T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 169, No. 5, June 6, 2005 777–787
The Rockefeller University Press$8.00
Essential roles of G
behaviors driving zebrafish convergence and
extension gastrulation movements
signaling in distinct cell
and Heidi Hamm
Diane S. Sepich,
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235
Department of Pediatrics, Northwestern University, Feinberg School of Medicine, Children’s Memorial Institute for Education and Research, Chicago, IL 60614
processes, however, their roles in vertebrate
gastrulation are largely unknown. Here, we show
that during zebrafish gastrulation, suppression of both
signaling by overexpressing dominant
negative proteins and application of antisense morpholino-
modified oligonucleotide translation interference disrupted
convergence and extension without changing embryonic
patterning. Analyses of mesodermal cell behaviors revealed
have been implicated in numerous cellularthat G
dorsalward migration during convergence independent
of noncanonical Wnt signaling. Furthermore, G
function cell-autonomously to mediate mediolateral cell
elongation underlying intercalation during notochord
extension, likely acting in parallel to noncanonical Wnt
signaling. These findings provide the first evidence that
have overlapping and essential roles in
distinct cell behaviors that drive vertebrate gastrulation.
are required for cell elongation and efficient
Gastrulation is a pivotal phase of vertebrate development during
which the body plan is established via a complex series of the
morphogenetic movements. Vertebrate gastrulation consists of
three main morphogenetic processes: epiboly, internalization of
presumptive mesendoderm, and convergence and extension
(C&E). In zebrafish gastrulae, C&E movements narrow the
germ layers mediolaterally and elongate them anteroposteriorly
to define embryonic axes. Mesodermal mediolateral cell inter-
calation, as well as directed dorsal and anterior cell migration
contribute to this morphogenetic processes (Warga and Kimmel,
1990; Trinkaus et al., 1992; Jessen et al., 2002; Glickman et al.,
2003; Ulrich et al., 2003). How this diversity of gastrulation
cell behaviors is generated remains poorly understood.
Noncanonical Wnt signaling, an equivalent of the planar
cell polarity signaling in
is a major regulator of the mediolateral cell polarization required
for cell intercalation in frog and fish, and fast dorsal migration
in fish gastrulae (Keller, 2002; Myers et al., 2002b; Wallingford
et al., 2002). Mutants of several genes involved in this path-
way, such as
; Topczewski et al., 2001),
Heisenberg et al., 2000) display shortened axis and defective
mesodermal cell polarization. Recent evidence indicates that
Heterotrimeric G proteins may participate in the Wnt/Ca
branch of the noncanonical pathway, which involves intracellular
release and activation of PKC (Sheldahl et al., 1999;
Malbon et al., 2001). The Wnt signaling pathway is activated
by the binding of Wnt ligands to the Frizzled receptors, which
have seven transmembrane domains, a structural characteristic
of G protein–coupled receptors (GPCRs). There is evidence
that like GPCRs, Frizzled receptors may activate G proteins to
mediate their signal transduction. In cultured cells, coupling of
the Frizzled receptor to G
(Liu et al., 1999; Liu et al., 2001; Ahumada et al., 2002). In
addition, it has been shown that G proteins are involved in Wnt
signaling pathways that mediate gastrulation. Expression of
pertussis toxin (which ADP-rybosylates G
uncouples them from their cognate receptors) disrupts tissue
seen with Xfz7 depletion. Moreover, PKC can rescue the defect
in tissue separation in both Xfz7-depleted and PTX-injected
; Jessen et al., 2002),
, and G
has been reported
gastrulation, an effect also
Correspondence to Lilianna Solnica-Krezel: lilianna.solnica-krezel@Vanderbilt.
edu; or Heidi Hamm: firstname.lastname@example.org
Abbreviations used in this paper: C&E, convergence and extension; dpf, days
postfertizilation; GPCR, G protein–coupled receptor; HEK, human embryonic
kidney; hpf, hours postfertizilation; LWR, length to width ratio; MO, morpholino-
modified oligonucleotide; Rok, Rho kinase; WT, wild-type.
The online version of this article contains supplemental material.
on March 18, 2013
Published May 31, 2005
Supplemental Material can be found at:
JCB • VOLUME 169 • NUMBER 5 • 2005778
embryos, suggesting that PTX-sensitive G proteins and PKC
are involved in
gastrulation movements (Winklbauer
et al., 2001). In addition, PKC
zled receptors, possibly through G proteins and Dishevelled to
regulate C&E movements in
noshita et al., 2003). Furthermore, G
canonical Wnt and PCP signaling in
al., 2005). Recently, it has been reported that G
may also play important roles in C&E movements. In
gastrulae, inhibition of G
signaling by overexpression of
(which sequester free G
that resulted from activation of Wnt11/Xfz7 (Penzo-Mendez et
al., 2003). In addition, inhibition of G
dorsal marginal zone resulted in gastrulation arrest. How-
ever, exactly which G
-proteins are involved in Wnt-PCP–
mediated gastrulation remains unknown.
G proteins consist of four classes: G
(Simon et al., 1991). G
vergent G protein family and have been implicated in numer-
ous cellular processes such as Rho-mediated cytoskeletal rear-
rangements, thereby affecting cell shape and migration (Buhl et
al., 1995; Gohla et al., 1999; Sugimoto et al., 2003). Studies in
indicate that G
trulation, as inactivation of the
impairs cell shape changes underlying mesoderm
internalization during gastrulation (Parks and Wieschaus,
1991). In mice, disruption of G
at midgestation, due to the failure of endothelial cells to form
an organized vascular system (Offermanns et al., 1997). In ad-
have been shown to induce primitive endoderm
formation in mouse F9 cells (Lee et al., 2004). However, the
role of G
in vertebrate gastrulation has not been analyzed.
Here, we used zebrafish as a model to investigate the role of
in early vertebrate embryogenesis. Using dominant nega-
tive receptor blocking peptides and antisense morpholino oligo-
nucleotides (MOs), we demonstrate that G
overlapping and essential roles in C&E. Cell movement analyses
show that G
signaling regulates slow dorsal migration of lat-
eral mesoderm cells independent of noncanonical Wnt signaling.
In the notochord, G
are required for mediolateral cell inter-
calation, acting cell-autonomously, and likely in parallel to nonca-
nonical Wnt signaling. Our studies for the first time suggest a cen-
tral role for G
signaling in generating the diversity of
gastrulation cell behaviors in vertebrate embryos.
are activated by Friz-
(Kuhl et al., 2001; Ki-
is required for both the
) rescued C&E defects
signaling in the
subunits are the most di-
signaling plays a role in gas-
gene led to embryonic death
Cloning and characterization of zebrafish
gna12 and gna13 genes
One gene encoding G
(referred to as
) encoding G
a and G
b share 81% identical and 91% similar
amino acid residues with each other, and have 90–93% se-
quence similarity to human G
shares 81% identical and 91% similar amino acid residues with
(Fig. S1, available at http://www.jcb.org/cgi/
) and two para-
were found in ze-
. The zebrafish G
In cultured cells, mammalian G
formation via a RhoA/Rho kinase (Rok)-dependent pathway
(Buhl et al., 1995; Gohla et al., 1998). To evaluate if zebrafish
have similar activities, wild-type (WT) and constitu-
tively active G
mutant proteins were transiently expressed
in human embryonic kidney (HEK) cells. Cells overexpressing
either WT human or zebrafish G
F) or constitutively active human or zebrafish G
and G) displayed stress fibers even in the absence of agonist
stimulation. Formation of stress fibers was blocked by pretreat-
ment with 10
M of Rok inhibitor, Y-27623 (Fig. 1 H; Uehata
et al., 1997). These results indicate that like their human coun-
terparts, zebrafish G
can promote actin rearrangements in
cultured cells, through a RhoA/Rok-dependent pathway.
induce stress fiber
(Fig. 1, C, E, and
(Fig. 1, D
Expression of zebrafish
Whole-mount in situ hybridization revealed that
transcripts are maternally deposited (Fig. 2, A–C).
