Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

Article (PDF Available)inThe Journal of Cell Biology 198(5):941-52 · September 2012with43 Reads
DOI: 10.1083/jcb.201203012 · Source: PubMed
Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions.
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Correspondence to Stephanie Woo:; or Didier Y.R.
Abbreviations used in this paper: DN, dominant negative; GEF, guanine nucleo-
tide exchange factor; MO, morpholino; PBD, p21-binding domain; ROI, region
of interest.
During the development of vertebrate organs, cells exhibit dis-
tinct morphologies and behaviors, such as cell migration, adhe-
sion, and proliferation, that are indicative of their particular cell
type and differentiation state. Although much work has been
done to identify and characterize the signals that induce specic
cell fates, how these developmental signals are translated into
characteristic cellular behaviors is poorly understood.
Cell migration is important for numerous processes, in-
cluding embryonic development, immune function, and wound
healing, as well as the progression of diseases such as meta-
static cancer. The mode of cell migration can be persistent, in
which cells migrate in the same general direction over time,
or nonpersistent, in which cells frequently change direction
(Pankov et al., 2005; Petrie et al., 2009). Not only do different
cell types exhibit different modes of migration, but the same cell
may also change the way it migrates at different developmental
stages (Bak and Fraser, 2003; Pézeron et al., 2008). These ob-
servations suggest that the type of migratory behavior is a marker
of differentiation, but its signicance is poorly understood.
Endodermal cells in the early zebrash embryo exhibit
multiple modes of migration and thus constitute an ideal model
for investigating how different migratory behaviors are regu-
lated. Just before gastrulation, high levels of Nodal signaling at
the blastoderm margin induce endoderm specication (Stainier,
2002; Zorn and Wells, 2009). As gastrulation begins, endodermal
cells undergo ingression and migrate between the yolk and
epiblast. Initially, cells migrate in a random walk pattern, result-
ing in the dispersal of endodermal cells across the yolk surface
in a discontinuous salt-and-pepper pattern (zeron et al., 2008).
By 90% epiboly, endodermal cells begin a second phase of mi-
gration characterized by convergent movements toward the em-
bryonic axis. Finally, these individual migratory cells must
adhere together to ultimately form the epithelial lining of the
gastrointestinal tract. These progressive changes in migration
behavior are likely subject to tight regulation. However, although
much work has been done to understand how developmental
signaling molecules induce differential gene expression during
endoderm differentiation and patterning (Stainier, 2002; Zorn
and Wells, 2009), the downstream cellular responses, including
migration, remain to be explored.
mbryo morphogenesis is driven by dynamic cell be-
haviors, including migration, that are coordinated
with fate specification and differentiation, but how
such coordination is achieved remains poorly understood.
During zebrafish gastrulation, endodermal cells sequen-
tially exhibit first random, nonpersistent migration fol-
lowed by oriented, persistent migration and finally collective
migration. Using a novel transgenic line that labels the
endodermal actin cytoskeleton, we found that these stage-
dependent changes in migratory behavior correlated
with changes in actin dynamics. The dynamic actin and
random motility exhibited during early gastrulation were
dependent on both Nodal and Rac1 signaling. We further
identified the Rac-specific guanine nucleotide exchange
factor Prex1 as a Nodal target and showed that it medi-
ated Nodal-dependent random motility. Reducing Rac1
activity in endodermal cells caused them to bypass the
random migration phase and aberrantly contribute to me-
sodermal tissues. Together, our results reveal a novel role
for Nodal signaling in regulating actin dynamics and mi-
gration behavior, which are crucial for endodermal mor-
phogenesis and cell fate decisions.
Nodal signaling regulates endodermal cell motility
and actin dynamics via Rac1 and Prex1
Stephanie Woo,
Michael P. Housley,
Orion D. Weiner,
and Didier Y.R. Stainier
Department of Biochemistry and Biophysics,
Developmental and Stem Cell Biology,
Institute for Human Genetics,
Liver Center,
Diabetes Center,
Institute for
Regeneration Medicine, and
Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158
© 2012 Woo et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub-
lication date (see After six months it is available under a
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as described at
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Supplemental Material can be found at:
JCB • VOLUME 198 • NUMBER 5 • 2012 942
we can track actin rearrangements with high resolution in living
embryos and gain further insights into the in vivo regulation of
cytoskeletal dynamics.
