , J. 1999 . Cadherins and catenins: role in signal transduction and tumor
progression. Cancer Metastasis Rev. 18 : 15 – 30 .
Betchaku , T. , and J.P. Trinkaus . 1986 . Programmed endocytosis during epiboly
of Fundulus heteroclitus. Am. Zool. 26 : 193 – 199 .
Buhl , A.M. , N.L. Johnson , N. Dhanasekaran , and G.L. Johnson . 1995 . G ? 12 and
G ? 13 stimulate Rho-dependent stress fi ber formation and focal adhesion
assembly. J. Biol. Chem. 270 : 24631 – 24634 .
Chan , A.M. , T.P. Fleming , E.S. McGovern , M. Chedid , T. Miki , and S.A.
Aaronson . 1993 . Expression cDNA cloning of a transforming gene en-
coding the wild-type G ? 12 gene product. Mol. Cell. Biol. 13 : 762 – 768 .
Chen , S. , E.J. Dell , F. Lin , J. Sai , and H.E. Hamm . 2004 . RACK1 regulates spe-
cifi c functions of G ? ? . J. Biol. Chem. 279 : 17861 – 17868 .
Cheng , J.C. , A.L. Miller , and S.E. Webb . 2004 . Organization and function of
microfi laments during late epiboly in zebrafi sh embryos. Dev. Dyn.
231 : 313 – 323 .
Cooper , M.S. , and L.A. D ’ Amico . 1996 . A cluster of noninvoluting endocytic
cells at the margin of the zebrafi sh blastoderm marks the site of embry-
onic shield formation. Dev. Biol. 180 : 184 – 198 .
Doitsidou , M. , M. Reichman-Fried , J. Stebler , M. Koprunner , J. Dorries , D.
Meyer , C.V. Esguerra , T. Leung , and E. Raz . 2002 . Guidance of primor-
dial germ cell migration by the chemokine SDF-1. Cell . 111 : 647 – 659 .
Gilchrist , A. , M. Bunemann , A. Li , M.M. Hosey , and H.E. Hamm . 1999 . A dominant-
negative strategy for studying roles of G proteins in vivo. J. Biol. Chem.
274 : 6610 – 6616 .
Gohla , A. , R. Harhammer , and G. Schultz . 1998 . The G-protein G ? 13 but not
G ? 12 mediates signaling from lysophosphatidic acid receptor via epider-
mal growth factor receptor to Rho. J. Biol. Chem. 273 : 4653 – 4659 .
Halbleib , J.M. , and W.J. Nelson . 2006 . Cadherins in development: cell adhesion,
sorting, and tissue morphogenesis. Genes Dev. 20 : 3199 – 3214 .
Hart , M.J. , X. Jiang , T. Kozasa , W. Roscoe , W.D. Singer , A.G. Gilman ,
P.C. Sternweis , and G. Bollag . 1998 . Direct stimulation of the gua-
nine nucleotide exchange activity of p115 RhoGEF by G ? 13 . Science .
280 : 2112 – 2114 .
Hsu , H.J. , M.R. Liang , C.T. Chen , and B.C. Chung . 2006 . Pregnenolone sta-
bilizes microtubules and promotes zebrafi sh embryonic cell movement.
Nature . 439 : 480 – 483 .
Inbal , A. , J. Topczewski , and L. Solnica-Krezel . 2006 . Targeted gene expression
in the zebrafi sh prechordal plate. Genesis . 44 : 584 – 588 .
Ishii , I. , N. Fukushima , X. Ye , and J. Chun . 2004 . Lysophospholipid receptors:
signaling and biology. Annu. Rev. Biochem. 73 : 321 – 354 .
Kane , D.A. , and R.M. Warga . 2004 . Teleost gastrulation. In Gastrulation: From
Cells to Embryos. C.D. Stern, editor. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY. 157 – 169.
Kane , D.A. , M. Hammerschmidt , M.C. Mullins , H.M. Maischein , M. Brand , F.J.
van Eeden , M. Furutani-Seiki , M. Granato , P. Haffter , C.P. Heisenberg ,
et al . 1996 . The zebrafi sh epiboly mutants. Development . 123 : 47 – 55 .