Accordingly, high levels of G
?12 and G?13 proteins were de-
tected at the 8 cell stage by immunohistochemistry using anti-
bodies that recognize the last 11 aa of G?12 or G?13 (not de-
picted). During blastula and gastrula stages, transcripts of all
Figure 1. Overexpression of human and zebrafish G?12/13 promoted stress
fiber formation. Stress fiber formation was determined in HEK 293 cells
transiently transfected with cDNAs encoding GFP (A and B), WT human
(C) or zebrafish (E and F), constitutively active human (D) or zebrafish (G
and H) G-proteins. Cells were stimulated with (B) or without (A, and C–H)
10 nM thrombin for 10 min, and then stained with 0.17 ?M Rhodamine-
phalloidin. 10 ?M Y-27632 was used to block Rho kinase activity (H).
of gna12, gna13a, and gna13b genes during zebrafish embryogenesis
was detected by whole mount in situ hybridization. All embryos are shown
in lateral view; animal pole is up for 8 cell (A–C), sphere stage (D–F), and
80% epiboly (G–I) embryos; dorsal is toward the right for 80% epiboly;
anterior to the left for embryos at 1 and 2 dpf (J–O).
Expression of Zebrafish gna12 and gna13 genes. Expression
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G?12/13 IN ZEBRAFISH GASTRULATION • LIN ET AL.779
three genes are present ubiquitously throughout embryo (Fig.
2, D–I). By 1–2 d postfertilization (dpf), the expression be-
comes confined to anterior body regions (Fig. 2, J–O).
Interference with G?12/13 function
disrupts gastrulation movements
To investigate the function of the zebrafish G?12 and G?13 pro-
teins in embryonic development, we used two strategies to in-
hibit G?12/13 signaling. First, we overexpressed peptides encod-
ing the last 11 or 50 COOH-terminal aa of both proteins,
referred to as CT-peptides. The COOH-terminal region of G
protein ? subunits has been shown to be a major binding site of
G proteins to their cognate receptors (Hamm, 1998). Overex-
pression of CT-peptides in cells or mice has been shown to
block competitively the receptor sites that normally bind to G
proteins, leading to specific blockade of the respective G? sig-
naling (Akhter et al., 1998; Gilchrist et al., 1999, 2001; Feld-
man et al., 2002; Arai et al., 2003). Strikingly, compared with
their control siblings, embryos injected with synthetic RNAs
encoding the CT-peptides (the last 11 aa) of G?12 or G?13
(G?12-CT, G?13-CT) exhibited shortened body axes with
broader notochords and somites (Fig. 3, C–F), as well as de-
layed epiboly (unpublished data). At 3 dpf, these embryos re-
mained shorter, and frequently displayed synophthalmia or cy-
clopia (Fig. 3, P–S), phenocopying defects often associated
with defective C&E movements in silberblick (wnt11) or trilo-
bite (strabismus) noncanonical Wnt signaling mutants (Heisen-
berg et al., 2000; Jessen et al., 2002). Examination of expres-
sion patterns of tissue specific markers confirmed that embryos
expressing G?12-CT or G?13-CT displayed broader neural plate
and notochord. Furthermore, prechordal mesendoderm was po-
sitioned more posteriorly with respect to the anterior edge of
the neural plate during early segmentation stage (Fig. 3, I–L),
suggestive of impaired anterior migration of this cell popula-
tion (Heisenberg et al., 2000; Topczewski et al., 2001; Jessen et
al., 2002; Marlow et al., 2002). Both severity and penetrance of
the observed phenotypes increased with the dose of G?12-CT or
G?13-CT RNAs. Whereas 15% of embryos injected with 0.5 ng
G?12-CT or G?13-CT RNA showed morphology consistent
with impaired C&E movements, 60% and 70% of embryos in-
jected with 1 and 1.5 ng G?12-CT or G?13-CT RNA showed
similar C&E defects, respectively (Fig. 3 M). These data sug-
gest that G?13 and G?12 both function in C&E movements. In
contrast, C&E defects were not observed in embryos injected
C&E during zebrafish gastrulation. (A–L) Embryos express-
ing the COOH-terminal peptides of G?s, G?12, or G?13
at 1–2 somite stage. (A–F) Images of live embryos. D,
dorsal; nc, notochord; white arrows point to the anterior
and posterior limits of the anteroposterior embryonic
axis; lateral view (A, C, and E); dorsal views, animal
pole up (B, D, and F). (G–L) Expression of prechordal
plate (hgg1, red), neural plate boundary (dlx3), rhom-
bomeres 3 and 5 (krox20), midline (shh), somites
(deltaC) markers. Dorsoanterior view (G, I, and K); dorsal
view (H, J, and L); (*), marks notochord; np, neural plate.
(M) Frequencies of embryos overexpressing CT-peptides
with C&E defects. Number on the top of the bar indicates
the number of injected embryos from several separate
experiments. (N and O) Confocal images of embryos
overexpressing HA-tagged G?12-CT or G?13-CT immuno-
stained with anti-HA antibody. White arrows indicate
enrichment of staining on the membrane. (P–S) WT (P
and R) and G?13-CT expressing (Q and S) embryos at 3
dpf. ey, eye; ventral (P and Q), and lateral views (R and S),
anterior to the left.
Dominant negative G?12/13 CT-peptides disrupt
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JCB • VOLUME 169 • NUMBER 5 • 2005780
with up to 2 ng RNA encoding G?s CT peptide (a different
class of G? subunit; Fig. 3, A, B, G, and H), suggesting the
specific involvement of G?12/13 in gastrulation. Similarly, over-
expression of the COOH-terminal 50 aa of G?12 or G?13, con-
jugated with HA-tag, produced similar gastrulation defects (not
depicted). Anti-HA immunohistochemistry showed these longer
peptides were robustly expressed in the cytosol, with slight en-
richment on the membrane (Fig. 3, N and O).
In a complementary approach, we used antisense MOs
(Nasevicius and Ekker, 2000) to block G?12/13 translation in ze-
brafish. The endogenous G?12 and G?13 in zebrafish blastulae
were detected predominantly on the cell membranes and in a
punctate pattern in the cytosol (Fig. 4, A and C). When em-
bryos were injected with MO (4 ng) targeting gna12 transcript,
the expression of G?12 was strongly reduced (Fig. 4 B). Like-
wise coinjection of MOs targeting gna13a and gna13b tran-
scripts (4 ng each) decreased the level of both G?13 proteins at
late blastula stage (Fig. 4 D). Together, these results demon-
strated that the MOs we designed effectively blocked gna12 or
gna13 translation. Injections of up to 20 ng/embryo of MO
against any single gna12/13 transcript (either gna13a or
gna13b alone or in combination, or gna12 alone) had no obvi-
ous effect on embryonic development probably due to func-
tional redundancy between G?12 and G?13 in zebrafish (Fig. 4
E and not depicted). However, when the embryos were injected
with a mixture of MOs against gna13a, gna13b, and gna12
(3MOs, 4 ng each), 76% of embryos showed impaired C&E
movements as judged by alteration of embryonic morphology
and gene expression patterns (Fig. 4, F and H; n ? 214). The
phenotypes resulting from MO interference were very similar
to those caused by overexpression of CT-peptides. To test
whether the above gastrulation defects are due to specific inter-
ference of MOs with G?12/13 function, we coinjected a sub-
threshold dose of human GNA13 RNA along with three MOs.
Because the MO targeted nucleotide sequence of the human
gene diverges from the sequences of zebrafish homologues, it
cannot be blocked by the MOs used here. Injection of 10–20 pg
human GNA13 RNA had no obvious effect on zebrafish mor-
phogenetic movements of epiboly and C&E (unpublished
data). However, when this amount of human GNA13 RNA was
coinjected with the combination of the three MOs, the percent-
age of embryos with gastrulation defects decreased from 76%
to 20% (Fig. 4 H). Whereas 75% of embryos (n ? 120) in-
jected with the three MOs exhibited a very short body axis, and
some degree of brain degeneration by 1 dpf, coexpression of
human G?13 largely suppressed the axis extension defects with
75% embryos showing an almost normal body length (Fig. 4,
I–K; n ? 138). As shown by morphometric analysis, embryos
coinjected with three MOs exhibited a reduced body length of
2015 ? 50 ?m (n ? 22), compared with embryos injected with
a single MO (2962 ? 10 ?m, n ? 10). By contrast, embryos
coinjected with human GNA13 RNA and three MOs showed
significantly restored body length (2432 ? 47 ?m, n ? 22, P ?