Endodermal cells exhibit progressive
changes in migratory behavior and actin
dynamics during gastrulation
A previous study has shown that endodermal cells undergo ran-
dom migration during early gastrulation but switch to conver-
gence movements in late gastrulation (Pézeron et al., 2008). We
rst conrmed that cells labeled by Tg(sox17:GFP-UTRN)
expression exhibit similar migration behaviors. We quantied
both the directional persistence of migration (dened as the
ratio of net over total distance traveled) as well as the mean in-
stantaneous velocity over 1-h intervals. During early stages
(shield to 75% epiboly), cells migrated relatively randomly, al-
though with a slight bias toward the dorsal side of the embryo
(Fig. 2 [A and B] and Video 3). However, during late stages
(90% epiboly to tailbud), endodermal cells moved with strong
persistence in the dorsal direction, which was accompanied by
a signicant increase in migration velocity (Fig. 2, D and E).
This switch from random to oriented migration was accompa-
nied by a change in cell shape (Fig. 2 [F–H] and Video 3). In
early stages, cells were mostly round with a few small lamelli-
podial protrusions (Fig. 2 F), but, by late stages, cells took on a
attened appearance with much broader lamellipodia (Fig. 2 G).
By tail bud stage, the converging endodermal cells began to ad-
here to each other to form the endodermal sheet (Video 4).
By tracking GFP-UTRN uorescence, we investigated
the actin cytoskeletal rearrangements that occur during these
changes in cell motility (Fig. 3 and Videos 1 and 2). First, we
determined the dynamics of the actin cytoskeleton at early (70%
epiboly) and late (90% epiboly) stages by measuring the persist-
ence of GFP-UTRN uorescence, focusing on the large uores-
cent patches that often marked lamellipodia-like protrusions
(Fig. 3, A and B). We found that these lamellipodia were rela-
tively transient at 70% epiboly but were signicantly more long
lived at 90% epiboly (Fig. 3 C). This result suggests that the en-
dodermal lamellipodia are more dynamic during early stages,
which likely contributes to the ability of the cells to rapidly
change migration direction. We also recorded the spatial orien-
tation of lamellipodia within the cell with respect to the embryonic
Cell migration involves the complex rearrangement of the
actin cytoskeleton, which is coordinated by numerous actin
regulatory proteins (Rottner and Stradal, 2011). The Rho family
of small GTPases, including RhoA, Rac1, and Cdc42, play sev-
eral well-characterized roles in regulating actin dynamics dur-
ing cell migration. For example, Cdc42 and Rac1 promote actin
polymerization to drive membrane protrusion at the leading
edge (Kozma et al., 1995; Wu et al., 2009), whereas RhoA in-
duces actomyosin contraction, which provides the force nec-
essary for cell translocation (Chrzanowska-Wodnicka and
Burridge, 1996). The majority of studies investigating the mo-
lecular mechanisms underlying these actin dynamics have pri-
marily used cells cultured on 2D or 3D substrates. However,
it is known that cell migration can differ markedly in vivo
(Yamada and Cukierman, 2007), but, until recently, it has been
difcult to study subcellular actin dynamics within living or-
ganisms. In this study, we used a novel transgenic zebrash line
in which F-actin is uorescently labeled specically in endoder-
mal cells. Using this line, we were able to track actin dynamics
and cell motility at high resolution within the developing
zebrash embryo. We found that Nodal signaling can affect actin
stability and retrograde ow in endodermal cells, which corre-
lated with Nodal-dependent changes in cell migration. We fur-
ther show that the effects of Nodal signaling on actin dynamics
and cell migration are mediated by Rac1 and that Nodal signal-
ing induces expression of the Rac activator Prex1. We found
that similar to Nodal and Rac1, Prex1 is also required for the
dynamic motility of endodermal cells and that it acts down-
stream of Nodal to drive random migration. Finally, we show
that perturbing Rac1 activity in endodermal cells results in their
aberrant contribution to mesodermal tissues, thereby revealing
the importance of regulated cell motility to cell fate decisions.