and a myc-tagged full-length Arhgef11, or myc-tagged Arhgef11 mutants
lacking the RGS domain ( ? RGS) or DH and PH domains ( ? DHPH; Panizzi
et al., 2007 ). After serum starvation overnight, cells were washed twice
with serum-free medium and lysed in PBS containing 1% Igapal, 0.2% de-
oxycholate, and protease inhibitors. For the GST pull-down assay, protein
extracts were incubated with glutathione – Sepharose beads (GE Health-
care). The presence of G ? 13 a in the lysates and precipitate was detected
by anti-G ? 13 antibody (1:1,000; Lin et al., 2005 ). For coimmunoprecipita-
tion of G ? 13 a with myc-tagged WT and mutant Arhgef11, the lysates were
incubated with mouse anti-myc antibody (1:100; Santa Cruz Biotechnol-
ogy, Inc.) overnight at 4 ° C. Protein-A – Sepharose was then added for 2 h
at 4 ° C. Immunoprecipitates were immunoblotted with anti-G ? 13 (1:1,000)
and anti-myc (1:1,000; Fitzgerald) antibodies to detect the presence of
G ? 13 a and the myc-tagged proteins. G ? 13 antibody was provided by
D. Manning (University of Philadelphia, Philadelphia, PA).
Embryos were fi xed at appropriate stages in 4% PFA/PBS/4% sucrose at
4 ° C overnight. For F-actin staining, Alexa Fluor 546 phalloidin (1:100;
Invitrogen) was used as described previously ( Koppen et al., 2006 ). In addi-
tion, the following primary antibodies were used: anti – E-cadherin (1:1,000;
Babb and Marrs, 2004 ), anti – ? -tubulin (DM1A, 1:300; EMD), and anti –
? -catenin (1:250; Sigma-Aldrich). Embryos were then mounted in 75%
glycerol in PBS for analysis by microscopy.
Quantifi cation of cell shape and alignment
Confocal images of EVL cells in Phalloidin-stained embryos were collected
using a 20 × /0.8 NA objective lens (Carl Zeiss, Inc.). LWRs (a ratio of the
longest to shortest axis of the cell) and the angle of the long axis of the cells
relative to a line parallel to the EVL margin were determined using Object-
Image software. The angle of the long cell axes relative to the EVL margin
was plotted in a half-Rose diagram (Vector Rose; PAZ software).
Cell scattering assays in vivo
At the 256-cell stage, a single cell at the animal pole was injected with
0.5% rhodamine-dextran ( Warga and Kane, 2003 ). Embryos were
mounted on bridged slides fi lled with 2% methylcellulose, incubated at
28 ° C, and photographed every hour for 3 h to monitor the scattering of the
labeled cells. To measure cell scattering, we exported the images to
Object-Image. The exterior-most outlines of the labeled cells were marked
and the areas encompassing the dispersed cells were calculated.
Live embryos for still photography were mounted in 1.5 – 2% methylcellu-
lose at 28.5 ° C, whereas fi xed embryos were mounted in 75% glycerol/
PBS. Embryos were photographed using 5 – 20 × objectives on an Axio-
phot2 microscope or a Stereomicroscope (Stereo Discovery V12) equipped
with an Axiocam digital camera (all from Carl Zeiss, Inc.). Axiovision
software was used to capture the images. Confocal images were collected
on a laser scanning inverted microscope (LSM 510; Carl Zeiss, Inc.) using
a 40 × /1.30 NA oil objective with zoom 2 or a 20 × /0.8 NA objective
using the LSM 510 software. The acquired images were exported and
edited using Photoshop (Adobe), and then compiled in Illustrator soft-
Data are presented as the mean ± SEM. Statistical analyses were per-
formed using unpaired Student ’ s t tests with 2 tails, unequal variance.
Online supplemental material
Fig. S1 shows that the distribution of ? -catenin and ? -catenin is not changed
in cells expressing G ? 13 a. Fig. S2 shows that G ? 13 a overexpression does
not promote cell proliferation. Fig. S3 shows the microtubules in WT control
embryos and embryos overexpressing G ? 13 a revealed by anti – ? -tubulin
staining. Online supplemental material is available at http://www.jcb
We thank L. Solnica-Krezel and H.E. Hamm laboratory members for helpful
discussions, Nick Echemendia for technical support, Christina Speirs for
critical comments, and Christine Blaumueller for proofreading the manu-
script. We acknowledge Joshua Clanton, Heidi Beck, and Amanda Brad-
shaw for excellent fi sh care. We are grateful to Dr. David Manning for the
G ? 13 antibody.
Confocal experiments were performed in the Vanderbilt University Med-
ical Center Cell Imaging Core facility (supported by National Institutes of
Health [NIH } grant 1S10RR015682). F. Lin is supported by the training grant
921 G ? Download full-text
12/13 REGULATE ZEBRAFISH EPIBOLY • Lin et al.