7.6 ? 10?9). However, only modest suppression of brain de-
generation was observed. These results indicate that the mor-
phogenetic defects are a specific consequence of the interfer-
ence with G?12/13 function, whereas the neural degeneration
phenotype might be a nonspecific defect, often associated with
MO injection (Nasevicius and Ekker, 2000). In addition, ze-
brafish and human G?13 share a conserved activity in gastrula-
tion. Interestingly, the effects of CT peptides and MOs were
synergistic. Although very few embryos showed C&E defects
when injected with moderate doses of either G?13-CT RNA
(9%, 500 pg, n ? 126) or gna13a-MO (0%, 5 ng, n ? 120),
coinjection of both resulted in 65% embryos displaying C&E
defects (65%, n ? 73). Comparable results were found when
embryos were coinjected with G?13-CT RNA and gna13b-MO
or G?12-CT RNA and gna12-MO (unpublished data).
Similar C&E defects were also observed in embryos
overexpressing WT G?13a, G?13b, or G?12 proteins, and oc-
curred in a dose-dependent manner (Fig. 4 G and not depicted).
Co-injection of G?12/13 specific MOs suppressed gastrulation
defects resulting from overexpression of G?12/13 (not depicted).
This indicates that the phenotypes caused by G?12/13 are due to
specific interference with their functions, and provides further
support for the effectiveness of these MOs. Together these re-
sults show that both reduction and excess G?12/13 function im-
pair the C&E gastrulation process.
Interference with G?12/13 function does
not alter cell fate specification during
Gastrulation defects might be a consequence of altered embry-
onic patterning and consequent changes in cell movements, or
might be due to defects in cell movements alone (Myers et al.,
2002b). Therefore, we tested whether dorsoventral patterning
is affected in G?12/13-CT or MO injected embryos by analyzing
expression of dorsoventral patterning genes, bmp4 and chordin
(Hammerschmidt and Mullins, 2002). Our results revealed that
bmp4 expression was not altered in early and late gastrulae in-
jected with three MOs (Fig. 4 M, n ?34, and not depicted).
Likewise, expression of chordin gene encoding a Bmp antago-
nist was confined to its normal dorsal expression domain dur-
ing early gastrulation in embryos injected with the combination
of 3MO (Fig. 4 O, n ? 32), or with RNAs encoding G?12/13-CT
peptides (not depicted). Moreover, embryos injected with CT
peptide RNA or 3MO displayed normal expression of several
cell type specific markers at late gastrulation, consistent with
normal cell fate specification (Fig. 3, I–L; Fig. 4 F). Finally,
cell tracing experiments revealed that the labeled lateral meso-
dermal cells acquired somitic fates in G?12/13-depleted embryos
(not depicted), consistent with their positions in the early gas-
trula (Sepich et al., 2000). Based on these results, we conclude
that morphogenetic defects observed in G?12/13 depleted em-
bryos are likely not associated with significant patterning or
cell fate changes during gastrulation.
G?12/13 are required for efficient directed
cell migration during early dorsal
Shortened anteroposterior and enlarged mediolateral dimen-
sions of the embryonic axes in G?12/13-depleted gastrulae could
be a consequence of defective C&E movements (Sepich et al.,
2000). Recent studies reveal that convergence movements in
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G?12/13 IN ZEBRAFISH GASTRULATION • LIN ET AL.781
zebrafish mesoderm are accomplished by a stereotyped se-
quence of cell behaviors, including slow and fast directed cell
migration (Jessen et al., 2002). To investigate if any of the con-
vergence cell behaviors were altered in G?12/13-depleted em-
bryos, we performed Nomarski time-lapse analyses in WT (6
embryos, 144 cells) and 3MO-injected (6 embryos, 134 cells)
embryos at midgastrulation. Analysis of total cell speed, ac-
counting for movement in all directions, revealed that in G?12/13-
depleted gastrulae, cells moved at a reduced speed (70% of
WT total speed; P ? 9.8 ? 10?27; Fig. 5 B). Interestingly, the
net dorsal speed of G?12/13-depleted cells was especially
strongly compromised, accounting only for 28% of the WT net
dorsal speed (P ? 2.1 ? 10?13, Fig. 5 B). Further analysis of
cell migration paths revealed that, similar to the WT cells,
G?12/13-depleted cells migrated predominantly dorsally (Fig. 6
A). However, compared with WT, these cells more frequently
changed their movement direction (Fig. 6, A and B), at the ex-
pense of movement in the dorsal direction (Fig. 6 C). To deter-
mine how efficiently WT and 3MO cells corrected their path
direction when they were off-course, we examined cells mov-
ing toward dorsal, animal, ventral or vegetal direction (? 15?)
and assayed the direction of their next step (Fig. 6, D–G). We
found that in WT embryos, cells moving dorsally largely main-
tained this direction in the next step. Moreover, WT cells that
had been moving in the animal or vegetal direction turned to-
ward dorsal in the next movement step, very few cells from
these populations moved away from dorsal. By contrast, equiv-
alent cell populations in embryos injected with 3MO were less
persistent in dorsal movement (Fig. 6 D). Moreover, when
these cells moved in animal or vegetal direction, they less fre-
quently corrected their movement toward dorsal compared
in dorsoventral patterning. (A–D) Confocal immunostaining images of
blastoderm injected with MOs against gna12 (B) or gna13a and gna13b
(D; 16 embryos, from two independent experiments). (E–G) In situ hybrid-
ization with hgg1 (red), dlx3, shh, and krox20 of embryos injected with
either a single MO (E; 1MO) or a combination of three MOs against
G?12/13 (F; 3MO), or embryos overexpressing G?13a (G; 100 pg) at 1
somite stage. Animal pole view, dorsal toward the bottom. np, neural
plate; D, dorsal. (H) Frequencies of embryos with C&E defects after the
injection of MOs alone or with human GNA13 RNA. Number on top of
the bar indicates the number of injected embryos. Data were compiled
from several separate experiments. (I–K) Group images of live embryos
injected with single (I; 1MO) or three (J; 3MO) MOs, or 3MO together
with human GNA13 RNA (K) at 25 h postfertilization (hpf). (L–O) Expression
of bmp4 (L and M; lateral view, dorsal on the right), and chordin (N and O,
animal view) in WT and 3MO-injected embryos at early gastrulation (6 hpf).
D, dorsal; AP, animal pole.
G?12/13-depleted embryos exhibit C&E defects without changes
migration. (A) Domains of C&E movements in zebrafish gastrulae (Myers et
al., 2002b). Yellow arrows in the dorsal region indicate strong extension
movements with little convergence. Light and dark blue arrows indicate
domains of slow and fast C&E, respectively. (B) Total and net dorsal speed of
lateral mesodermal cells at 80% epiboly were determined in WT and 3MO-
injected embryos. Net speed in these experiments is somewhat higher than
previously reported for this domain (Jessen et al., 2002). (C) A schematic
representation of the method used to measure cell shape (LWR, length-to-
width ratio). (D) LWR of lateral mesodermal cells in WT, 1MO-, or 3MO-
injected embryos at 80% epiboly (8.5 hpf) and tailbud (TB, 10 hpf) stages.
G?12/13 signaling is required for efficient directed dorsal
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JCB • VOLUME 169 • NUMBER 5 • 2005 782
with WT cells (Fig. 6, E and F). Cells moving ventrally made
large turns, and no clear difference is apparent between WT
and 3MO cells (Fig. 6 G). Consequently, cells in G?12/13-
depleted gastrulae migrated less efficiently toward dorsal. In
support of the notion that this defect is specific to the depletion
of G?12/13 proteins, cells in embryos injected with an equivalent
dose of gna13a-MO (12 ng) migrated dorsally at comparable
speed to WT cells (unpublished data). Together, these studies
revealed that G?12/13 function is required for directed dorsal mi-
gration underlying the early, slow convergence movements of
lateral mesodermal cells.