expression labels
F-actin in endodermal cells
To investigate the molecular mechanisms underlying endoderm
migration in vivo, we generated a transgenic line in which the
endoderm-specic sox17 promoter drives expression of a uor-
escent actin probe consisting of the F-actin–binding domain of
Utrophin (Burkel et al., 2007) fused to GFP (Tg(sox17:GFP-
UTRN)). Tg(sox17:GFP-UTRN) expression readily labels
actin-rich structures in vivo, including lamellipodia, lopodia,
retraction bers, dorsal rufes, actin bundles, and cleavage fur-
rows of dividing cells (Fig. 1 and Videos 1–4). Cells often con-
tained multiple sites of GFP-UTRN uorescence, suggesting
that actin polymerization is not restricted to a single leading
edge. To examine actin dynamics during active migration, we
imaged Tg(sox17:GFP-UTRN) gastrulae by time-lapse spinning-
disk confocal microscopy (Videos 1 and 2). We observed
that GFP-UTRN uorescence rapidly accumulated in protru-
sive areas of cells, presumably a result of actin polymerization,
and rapidly disappeared at sites of membrane retraction. Within
the larger protrusions, we sometimes observed uorescent par-
ticles streaming back toward the cell center, indicative of retro-
grade ow (arrow in Video 1). Thus, using this transgenic line,
Figure 1. Tg(sox17:GFP-UTRN) expression labels actin-based struc-
tures in endodermal cells. (A–C) Images were taken from lateral mar-
ginal regions of gastrulating zebrafish embryos (6–9 h after fertilization).
(A and B) Fluorescence from Tg(sox17:GFP-UTRN) expression accumulates
in actin-based structures such as lamellipodia (arrow in A) and retraction
fibers (arrowhead in A) as well as cytokinetic furrows (arrow in B).
(C) More detailed actin organization including actin bundles can be seen
at higher magnification. Bars, 10 µm.
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943Regulation of endoderm migration by Nodal and Rac1 • Woo et al.
are less clear, although one study suggests that Nodal signaling
may promote random migration of mesendodermal cells (Pézeron
et al., 2008). Nodal is a member of the TGF- superfamily of
signaling proteins that is required for the specication of endo-
derm and mesoderm (Feldman et al., 1998; Stainier, 2002).
Classically, the role of Nodal signaling during endoderm devel-
opment has been to induce the expression of endoderm-specic
transcription factor genes (Alexander and Stainier, 1999; Reiter
et al., 2001; Poulain and Lepage, 2002; Stainier, 2002; Zorn and
Wells, 2009). To determine whether Nodal signaling regulates
the migration of endodermal cells in addition to its role in endo-
dermal fate specication, we treated Tg(sox17:GFP-UTRN)
embryos with the Nodal receptor/Alk4/5/7 inhibitor SB-505124
(Fig. 4; Hagos and Dougan, 2007). To focus on events sub-
sequent to endoderm specication, inhibitor treatment started
at 5 h after fertilization, which does not appear to interfere with
the onset of endodermal marker gene expression (Fig. S1, A–D).
We found that treatment with 50 µM SB-505124 signicantly
slowed migration velocity and increased migration persistence
at early stages (70% epiboly) compared with DMSO-treated
control (Fig. 4 [A–D] and Video 5). Nodal receptor inhibition
also induced changes in actin dynamics. In particular, we found
that SB-505124 treatment signicantly increased lamellipodia
lifetime and slowed the rate of retrograde ow (Fig. 4, E–J).
However, we did not detect any directional bias in lamellipodia
formation (unpublished data), suggesting that although Nodal
inhibition can promote migration persistence, it likely does not
provide guidance information.
Nodal signaling promotes Rac1 activity
in endodermal cells
Our results suggest that Nodal signaling can regulate actin
dynamics, but there are no known cytoskeletal regulators in
the Nodal signaling pathway. To identify a link between Nodal
and the actin cytoskeleton, we focused on the Rho family
GTPase Rac1 as a candidate. Rac1 has well-characterized roles
in many aspects of cell migration, including promoting actin
polymerization and lamellipodia formation (Ridley et al., 1992).