Thisse , C. , and B. Thisse . 1998 . High resolution whole-mount in situ hybridiza-
tion. The Zebrafi sh Science Monitor . 5:8 – 9.
Trinkaus , J.P. 1951 . A study of the mechanism of epiboly in the egg of Fundulus
heteroclitus . J. Exp. Zool. 118 : 269 – 320 .
Trinkaus , J.P. 1984 . Mechanism of Fundulus epiboly: A current view. Am. Zool.
24 : 673 – 688 .
Trinkaus , J.P. 1993 . The yolk syncytial layer of Fundulus : its origin and history
and its signifi cance for early embryogenesis. J. Exp. Zool. 265 : 258 – 284 .
Voyno-Yasenetskaya , T.A. , A.M. Pace , and H.R. Bourne . 1994 . Mutant ? sub-
units of G 12 and G 13 proteins induce neoplastic transformation of Rat-1
fi broblasts. Oncogene . 9 : 2559 – 2565 .
Warga , R.M. , and C.B. Kimmel . 1990 . Cell movements during epiboly and gas-
trulation in zebrafi sh. Development . 108 : 569 – 580 .
Warga , R.M. , and D.A. Kane . 2003 . One-eyed pinhead regulates cell motility
independent of Squint/Cyclops signaling. Dev. Biol. 261 : 391 – 411 .
Wilkins , S.J. , S. Yoong , H. Verkade , T. Mizoguchi , S.J. Plowman , J.F. Hancock ,
Y. Kikuchi , J.K. Heath , and A.C. Perkins . 2008 . Mtx2 directs zebrafi sh
morphogenetic movements during epiboly by regulating microfi lament
formation. Dev. Biol. 314 : 12 – 22 .
Xu , J. , F. Wang , A. Van Keymeulen , P. Herzmark , A. Straight , K. Kelly , Y.
Takuwa , N. Sugimoto , T. Mitchison , and H.R. Bourne . 2003 . Divergent
signals and cytoskeletal assemblies regulate self-organizing polarity in
neutrophils. Cell . 114 : 201 – 214 .
Zalik , S.E. , E. Lewandowski , Z. Kam , and B. Geiger . 1999 . Cell adhesion and
the actin cytoskeleton of the enveloping layer in the zebrafi sh embryo
during epiboly. Biochem. Cell Biol. 77 : 527 – 542 .
Kane , D.A. , K.N. McFarland , and R.M. Warga . 2005 . Mutations in half baked/
E-cadherin block cell behaviors that are necessary for teleost epiboly.
Development . 132 : 1105 – 1116 .
Kaplan , D.D. , T.E. Meigs , and P.J. Casey . 2001 . Distinct regions of the cadherin
cytoplasmic domain are essential for functional interaction with G ? 12 and
? -catenin. J. Biol. Chem. 276 : 44037 – 44043 .
Kimmel , C.B. , W.W. Ballard , S.R. Kimmel , B. Ullmann , and T.F. Schilling .
1995 . Stages of embryonic development of the zebrafi sh. Dev. Dyn.
203 : 253 – 310 .
Koppen , M. , B.G. Fernandez , L. Carvalho , A. Jacinto , and C.P. Heisenberg .
2006 . Coordinated cell-shape changes control epithelial movement in
zebrafi sh and Drosophila . Development . 133 : 2671 – 2681 .
Kozasa , T. , X. Jiang , M.J. Hart , P.M. Sternweis , W.D. Singer , A.G. Gilman , G.
Bollag , and P.C. Sternweis . 1998 . p115 RhoGEF, a GTPase activating
protein for G ? 12 and G ? 13 . Science . 280 : 2109 – 2111 .
Lachnit , M. , E. Kur , and W. Driever . 2008 . Alterations of the cytoskeleton in all
three embryonic lineages contribute to the epiboly defect of Pou5f1/Oct4
defi cient MZspg zebrafi sh embryos. Dev. Biol. 315 : 1 – 17 .
Leptin , M. 2005 . Gastrulation movements: the logic and the nuts and bolts. Dev.
Cell . 8 : 305 – 320 .
Lin , F. , D.S. Sepich , S. Chen , J. Topczewski , C. Yin , L. Solnica-Krezel , and H.
Hamm . 2005 . Essential roles of G ? 12/13 signaling in distinct cell behaviors
driving zebrafi sh convergence and extension gastrulation movements.