Next, we aimed to identify the cellular basis of this
movement defect. We analyzed the shapes (length to width ra-
tio [LWR], where the length and width represent the largest
measurable distances along each cellular axis, Fig. 5 C) of
G?12/13-depleted lateral mesodermal cells at mid- and late-
gastrulation. In WT embryos at midgastrulation, lateral meso-
dermal cells exhibited an average LWR of 1.41 ? 0.28 (250
cells, 6 embryos), and became elongated at late gastrulation
with LWR of 1.72 ? 0.4 (487 cells, 16 embryos, P ? 3.2 ?
10?32; Fig. 5 D), in agreement with previous findings (Top-
czewski et al., 2001; Jessen et al., 2002). Cells in embryos in-
jected with a high dose of a single MO (12 ng, any of the
three) exhibited shapes not significantly different from those
observed in WT embryos at equivalent stages, at midgastrula-
tion (with LWR of 1.4 ? 0.26, 182 cells, 6 embryos; P ? 0.7)
and at tailbud (with LWR of 1.67 ? 0.28, 102 cells, 6 em-
bryos, P ? 0.2; Fig. 5 D). In contrast, in embryos injected with
the combination of three MOs against gna12/13 transcripts,
mesodermal cells were significantly less elongated at midgas-
trulation with LWR of 1.2 ? 0.14 (443 cells, 9 embryos, P ?
7.0 ? 10?24). At late gastrulation, these cells remained
rounder than equivalent cells in the control embryos with
LWR of 1.43 ? 0.27 (158 cells, 6 embryos, P ? 4.3 ? 10?24;
Fig. 5 D). These results revealed that G?12/13 signaling medi-
ates cell elongation associated with the early convergence
movements. Notably, the requirement for G?12/13 function for
moderate elongation of lateral mesodermal cells is manifest al-
ready at midgastrulation, and thus before the noncanonical
Wnt signaling is thought to become essential during conver-
gence (Jessen et al., 2002). Collectively, these results indicate
that G?12/13 function is required for normal cell elongation and
effective directed migration during C&E movements.
Interference with G?12/13 function
disrupts mediolateral cell intercalation
Mediolaterally oriented intercalation of cells at the dorsal mid-
line drives robust axial extension in zebrafish (Crawford et al.,
2003; Glickman et al., 2003) and Xenopus gastrulae (Shih and
Keller, 1992). During gastrulation, notochord precursor cells
elongate, align mediolaterally and intercalate between one an-
other to lengthen the notochord anteroposteriorly and narrow
its mediolateral dimension, decreasing from the initial width of
4–5 to 1–2 cells (Glickman et al., 2003). To investigate whether
G?12/13 signaling is required for mediolateral intercalation of
midline cells, we analyzed shape (LWR) and orientation of no-
tochord cells at the 4 and 6 somite stage. We found that at the 4
somite stage, the WT notochord was one to two cells wide, and
cells were aligned mediolaterally (at an average angle of 6 ? 5?
relative to a line perpendicular to the embryonic axis as repre-
sented by the notochord) and were well elongated with LWR of
3.24 ? 1.19 (166 cells, 6 embryos; Fig. 7, A–C). At the 6
somite stage, notochord cells were further elongated with LWR
of 4.84 ? 1.92 and aligned mediolaterally with an angle of 4 ?
3? (241 cells, 8 embryos; Fig. 7, A and B). In contrast, in G?12/13-
depleted embryos at the 4 somite stage, the notochord was
two or three cells wide revealing an intercalation defect, and
these cells were rounder, exhibiting an average LWR of 2.25 ?
0.79 (254 cells, 9 embryos, P ? 3.3 ? 10?18; Fig. 7, A and D),
however, these cells still aligned mediolaterally but at a slightly
greater angle of 11 ? 12? (P ? 4.2? 10?9; Fig. 7, B and D). At
the 6 somite stage, notochord cells in G?12/13-depleted embryos
continued to show impaired elongation and orientation defects
with LWR of 2.95 ? 1.2 and angle of 8 ? 8? relative to the me-
Nomarski time-lapse analyses were performed on lateral mesodermal cells
of WT and 3MO-injected embryos at 80% epiboly. (A) Representative cell
shape changes and migration paths of a few cells from a single embryo.
Cell shape was drawn for these selected cells at every 2.5-min interval.
(B) Movement direction change in degrees. (Inset) A–C represent positions
of the cell analyzed. A cell establishes a direction from position A to B,
then moves to position C. Movement direction change presents angle
change of position B to C. (C–G) Actual directions of cell movement (every
60-s interval, grouped into 30? sectors) shown in percentage of cells in
WT (blue line) and 3MO-injected embryos (violet line). Directions of all
cell movement events throughout the time lapse (C; 3,937 events in WT,
626 events in 3MO-injected embryo). Cell movement directions during the
next 60-s interval of the population of cells moving toward dorsal (D; WT,
1,139 events; 3MO, 74 events), animal (F; WT, 149 events; 3MO, 35
events), vegetal (E; WT, 151 events; 3MO, 62 events) or ventral (G; WT,
62 events; 3MO, 35 events) direction (? 15?). Insets: starting directions.
An, animal; Vg, vegetal; D, dorsal; V, ventral.
G?12/13 signaling is essential for error-free directed migration.
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G?12/13 IN ZEBRAFISH GASTRULATION • LIN ET AL.783
diolateral axis (202 cells, 6 embryos, P ? 5.1? 10?31; Fig. 7, A
and B). These results indicate G?12/13 signaling is also required
for elongation and intercalation of axial mesodermal cells, and
consequently for C&E of embryonic axis.
To test the cell-autonomy of G?12/13 function in noto-
chord C&E, we transplanted cells at blastula stage from do-
nors injected with rhodamine-dextran alone or with 3MOs
into WT hosts injected with mgfp RNA and 3MOs or mgfp
RNA alone, and determined shape and orientation of cell
bodies in notochord at 4 somite stage. We found that trans-
planted G?12/13-depleted cells exhibited rounder shapes (with
LWR of 2.50 ? 1.21, 39 cells, 7 embryos, P ? 4.49 ? 10?9)
and more random orientation (with angle of 13.91 ? 11.66?,
P ? 7.0 ? 10?4) even in WT environment (with LWR of
4.01 ? 1.47 and angle of 6.84 ? 6.66?, 167 cells; Fig. 7, K,
M, and N). Conversely, WT cells displayed normal elon-
gated shape and orientation (with LWR of 4.15 ? 1.26 and
angle of 7.43 ? 5.89?, 43 cells, 5 embryos; Fig. 7, K, M, and
N) even in G?12/13 signaling-depleted hosts (with LWR of
2.45 ? 0.8 and angle of 13.21 ? 11.65?, 157 cells, P ? 3.3 ?
10?11 for LWR and P ? 1.6 ? 10?5 for angle compared with
WT cells; Fig. 7, L–N). These results reveal a cell-autono-
mous requirement for G?12/13 in mediolateral cell elongation
during C&E of notochord.
The relationship between G?12/13 and the
noncanonical Wnt signaling during
The noncanonical Wnt signaling pathway mediates mediolat-
eral cell polarization underlying normal C&E movements
(Keller, 2002; Myers et al., 2002b; Wallingford et al., 2002).
The morphological changes in embryos with altered G?12/13
signaling are strikingly similar to those reported for mutants of
slb (wnt11) and knypek (glypican4/6) that resulted from the dis-
ruption of the noncanonical Wnt signaling (Heisenberg et al.,
2000; Topczewski et al., 2001). Moreover, G?12/13-depleted
mesodermal cells also exhibited impaired cell elongation dur-
ing late gastrulation (Fig. 5), similar to embryos overexpress-
ing dominant negative Rok2 and trilobite and knypek mutants
(Topczewski et al., 2001; Jessen et al., 2002; Marlow et al.,
2002). However, our studies showed that G?12/13 are required
for two types of directed cell migration for which noncanonical
signaling does not appear to be required: early slow conver-
gence and epiboly (Fig. 5; unpublished data), suggesting that
required for efficient mediolateral intercalation
and interacts with Wnt signaling. (A and B)
LWR of notochord cells (A) and the angle of
the long axis of notochord cells relative to a
line perpendicular to the embryonic axis (B) in
WT or knym119 embryos injected with mgfp
RNA alone (control) or with 3MOs at 4 and 6
somite stages. (C–F) Representative images of
notochord of WT or knym119 embryos at 4
somite stage injected with mgfp RNA alone or
with 3MOs. Yellow arrows indicate notochord
boundary and a few notochord cells in each
group were artificially filled with light blue for
illustration, their membrane were outlined with
violet lines. (G–J) 1-somite knym119 embryos
(G and H) and knym119 embryos injected with
3MOs (I and J) were labeled with hgg1, dlx3,
krox20, and shh. Dorsoanterior view (G and I);
lateral view (H and J); (*), marks notochord.