The characteristics of endodermal cells during early gastrulation—
in particular, weak directionality and short-lived, nonoriented
protrusions—are strikingly similar to cells expressing constitu-
tively active forms of Rac1 (Pankov et al., 2005; Woo and
Gomez, 2006). Moreover, expression levels of Rac1 were
shown to be sufcient to modulate the migration persistence
of broblasts in vitro, with high levels promoting random mi-
gration and low levels facilitating persistent migration
(Pankov et al., 2005).
First, we determined whether Rac1 was required for early
random migration by overexpressing dominant-negative (DN)
Rac1 in Tg(sox17:GFP-UTRN) embryos. Injection of large
amounts of DN Rac1 mRNA (10 pg) resulted in cessation of all
cell movements (unpublished data). However, a low dose of
DN Rac1 mRNA (2 pg) only moderately inhibited endodermal
migration speed but signicantly increased migration persis-
tence at 70% epiboly, similar to what was observed with Nodal
receptor inhibition (Fig. 4, K–N). This low dose of DN Rac1
expression did not appear to affect expression of the endodermal
axes (dorsal, ventral, animal, or vegetal; Fig. 3 D). At 70%
epiboly, lamellipodia oriented at similar frequencies toward the
dorsal, ventral, or vegetal directions but were less likely to
occur toward the animal pole. However, at 90% epiboly, lamel-
lipodia formation was signicantly more biased in the dorsal
direction (P = 0.00163 by
test). Thus, the preferential initia-
tion and persistence of dorsally oriented actin polymerization
likely underlie the dorsal-directed movement of endodermal
cells at late stages.
A study of migratory cells in vitro has shown that the
rate of retrograde ow decreases as protrusion persistence in-
creases (Lim et al., 2010). Therefore, we used kymography
(Batchelder et al., 2011) to determine whether retrograde ow
within protrusions varied from early to late stages (Fig. 3, E–I).
We found that the rate of retrograde ow within endodermal
cells was signicantly faster during early compared with late
stages (Fig. 3 I), correlating with the shift from random to ori-
ented migration.
Nodal signaling promotes random migration
and actin dynamics during early stages
A study has reported that the dorsally oriented migration of en-
dodermal cells during late gastrulation depends on the chemo-
kine Cxcl12b and its receptor Cxcr4a (Mizoguchi et al., 2008).
In contrast, the mechanisms controlling early random migration
Figure 2. Endodermal cells exhibit changes in migratory behavior and
cell shape as embryos progress through gastrulation. (A–C) Represen-
tative migration tracks over a 1-h period of endodermal cells at shield
(6 h after fertilization; A), 75% epiboly (8 h after fertilization; B), and
90% epiboly (9 h after fertilization; C). Dorsal is to the right. Bars, 25 µm.
(D and E) Quantification of migration persistence (D) and instantaneous
velocity (E) shows that migration persistence and speed increase as gas-
trulation proceeds. Shield, n = 49 cells; 75% epiboly, n = 74 cells; 90%
epiboly, n = 95 cells. (F and G) Representative images of endodermal cells
from Tg(sox17:GFP-UTRN) embryos at 70% epiboly (F) and 90% epiboly
(G). (H) Quantification of circularity shows that cells are significantly more
rounded at 70% epiboly. a.u., arbitrary units. 70% epiboly, n = 63 cells;
90% epiboly, n = 66 cells. All error bars represent SEM. *, P < 0.05.
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JCB • VOLUME 198 • NUMBER 5 • 2012 944
motility was assessed starting at 70% epiboly. Importantly,
transplanted sox32-overexpressing cells display biphasic mi-
gration behaviors similar to those of endogenous endodermal
cells, switching from random to persistent migration between
early/mid and late gastrulation (Fig. S2, A–D). These cells also
undergo the corresponding changes in cell shape (Fig. S2, E and F).