J. Cell Biol. 169 : 777 – 787 .
Lloyd , B. , Q. Tao , S. Lang , and C. Wylie . 2005 . Lysophosphatidic acid signal-
ing controls cortical actin assembly and cytoarchitecture in Xenopus em-
bryos. Development . 132 : 805 – 816 .
McFarland , K.N. , R.M. Warga , and D.A. Kane . 2005 . Genetic locus half baked is
necessary for morphogenesis of the ectoderm. Dev. Dyn. 233 : 390 – 406 .
Meigs , T.E. , T.A. Fields , D.D. McKee , and P.J. Casey . 2001 . Interaction of G ? 12
and G ? 13 with the cytoplasmic domain of cadherin provides a mechanism
for beta -catenin release. Proc. Natl. Acad. Sci. USA . 98 : 519 – 524 .
Meigs , T.E. , M. Fedor-Chaiken , D.D. Kaplan , R. Brackenbury , and P.J. Casey .
2002 . G ? 12 and G ? 13 negatively regulate the adhesive functions of cad-
herin. J. Biol. Chem. 277 : 24594 – 24600 .
Montero , J.A. , L. Carvalho , M. Wilsch-Brauninger , B. Kilian , C. Mustafa , and
C.P. Heisenberg . 2005 . Shield formation at the onset of zebrafi sh gastru-
lation. Development . 132 : 1187 – 1198 .
Panizzi , J.R. , J.R. Jessen , I.A. Drummond , and L. Solnica-Krezel . 2007 . New
functions for a vertebrate Rho guanine nucleotide exchange factor in cili-
ated epithelia. Development . 134 : 921 – 931 .
Radhika , V. , and N. Dhanasekaran . 2001 . Transforming G proteins. Oncogene .
20 : 1607 – 1614 .
Riobo , N.A. , and D.R. Manning . 2005 . Receptors coupled to heterotrimeric G
proteins of the G ? 12 family. Trends Pharmacol. Sci. 26 : 146 – 154 .
Rohde , L.A. , and C.P. Heisenberg . 2007 . Zebrafi sh gastrulation: cell movements,
signals, and mechanisms. Int. Rev. Cytol. 261 : 159 – 192 .
Sadot , E. , I. Simcha , M. Shtutman , A. Ben-Ze ’ ev , and B. Geiger . 1998 . Inhibition
of beta-catenin-mediated transactivation by cadherin derivatives. Proc.
Natl. Acad. Sci. USA . 95 : 15339 – 15344 .
Schulte-Merker , S. , F.J. van Eeden , M.E. Halpern , C.B. Kimmel , and C.
Nusslein-Volhard . 1994 . no tail (ntl) is the zebrafi sh homologue of the
mouse T (Brachyury) gene. Development . 120 : 1009 – 1015 .
Shimizu , T. , T. Yabe , O. Muraoka , S. Yonemura , S. Aramaki , K. Hatta , Y.K. Bae ,
H. Nojima , and M. Hibi . 2005 . E-cadherin is required for gastrulation cell
movements in zebrafi sh. Mech. Dev. 122 : 747 – 763 .
Solnica-Krezel , L. 2005 . Conserved patterns of cell movements during verte-
brate gastrulation. Curr. Biol. 15 : R213 – R228 .
Solnica-Krezel , L. 2006 . Gastrulation in zebrafi sh – all just about adhesion? Curr.
Opin. Genet. Dev. 16 : 433 – 441 .
Solnica-Krezel , L. , and W. Driever . 1994 . Microtubule arrays of the zebra-
fi sh yolk cell: organization and function during epiboly. Development .
120 : 2443 – 2455 .
Solnica-Krezel , L. , A.F. Schier , and W. Driever . 1994 . Effi cient recovery
of ENU-induced mutations from the zebrafi sh germline. Genetics .
136 : 1401 – 1420 .
Strahle , U. , and S. Jesuthasan . 1993 . Ultraviolet irradiation impairs epiboly in
zebrafi sh embryos: evidence for a microtubule-dependent mechanism of
epiboly. Development . 119 : 909 – 919 .
Tao , Q. , B. Lloyd , S. Lang , D. Houston , A. Zorn , and C. Wylie . 2005 . A novel
G protein-coupled receptor, related to GPR4, is required for assembly
of the cortical actin skeleton in early Xenopus embryos. Development .
132 : 2825 – 2836 .
Tao , Q. , S. Nandadasa , P.D. McCrea , J. Heasman , and C. Wylie . 2007 . G-protein-
coupled signals control cortical actin assembly by controlling cadherin
expression in the early Xenopus embryo. Development . 134 : 2651 – 2661 .