Black lines indicate the width of the neural
plate; red arrows show prechordal plate and
red arrowheads point the most anterior and
posterior extent of the prechordal plate, which
is abnormally elongated in 3MO-injected
embryos. (K and L) Representative confocal
images of transplanted WT (K) and 3MO-
injected (L) donor-derived notochord cells
(red), surrounded by mGFP labeled host cells
(green). (M and N) LWR and the angle of trans-
planted and donor notochord cells with respect
to a line perpendicular to the embryonic axis.
G?12/13 signaling is autonomously
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JCB • VOLUME 169 • NUMBER 5 • 2005784
G?12/13 mediate these movements independent of Wnt signal-
ing. To elucidate the functional relationship between G?12/13
signaling and noncanonical Wnt signaling during gastrulation,
we analyzed the effect of modulation of G?12/13 signaling on
noncanonical Wnt signaling mutant phenotypes.
The small GTPase Rho, is the main effector of G?12 and
G?13 (Buhl et al., 1995) and is also implicated in noncanonical
Wnt signaling (Habas et al., 2001). Accordingly, Rho down-
stream mediator Rok2 can partially suppress the slb (wnt11)
gastrulation defects (Marlow et al., 2002). To address whether
zebrafish G?12/13 can modulate Wnt11 signaling during gastru-
lation, we injected RNAs encoding G?13 to enhance, or G?13-
CT to inhibit the function of G?13, in homozygous slb
(wnt11)tz216/tz216 embryos (Heisenberg et al., 2000). However,
neither various levels of excess nor deficit of G?13 signaling
could suppress the gastrulation defects in slb (wnt11)?/? em-
bryos. Rather, both perturbations of G?13 signaling exacerbated
the slb (wnt11)?/? phenotype (unpublished data). We also per-
formed molecular epitasis experiments by injecting embryos
obtained from knym119 heterozygotes, carrying a null mutation
in the glypican 4/6 gene (Topczewski et al., 2001), with a small
dose of synthetic RNAs encoding G?13 or MOs against gna13
or three MOs against both gna13 and gna12. Neither overex-
pression of G?13 nor down-regulation of G?13 signaling sup-
pressed kny(?/?) C&E defects. Instead, depletion of G?12/13 sig-
naling by injection with three MOs enhanced the kny(?/?)
defects of neuroectoderm convergence and of anterior pre-
chordal mesendoderm migration (Fig. 7, G–J). Finally, the ex-
pression pattern of wnt11, kny, or tri was unchanged in em-
bryos with excess or deficit of G?12/13 signaling (unpublished
data). These results are consistent with the notion that G?12/13
and noncanonical Wnt signaling functionally interact during
zebrafish gastrulation, likely acting in parallel pathways.
To address the cellular basis of the exacerbated C&E de-
fects in kny mutants with compromised G?12/13 signaling, we
focused on cell intercalation that drives notochord convergence
and extension (see above; Glickman et al., 2003). Our previous
studies showed that axial mesoderm C&E is impaired in kny
mutants (Topczewski et al., 2001), although the underlying cell
defects have not been investigated. Compared with WT em-
bryos, notochord cells in kny mutants showed impaired elonga-
tion and orientation at the 4 somite stage with LWR of 2.37 ?
0.8 and angle of 12 ? 11? (269 cells, 8 embryos) and at 6
somite stage with LWR of 3.16 ? 1.2 and angle of 11 ? 9?
(256 cells, 7 embryos; Fig. 7, A, B, and E). These results sug-
gest that kny not only mediates lateral cell elongation, it also
contributes to midline cell alignment. To test whether G?12/13
signaling functions in addition to kny in midline cells, embryos
obtained from kny heterozygous parents were injected with
3MOs and notochord cells from kny homozygous mutant em-
bryos were analyzed. Notably, deficit of G?13 signaling in kny
homozygous mutant embryos exacerbated defects in notochord
cells relative to kny mutant embryos. At 4 somite stage, the no-
tochord cells were much rounder with LWR of 1.48 ? 0.35
(220 cells, 7 embryos, P ? 1.7? 10?44 compared with kny em-
bryos) and lacked proper mediolateral alignment with an angle
of 30 ? 25? (P ? 9.3? 10?20; Fig. 7, A, B, and F). Similar re-
sults were found in embryos at 6 somite stage (Fig. 7, A and B).
Moreover, we also performed molecular epistasis experiments
by injecting embryos obtained from kny heterozygotes with a
small dose of gna13a RNA or MOs against gna13 or 3MOs
against both gna13 and gna12. Overexpression of G?13 did not
suppress kny C&E defects (not depicted), whereas depletion of
G?12/13 signaling by injection with 3MO enhanced these de-
fects (Fig. 7, G–J).
Collectively, these results suggest that G?12/13 do not sim-
ply act as the downstream effectors of slb or kny to mediate
C&E movements. However, G?12/13 appears to functionally in-
teract with noncanonical Wnt signaling to influence cell move-
ments, likely acting through a parallel pathway.
In this study, we have provided evidence that G?12 and G?13
play overlapping and essential roles during zebrafish gastrula-
tion. Thus, overexpression of G?12/13 CT-peptides to uncouple
G?12/13 from their cognate receptors, or reducing the level of
G?12/13 by MO translation interference resulted in C&E defects.
Overexpression of either G?12 or G?13 CT-peptides caused
C&E defects with similar efficiency suggests that signal trans-
duction through both G?12 and G?13 is essential for these gas-
trulation movements. However, based on several consider-
ations, we believe that G?12 and G?13 are functionally
redundant in zebrafish gastrulation. First, our MO translation
interference experiments showed that gastrulation defects were
only observed in embryos injected with the combination of the
three MOs to inhibit protein synthesis of G?12, G?13a, and
G?13b, but not in embryos injected with the same amount of
any single MO. Second, it is well established that G?12 and
G?13 can regulate similar physiological processes via similar
signaling pathways (Dhanasekaran and Dermott, 1996; Sah et
al., 2000). Indeed, we showed previously that either G?12 or
G?13 CT-peptide could inhibit thrombin receptor-stimulated
stress fiber formation in HMEC cells (Gilchrist et al., 2001).
Similar results were reported for LPA-mediated stress fiber
formation in fibroblasts (Sugimoto et al., 2003). It is possible
that G?12 and G?13 interact with the same receptors in zebrafish
gastrulae to regulate C&E. If their binding sites on the recep-
tors are overlapping, it is conceivable that either G?12 or G?13
CT-peptide is sufficient to block completely signal transduc-
tion from both G?12 and G?13. Finally, the functional redun-
dancy of G?12 and G?13 in vertebrates is also supported by the
findings that compound G?13 and G?12 mutant mice die earlier
(e8.5) than G?13 null mice (e9.5), whereas G?12 knockout
mice are viable (Offermanns, 2001). Together, these findings
strongly argue for overlapping functions of G?12 and G?13 in
In vivo time-lapse analyses revealed that G?12/13 signal-
ing is required for several distinct gastrulation cell behaviors,
including dorsalward migration and intercalation during C&E
movements. However, G?12/13 signaling does not appear to act
as a general motility factor. Indeed, mesendoderm internaliza-
tion occurred without any obvious defects in G?12/13-depleted
gastrulae (unpublished data). Therefore, we conclude that G?12/13
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Published May 31, 2005
G?12/13 IN ZEBRAFISH GASTRULATION • LIN ET AL.785
signaling affects only a subset of morphogenetic events in
zebrafish gastrula. We cannot, however exclude the possibility
that there is a residual activity in embryos injected with MOs
and or CT-peptides, permitting other morphogenetic processes
that require lower levels of G?12/13 signaling.