However, when transplanted cells coexpressed DN Rac1, we
found that directional persistence signicantly increased during
marker genes sox17 and sox32 (Fig. S1, E–H), suggesting that
the effects on endodermal motility were not a result of mis-
specication. To determine whether Rac1 was required cell
autonomously within endodermal cells to promote dynamic migra-
tion, we performed cell transplantation experiments. Donor en-
dodermal cells were generated by overexpression of sox32
either alone or combined with DN Rac1. Cells were transplanted
into wild-type host embryos at 4–5 h after fertilization, and cell
Figure 3. Actin dynamics within endodermal cells
change from early to late gastrulation. (A and B)
Actin dynamics were analyzed by tracking la-
mellipodia through accumulations in GFP-UTRN
fluorescence. Representative lamellipodia are high-
lighted in red in B from the cells in A. Bars, 25 µm.
(C) Lamellipodial lifetime increases during late
gastrulation. Early (70% epiboly), n = 523 lamel-
lipodia from 45 cells; late (90% epiboly), n = 665
lamellipodia from 77 cells. (D) Orientation of la-
mellipodia formation with respect to the embryonic
axes. V, ventral; A, animal; Vg, vegetal; D, dorsal.
Lamellipodia formation is biased toward the dor-
sal direction during late gastrulation (P = 0.00163
test). Early (70% epiboly), n = 45 cells; late
(90% epiboly), n = 77 cells from two independent
experiments. (E–I) Analysis of retrograde flow. Ky-
mographs in F and H were generated along the red
lines shown in E and G, respectively. Time is plotted
horizontally, and the direction of membrane protru-
sion is oriented toward the top of the images. Red
lines in F and H highlight retrograde-moving actin
structures, which form streaks in the kymographs.
The slope of these streaks was used to calculate
the rate of retrograde flow (I), which decreases in
late gastrulation. Early (70% epiboly), n = 12 cells;
late (90% epiboly), n = 15 cells. Bars: (E–H) 10 µm;
(F and H) 5 µm. All error bars represent SEM.
*, P < 0.05.
Figure 4. Cell migration and actin dynamics during early
gastrulation depend on Nodal and Rac1 signaling. (A and
B) Representative migration tracks over a 1-h period from
embryos treated with DMSO carrier (A) and 50 µM Nodal
receptor inhibitor SB-505124 (SB; B). Dorsal is to the
right. Bars, 25 µm. (C and D) Quantification of migration
persistence and instantaneous velocity shows that Nodal
inhibition leads to significantly increased migration per-
sistence and reduced migration velocity. DMSO, n = 74
cells; SB-505124, n = 48 cells. (E) Nodal inhibition in-
creases lamellipodial lifetime. DMSO, n = 191 lamelli-
podia from 28 cells; SB-505124, n = 324 lamellipodia
from 46 cells. (F–J) Nodal inhibition slows retrograde
flow. Kymographs in G and I were generated along the
red lines shown in F and H, respectively. Time is plotted
horizontally, and the direction of membrane protrusion is
oriented toward the top of the images. Bars: (F and H)
10 µm; (G and I) 5 µm. The rate of the retrograde flow is
quantified in J. DMSO, n = 9 cells; SB-505124, n = 5 cells.
(K and L) Representative migration tracks over a 1-h period
from control embryos (K) and embryos expressing DN
Rac1 (L). Bars, 25 µm. (M and N) Quantification of migra-
tion persistence and instantaneous velocity from control
(Ctrl) embryos and embryos expressing DN Rac1. Loss of
Rac1 activity significantly increases migration persistence
and moderately reduces migration velocity. Control, n =
76 cells; DN Rac1, n = 98 cells. All error bars represent
SEM. *, P < 0.05.
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945Regulation of endoderm migration by Nodal and Rac1 • Woo et al.
localized to membrane protrusions. Collectively, these re-
sults suggest that Nodal signaling promotes Rac1 activation
to induce membrane protrusions.
Using the same RFP-PBD assay, we also investigated
whether a drop in Rac1 activity accompanies the switch from
random to persistent migration in wild-type gastrulae (Fig. S4 I).