We demonstrated that in G?12/13-depleted embryos, me-
diolateral cell elongation of slowly migrating cells at midgastru-
lation is impaired, whereas this cell behavior is normal in the tri
mutant (Jessen et al., 2002). Moreover, our preliminary analy-
ses of lateral mesodermal cells in embryos overexpressing G?13
revealed normal cell elongation (unpublished data). This sug-
gests that the role of G?12/13 signaling in the regulation of cell
elongation is distinct from that of noncanonical Wnt signaling,
where either increased or decreased pathway activity impairs
elongation of mesodermal cells (Wallingford et al., 2000). Con-
sistent with this hypothesis, our mosaic analyses indicate that
mediolateral cell elongation requires only cell-autonomous
G?12/13 activities, whereas noncanonical Wnt signaling regu-
lates cell elongation both cell-autonomously and nonautono-
mously (Jessen et al., 2002; Marlow et al., 2002). In addition,
ectopic G?12/13 activity cannot suppress the kny?/? and slb?/?
phenotypes (Fig. 5 and not depicted) and Fz2/7 morphant pheno-
types (not depicted). Collectively, these results strongly argue
that G?12/13 do not act as components of a linear noncanonical
Wnt signaling pathway to mediate cell polarization. Identifica-
tion of the ligands and receptors that regulate gastrulation be-
haviors acting upstream of G?12/13 will be our next main focus.
Recent studies indicate that a number of distinct cell be-
haviors contribute to vertebrate gastrulation (Elul and Keller,
2000; Jessen et al., 2002; Myers et al., 2002b; Montero et al.,
2003). How is this diversity of cell behaviors generated? In
some gastrula regions, cells are engaged in more than one cell
behavior, suggesting that cells are competent to respond to
several cues. Our work implicates G?12/13 as key mediators of
many different gastrulation cell behaviors: slow and fast dor-
sal convergence in lateral regions and cell intercalation in
dorsal regions (Figs. 5 and 7; Jessen et al., 2002; Myers et al.,
2002b; Glickman et al., 2003). Given that G?12/13 can be acti-
vated by a variety of GPCR/ligands, it is tempting to hypothe-
size that these proteins may underlie interaction with signals
in different regions of fish gastrulae to generate the distinct
gastrulation cell behaviors. In the lateral region, cells become
influenced by a dorsally provided attracting system that ini-
tiates convergence movements. Evidence suggests that
?-catenin activates the STAT3 pathway in the dorsal gastrula
organizer to produce a long range dorsal attractant, which
could interact with G?12/13 signaling to mediate slow dorsal
convergence movements (Yamashita et al., 2002). In the dor-
solateral region, G?12/13 may also interact with the noncanoni-
cal Wnt signaling to generate high mediolateral elongation
underlying fast dorsal migration. Finally in the dorsal region,
G?12/13 and noncanonical Wnt signaling could interact with
yet to be identified regulators to mediate intercalation behav-
ior. In conclusion, we establish a central role for G?12 and
G?13 proteins in mediating several distinct cell behaviors that
drive vertebrate gastrulation. Identification of extracellular
cues that are integrated by G?12/13 to mediate individual gas-
trulation cell behaviors is an important future goal for this re-
search into the molecular mechanisms of morphogenesis.
Materials and methods
WT zebrafish of AB*, India, TL and hybrid backgrounds, slb(tz216/tz216,
knym119 (Heisenberg et al., 2000; Topczewska et al., 2001), zebrafish
strains were maintained as described previously (Solnica-Krezel et al.,
1994). Embryos were obtained from natural mating and staged accord-
ing to morphology as described previously (Kimmel et al., 1995).
Cloning zebrafish gna12/13 and generation of G?12/13 COOH-terminal
Zebrafish gna12 and gna13 cDNAs were cloned by RT-PCR and then sub-
cloned into the pCS2 expression vector. The conserved glutamine at resi-
due 226 of G?13a was changed to leucine to generate a constitutively ac-
tive form of G? protein (Katoh et al., 1998) using the QuikChange
mutagenesis kit (Stratagene). To generate constructs encoding the last 11
COOH-terminal aa of G?S (QRMHLRQYELL), G?12 (LQENLKDIMLQ), and
G?13 (LHDNLKQLMLQ), two synthetic short complimentary oligonucle-
otides encoding the peptide sequences were obtained for each gene. The
forward and reverse oligonucleotides were annealed, and subcloned into
pCS2 vector. These constructs were designated as G?s-CT, G?12-CT, and
G?13-CT (one peptide was used to block function of both G?13a and
G?13b because they have identical COOH-terminal sequences). Longer
forms of CT peptides encoding the last 50 aa of COOH termini of G?13
and G?12, which included a HA-tag at the NH2-terminus, were constructed
by PCR. All constructs were verified by DNA sequencing.
Live embryos for still photography were mounted in 1.5–2% methylcellu-
lose at 28?C, whereas embryos processed for whole-mount in situ hybrid-
ization were mounted in 75% glycerol/PBT. Embryos were photographed
using an Axiophot2 microscope (Carl Zeiss MicroImaging, Inc.) and an
Axiocam digital camera (Carl Zeiss MicroImaging, Inc.). For confocal im-
aging, embryos were mounted in 75% glycerol/PBT, and a laser scanning
inverted microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.) with
a 40? lens and 2? digital Zoom was used. All images acquired were
compiled and edited using Adobe Photoshop and Illustrator software.
In situ hybridization
Sense and antisense RNA probes for gna12, gna13a, and gna13b were
synthesized using the NH2-terminal EST clones as templates. Antisense RNA
probes hgg1, dlx3, krox20, shh, deltaC, ntl, bmp4, and chordin were syn-
thesized as described previously (Jessen et al., 2002). Whole-mount in situ
hybridization was performed as described previously (Thisse and Thisse,
1998), except that BM purple (Roche) was used for the chromogenic reac-
tion. Sense probes produced no signal.
Cell culture stress fiber formation assay
HEK cells were transiently transfected with GFP or with G protein con-
structs as indicated. To block Rok activity, ROCK inhibitor, Y-27623 was
added at 10 ?M to media after transfection (Uehata et al., 1997). Stress
fiber formation assay was performed as described previously (Gilchrist et
al., 2001). Anti-G?12 or G?13 antibodies (1:100) generated against the
last 11 AAs of human G?12 or G?13 (Hallak et al., 1994) were used to
identify the G protein–expressing cells. Cells were mounted in Vectashield
mounting medium (Vector Laboratories) and confocal images were ac-
quired as described in Microscopy.
Zebrafish embryos were fixed in 4%PFA/PBS/4% sucrose at shield and
whole mount immunohistochemistry was performed as described previ-
ously (Topczewska et al., 2001). Primary anti-G?12 or G?13 antibodies
and Cy2-conjugated pAb (1:100) were used. No signal was detected
when the primary antibodies were preincubated with the peptides encod-
ing the COOH-terminal 11 residues of the G?12 or G?13, respectively, or
when only the secondary antibody was used. Confocal images were
RNA and antisense MO injections
Capped sense RNAs encoding the G?13, G?12, G?S CT-peptides, mGFP
(Wallingford et al., 2000) or a full-length zebrafish G?13a and human
on March 18, 2013
Published May 31, 2005
JCB • VOLUME 169 • NUMBER 5 • 2005786
G?13 were synthesized using mMessage Machine system (Ambion). RNAs
were injected into embryos at 1–2 cell stage.
Antisense MOs (Gene-Tools) targeted against the zebrafish gna12,
gna13a, and gna13b transcripts were designed according to the manu-
facturer’s suggestions and injected into embryos at 1 cell stage. Two dis-
tinct MOs were designed to target either the sequences overlapping the
ATG initiation codon (MO1) or the 5? untranslated sequences (MO2) of
gna13a transcript. For gna13b and gna12, one MO against sequence
overlapping the translation start site was designed for each transcript.
Time-lapse and cell shape analysis
Nomarski time-lapse images of lateral gastrula mesodermal cells at mid-
gastrulation (80% epiboly) were collected as described previously (Myers
et al., 2002a). Dechorionated zebrafish embryos were mounted in 0.8–1%
low melting point agarose in 30% Danieau’s buffer (100% Danieau’s
buffer: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM CaCl2, 5
mM Hepes, pH 7.6), to view the lateral mesoderm (90? from the dorsal
midline). The microscope room was maintained at 28?C during record-
ings. Single focal plane time-lapse recordings were collected at 30-s inter-
vals using DIC optics and a 20? objective (0.5 NA Plan Neofluor) on an
Axiophot2 microscope (Carl Zeiss MicroImaging, Inc.) and an Axiocam
digital camera (Carl Zeiss MicroImaging, Inc.). Images were analyzed us-
ing Object-Image software (Norbert Vischer, http://simon.bio.uva.nl/
object-image.html). Data was exported to Excel (Microsoft) where cell mi-
gration speed, path, direction, turning angle, and LWRs were determined
according to Topczewski et al. (2001). The direction of movements of lat-
eral mesodermal cells was determined at 30-s intervals and change in
movement direction was calculated. A turn was defined as a change in di-
rection of ?60?. We also determined the actual direction of lateral mesen-
dodermal cells at every 60-s interval.
To investigate shape of notochord cells, WT embryos or embryos
obtained from knym119 heterozygous parents were injected with mgfp RNA
(Wallingford et al., 2000) alone or with 3MOs, and were then fixed in
4% PFA. Images of notochord were collected on a confocal microscope
and LWR of notochord cells and the angle of the long axis relative to a
line perpendicular to the embryonic axis as represented by the notochord
were analyzed using Object-Image software.
For cell autonomy analyses, cells from embryos injected with dextran-Rho-
damine or together with 3MO were transplanted into host embryos in-
jected with mgfp RNA and 3MO or mgfp RNA alone at 1K-dome stage
(mgfp RNA was injected to visualize cell shape), as described previously
(Jessen et al., 2002). Host embryos were then fixed in 4% PFA at 4s stage
and stained with anti-Rhodamine, then LWR and the angle of the long axis
of notochord cells relative to a line perpendicular to the embryonic axis
Data are presented as the mean ? 1 SD. Statistical analyses were per-
formed using unpaired t tests unequal variance. In all analyses, the aster-
isk indicates P ? 0.001.
GenBank/EMBL/DDBJ accession nos. for the zebrafish gna12, gna13a, and
gna13b are AY386359, AY386360, and AY386361, respectively.
Online supplemental material
Fig. S1 shows the sequence alignment of human and zebrafish G?12/13.
Two synthetic short complementary oligonucleotides encoding the peptide
sequences were obtained for gna12, gna13, and gnaS. Online supple-
mental material is available at http://www.jcb.org/cgi/content/full/
We thank L. Solnica-Krezel and H. Hamm lab members for discussions and J.
Jessen, T.P. Wilm, and A. Inbal for critical comments. We acknowledge J.
Clanton and A. Bradshaw for excellent fish care. We are grateful to Dr.
David Manning (University of Philadelphia) for G?12 and G?13 antibodies, to
C.-P. Heisenberg for slb mutant fish, to P. Ingham, M. Ekker, N. Ueno, C.
and B. Thisse, M. Halpern, and J. Lewis for probes. Confocal experiments
were performed in VUMC Cell Imaging Core facility (supported by National
Institutes of Health [NIH] grant 1S10RR015682).
D.S. Sepich is supported by NIH Vascular Biology Training grant
(T32HL07751). The work in L. Solnica-Krezel lab is supported by NIH
GM55101 grant. H. Hamm lab is supported by NIH grants EY10291 and
Submitted: 19 January 2005
Accepted: 26 April 2005
Ahumada, A., D.C. Slusarski, X. Liu, R.T. Moon, C.C. Malbon, and H.Y.
Wang. 2002. Signaling of rat Frizzled-2 through phosphodiesterase and
cyclic GMP. Science. 298:2006–2010.
Akhter, S.A., L.M. Luttrell, H.A. Rockman, G. Iaccarino, R.J. Lefkowitz, and
W.J. Koch. 1998. Targeting the receptor-G?q 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 extracellular signal-regulated kinase activation. Mol. Pharmacol.
Buhl, A.M., N.L. Johnson, N. Dhanasekaran, and G.L. Johnson. 1995. G?12 and
G?13 stimulate Rho-dependent stress fiber formation and focal adhesion
assembly. J. Biol. Chem. 270:24631–24634.
Crawford, B.D., C.A. Henry, T.A. Clason, A.L. Becker, and M.B. Hille. 2003.
Activity and distribution of paxillin, focal adhesion kinase, and cadherin
indicate cooperative roles during zebrafish morphogenesis. Mol. Biol.
Dhanasekaran, N., and J.M. Dermott. 1996. Signaling by the G?12 class of G
proteins. Cell. Signal. 8:235–245.
Elul, T., and R. Keller. 2000. Monopolar protrusive activity: a new morpho-
genic cell behavior in the neural plate dependent on vertical interactions
with the mesoderm in Xenopus. Dev. Biol. 224:3–19.
Feldman, D.S., A.M. Zamah, K.L. Pierce, W.E. Miller, F. Kelly, A. Rapacci-
uolo, H.A. Rockman, W.J. Koch, and L.M. Luttrell. 2002. Selective inhi-
bition of heterotrimeric G?s signaling. Targeting the receptor-G protein
interface using a peptide minigene encoding the G?s carboxyl terminus.
J. Biol. Chem. 277:28631–28640.
Gilchrist, A., M. Bunemann, A. Li, M.M. Hosey, and H.E. Hamm. 1999. A domi-
nant-negative strategy for studying roles of G proteins in vivo. J. Biol.
Gilchrist, A., J.F. Vanhauwe, A. Li, T.O. Thomas, T. Voyno-Yasenetskaya,
and H.E. Hamm. 2001. G? minigenes expressing C-terminal peptides
serve as specific inhibitors of thrombin-mediated endothelial activation.
J. Biol. Chem. 276:25672–25679.
Glickman, N.S., C.B. Kimmel, M.A. Jones, and R.J. Adams. 2003. Shaping the
zebrafish notochord. Development. 130:873–887.
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.
Gohla, A., S. Offermanns, T.M. Wilkie, and G. Schultz. 1999. Differential in-
volvement of G?12 and G?13 in receptor-mediated stress fiber formation.
J. Biol. Chem. 274:17901–17907.
Habas, R., Y. Kato, and X. He. 2001. Wnt/Frizzled activation of Rho regulates
vertebrate gastrulation and requires a novel Formin homology protein
Daam1. Cell. 107:843–854.
Hallak, H., L. Muszbek, M. Laposata, E. Belmonte, L.F. Brass, and D.R. Man-
ning. 1994. Covalent binding of arachidonate to G protein ? subunits of
human platelets. J. Biol. Chem. 269:4713–4716.
Hamm, H.E. 1998. The many faces of G protein signaling. J. Biol. Chem. 273:
Hammerschmidt, M., and M.C. Mullins. 2002. Dorsoventral patterning in the
zebrafish: Bone morphogenetic proteins and beyond. In Pattern Forma-
tion in Zebrafish. L. Solnica-Krezel, editor. Springer-Verlag, Berlin
Heisenberg, C.P., M. Tada, G.J. Rauch, L. Saude, M.L. Concha, R. Geisler,
D.L. Stemple, J.C. Smith, and S.W. Wilson. 2000. Silberblick/Wnt11
mediates convergent extension movements during zebrafish gastrulation.
Jessen, J.R., J. Topczewski, S. Bingham, D.S. Sepich, F. Marlow, A. Chan-
drasekhar, and L. Solnica-Krezel. 2002. Zebrafish trilobite identifies
new roles for Strabismus in gastrulation and neuronal movements. Nat.
Cell Biol. 4:610–615.
Katanaev, V.L., R. Ponzielli, M. Semeriva, and A. Tomlinson. 2005. Trimeric G
protein-dependent Frizzled signaling in Drosophila. Cell. 120:111–122.
Katoh, H., J. Aoki, Y. Yamaguchi, Y. Kitano, A. Ichikawa, and M. Negishi.
1998. Constitutively active G?12, G?13 and G?q induce Rho-dependent
neurite retraction through different signaling pathways. J. Biol. Chem.