Surprisingly, we found that levels of Rac1 signicantly in-
creased during late gastrulation. One likely explanation is the
onset of Cxcl12a–Cxcr4 chemokine signaling at this stage
(Mizoguchi et al., 2008), which is known to activate Rac1
(Xu et al., 2012).
The Rac–guanine nucleotide exchange
factor (GEF)
is a Nodal target gene
and is required for random migration
Small GTPases such as Rac1 are activated by GEFs, which
promote the dissociation of GDP, allowing GTP to bind. TGF-1
has been shown to induce the expression of the Rho-GEF
NET1, leading to increased RhoA activity and actin stress
ber formation (Shen et al., 2001). Therefore, we hypothesized
that Nodal might similarly regulate expression of a Rac-GEF
to control Rac1 activity. To identify endodermally enriched
Nodal target genes, we performed microarray analysis using
Tg(sox17:GFP) embryos treated with SB-505124 to inhibit
Nodal signaling or overexpressing a constitutively active form
of the acvr1b Nodal receptor (taram-a*). Of the genes identi-
ed, three were Rac-specic GEFs: arhgef25b, prex1, and
tiam1 (Fig. S3 A). We veried these candidates by quantita-
tive real-time PCR and found that only prex1 expression was
consistently Nodal responsive (Figs. 6 A and S3 B). When
embryos were treated with SB-505124, prex1 expression was
down-regulated 2.8 ± 0.45 fold compared with DMSO-treated
control. Correspondingly, when Nodal signaling was activated
by expression of the constitutively active receptor taram-a*,
prex1 expression increased 2.85 ± 0.5 fold compared with that
in embryos expressing a control RNA.
Prex1 was initially identied in neutrophils as a protein
required for phosphatidylinositol (3,4,5)-trisphosphate (PIP
induced Rac activation (Welch et al., 2002). It consists of
a RhoGEF domain, a pleckstrin homology domain, two DEP
(dishevelled, Egl-10, and pleckstrin) domains, two PDZ do-
mains, and a C-terminal region with signicant similarity to
inositol polyphosphate-4-phosphatase but that is apparently
catalytically inactive. Prex1 is synergistically activated by PIP
and G (Welch et al., 2002; Barber et al., 2007; Zhao et al.,
2007) and is important for neutrophil function (Welch et al.,
2005), neurite formation (Waters et al., 2008), and motility of
breast cancer cells (Sosa et al., 2010). By in situ hybridization,
we found that at 70% epiboly, when endodermal cells are un-
dergoing random migration, prex1 appears to be most highly
expressed within the endoderm (Fig. 6 B).
We determined whether Prex1 functions as a Rac-GEF
in zebrash endodermal cells by examining the effects of mor-
pholino (MO)-mediated knockdown of Prex1 on Rac1 activity
(Fig. 6, C–E). Using the same aforementioned PBD uorescence
assay, we found that Prex1 knockdown resulted in a signicant
decrease in Rac1 activity (Fig. 6 E). We also examined the
early stages, whereas migration velocity was signicantly
slower, suggesting that Rac1 acts cell autonomously to regulate
endoderm migration (Fig. S2, G–J).