Keller, R. 2002. Shaping the vertebrate body plan by polarized embryonic cell
movements. Science. 298:1950–1954.
on March 18, 2013
Published May 31, 2005
G?12/13 IN ZEBRAFISH GASTRULATION • LIN ET AL.787 Download full-text
Kimmel, C.B., W.W. Ballard, S.R. Kimmel, B. Ullmann, and T.F. Schilling.
1995. Stages of embryonic development of the zebrafish. Dev. Dyn.
Kinoshita, N., H. Iioka, A. Miyakoshi, and N. Ueno. 2003. PKC? is essential for
Dishevelled function in a noncanonical Wnt pathway that regulates
Xenopus convergent extension movements. Genes Dev. 17:1663–1676.
Kuhl, M., K. Geis, L.C. Sheldahl, T. Pukrop, R.T. Moon, and D. Wedlich. 2001.
Antagonistic regulation of convergent extension movements in Xenopus
by Wnt/?-catenin and Wnt/Ca2? signaling. Mech. Dev. 106:61–76.
Lee, Y.N., C.C. Malbon, and H.Y. Wang. 2004. G?13 signals via p115RhoGEF
cascades regulating JNK1 and primitive endoderm formation. J. Biol.
Liu, T., A.J. DeCostanzo, X. Liu, H. Wang, S. Hallagan, R.T. Moon, and C.C.
Malbon. 2001. G protein signaling from activated rat Frizzled-1 to the
?-catenin-Lef-Tcf pathway. Science. 292:1718–1722.
Liu, X., T. Liu, D.C. Slusarski, J. Yang-Snyder, C.C. Malbon, R.T. Moon, and
H. Wang. 1999. Activation of a Frizzled-2/?-adrenergic receptor chi-
mera promotes Wnt signaling and differentiation of mouse F9 teratocar-
cinoma cells via G?13 and G?t. Proc. Natl. Acad. Sci. USA. 96:14383–
Malbon, C.C., H. Wang, and R.T. Moon. 2001. Wnt signaling and heterotri-
meric G-proteins: strange bedfellows or a classic romance? Biochem.
Biophys. Res. Commun. 287:589–593.
Marlow, F., J. Topczewski, D. Sepich, and L. Solnica-Krezel. 2002. Zebrafish
Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and ef-
fective convergence and extension movements. Curr. Biol. 12:876–884.
Montero, J.A., B. Kilian, J. Chan, P.E. Bayliss, and C.P. Heisenberg. 2003. Phos-
phoinositide 3-kinase is required for process outgrowth and cell polariza-
tion of gastrulating mesendodermal cells. Curr. Biol. 13:1279–1289.
Myers, D.C., D.S. Sepich, and L. Solnica-Krezel. 2002a. Bmp activity gradient
regulates convergent extension during zebrafish gastrulation. Dev. Biol.
Myers, D.C., D.S. Sepich, and L. Solnica-Krezel. 2002b. Convergence and ex-
tension in vertebrate gastrulae: cell movements according to or in search
of identity? Trends Genet. 18:447–455.
Nasevicius, A., and S.C. Ekker. 2000. Effective targeted gene “knockdown” in
zebrafish. Nat. Genet. 26:216–220.
Offermanns, S. 2001. In vivo functions of heterotrimeric G-proteins: studies in
G?-deficient mice. Oncogene. 20:1635–1642.
Offermanns, S., V. Mancino, J.P. Revel, and M.I. Simon. 1997. Vascular system
defects and impaired cell chemokinesis as a result of G?13 deficiency.
Parks, S., and E. Wieschaus. 1991. The Drosophila gastrulation gene concertina
encodes a G?-like protein. Cell. 64:447–458.
Penzo-Mendez, A., M. Umbhauer, A. Djiane, J.C. Boucaut, and J.F. Riou. 2003.
Activation of G?? signaling downstream of Wnt-11/Xfz7 regulates
Cdc42 activity during Xenopus gastrulation. Dev. Biol. 257:302–314.
Sah, V.P., T.M. Seasholtz, S.A. Sagi, and J.H. Brown. 2000. The role of Rho in
G protein-coupled receptor signal transduction. Annu. Rev. Pharmacol.
Sepich, D.S., D.C. Myers, R. Short, J. Topczewski, F. Marlow, and L. Solnica-
Krezel. 2000. Role of the zebrafish trilobite locus in gastrulation move-
ments of convergence and extension. Genesis. 27:159–173.
Sheldahl, L.C., M. Park, C.C. Malbon, and R.T. Moon. 1999. Protein kinase C
is differentially stimulated by Wnt and Frizzled homologs in a G-pro-
tein-dependent manner. Curr. Biol. 9:695–698.
Shih, J., and R. Keller. 1992. Cell motility driving mediolateral intercalation in
explants of Xenopus laevis. Development. 116:901–914.
Simon, M.I., M.P. Strathmann, and N. Gautam. 1991. Diversity of G proteins in
signal transduction. Science. 252:802–808.
Solnica-Krezel, L., A.F. Schier, and W. Driever. 1994. Efficient recovery of
ENU-induced mutations from the zebrafish germline. Genetics. 136:
Sugimoto, N., N. Takuwa, H. Okamoto, S. Sakurada, and Y. Takuwa. 2003.
Inhibitory and stimulatory regulation of Rac and cell motility by the
G?12/13-Rho and G?i pathways integrated downstream of a single G
protein-coupled sphingosine-1-phosphate receptor isoform. Mol. Cell.
Thisse, C., and B. Thisse. 1998. High resolution whole-mount in situ hybrid-
ization. ZFIN Zebrafish Science Monitor. 5:8–9.
Topczewska, J.M., J. Topczewski, A. Shostak, T. Kume, L. Solnica-Krezel, and
B.L. Hogan. 2001. The winged helix transcription factor Foxc1a is es-
sential for somitogenesis in zebrafish. Genes Dev. 15:2483–2493.
Topczewski, J., D.S. Sepich, D.C. Myers, C. Walker, A. Amores, Z. Lele, M.
Hammerschmidt, J. Postlethwait, and L. Solnica-Krezel. 2001. The ze-
brafish glypican knypek controls cell polarity during gastrulation move-
ments of convergent extension. Dev. Cell. 1:251–264.
Trinkaus, J.P., M. Trinkaus, and R. Fink. 1992. On the convergent cell move-
ments of gastrulation in Fundulus. J. Exp. Zool. 261:40–61.
Uehata, M., T. Ishizaki, H. Satoh, T. Ono, T. Kawahara, T. Morishita, H. Ta-
makawa, K. Yamagami, J. Inui, M. Maekawa, and S. Narumiya. 1997.
Calcium sensitization of smooth muscle mediated by a Rho-associated
protein kinase in hypertension. Nature. 389:990–994.
Ulrich, F., M.L. Concha, P.J. Heid, E. Voss, S. Witzel, H. Roehl, M. Tada, S.W.
Wilson, R.J. Adams, D.R. Soll, and C.P. Heisenberg. 2003. Slb/Wnt11
controls hypoblast cell migration and morphogenesis at the onset of
zebrafish gastrulation. Development. 130:5375–5384.
Wallingford, J.B., B.A. Rowning, K.M. Vogeli, U. Rothbacher, S.E. Fraser, and
R.M. Harland. 2000. Dishevelled controls cell polarity during Xenopus
gastrulation. Nature. 405:81–85.
Wallingford, J.B., S.E. Fraser, and R.M. Harland. 2002. Convergent extension:
the molecular control of polarized cell movement during embryonic
development. Dev. Cell. 2:695–706.
Warga, R.M., and C.B. Kimmel. 1990. Cell movements during epiboly and gas-
trulation in zebrafish. Development. 108:569–580.
Winklbauer, R., A. Medina, R.K. Swain, and H. Steinbeisser. 2001. Frizzled-7
signalling controls tissue separation during Xenopus gastrulation. Nature.
Yamashita, S., C. Miyagi, A. Carmany-Rampey, T. Shimizu, R. Fujii, A.F.
Schier, and T. Hirano. 2002. Stat3 controls cell movements during ze-
brafish gastrulation. Dev. Cell. 2:363–375.
on March 18, 2013
Published May 31, 2005