Next, we determined whether Nodal signaling regulates
Rac1 activity (Fig. 5). To visualize Rac1 activity, we ex-
pressed a uorescent probe consisting of the Rac1-binding
domain of p21-activated kinase tagged to an RFP (RFPp21-
binding domain [PBD]; Srinivasan et al., 2003; Miller and
Bement, 2009). Because detection of RFP-PBDuorescence
is facilitated by mosaic expression, we transplanted small
groups of RFP-PBD–expressing endodermal cells into unla-
beled hosts. To control for variation in cell size or shape, donor
cells were colabeled with Alexa Fluor 647–conjugated
10,000–molecular weight dextran (A647-dextran) as a vol-
ume marker, and Rac1 activity was determined as the ratio of
the RFP-PBD signal relative to the A647-dextran signal. We
found that active Rac1 was enriched along the cell periphery
and concentrated within actively protruding areas of endo-
dermal cells (Fig. 5 C and Video 6). This observation is con-
sistent with previous in vitro studies showing that active
Rac1 localizes to the cell membrane and leading edge (Kraynov
et al., 2000; Srinivasan et al., 2003). Treatment with SB-
505124 resulted in a global decrease in active Rac1 com-
pared with DMSO-treated control (Fig. 5 G). We also
measured the area of regions within cells in which the ratio
of RFP-PBD to A647-dextran was >1.0 (Fig. 5 H), as these
regions often corresponded to membrane protrusions. These
regions were signicantly reduced in size upon inhibitor
treatment, suggesting that active Rac1 was no longer differentially
Figure 5. Nodal signaling regulates Rac1 activity. (A–F) Visualization of
Rac1 activity in embryos treated with DMSO (A–C) and SB-505124 (SB;
D–F). A fluorescent Rac1 probe, RFP-PBD, was expressed in endodermal
cells (A and D). Cells were colabeled with fluorescent dextran (B and E) as
a volume marker. Rac1 activity was determined by generating ratiometric
images between the RFP-PBD and dextran signals and was pseudocolored
based on ratio value (C and F). Warmer colors indicate enrichment of
PBD relative to dextran. Bars, 10 µm. (G) Quantification of the mean ratio
of PBD to dextran indicates that Nodal inhibition reduces Rac1 activity.
DMSO, n = 121 cells; SB-505124, n = 125 cells. All error bars represent
SEM. *, P < 0.05. (H) Measurement of the size of cell regions where
the PBD/dextran ratio is >1.0. Area of Rac1 activation is dramatically
reduced upon Nodal inhibition.
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JCB • VOLUME 198 • NUMBER 5 • 2012 946
effects of Prex1 on endodermal motility during early stages by
injecting Prex1 MO into Tg(sox17:GFP-UTRN) embryos
(Fig. 6, F–I). In these MO-injected embryos, we observed some
GFP-UTRN–labeled cells positioned in the cell layers away
from the yolk surface (Video 9), suggesting that reduction in
Prex1 levels leads to defects in internalization or other epiboly
movements. Notably, we did not observe these effects with DN
Rac1 expression. As these supercial cells appeared rounded
and immobile, we excluded them from subsequent analysis and
restricted our measurements to the cells that were positioned at
the yolk surface. Similar to the observations with both Nodal
inhibition and DN Rac1 expression, we found that Prex1 knock-
down signicantly increased migration persistence (Fig. 6 H)
and decreased migration velocity (Fig. 6 I).
Next, we examined whether Prex1 acts downstream of
Nodal to promote random migration of endodermal cells by
determining whether overexpressing Prex1 was able to rescue
the effects of Nodal inhibition on cell motility (Fig. 6, J and K).
Embryos injected with 500 pg Prex1 mRNA or an equivalent
amount of mCherry mRNA as a control were treated with
50 µM SB-505124 at 5 h after fertilization, and cell motility was
assessed at 7 h after fertilization. As we previously observed,
control-injected embryos treated with Nodal inhibitor exhibited
increased directional persistence and decreased migration
velocity. Overexpression of Prex1 rescued the effects on direc-
tionality and partially rescued the effects on migration velocity,
suggesting that Prex1 at least partially mediates signaling down-
stream of Nodal to control endodermal cell motility.
All together, these results suggest that prex1 is an
endodermally expressed Nodal target gene that activates Rac1
and mediates the Nodal-dependent dynamic motility of endo-
dermal cells.
Random migration is required to maintain
endodermal identity
It is not clear how an initial phase of random migration con-
tributes to subsequent steps of endodermal morphogenesis.
To address this question, we expressed low levels of DN
Rac1 to bypass the random migration phase and promote pre-
cocious persistent migration and then assessed the effects on
later stages of endoderm development (Fig. 7). Control endo-
dermal donor cells labeled by Tg(sox17:dsRed) expression
were transplanted together with DN Rac1–expressing cells
labeled by Tg(sox17:GFP) expression into unlabeled wild-
type hosts before gastrulation (4–5 h after fertilization). The
distribution of GFP- and dsRed-labeled cells was then as-
sessed at 22–24 h after fertilization. We found that the major-
ity of both control and Rac1-deficient cells were located
within the gut tube and pharyngeal endoderm (Fig. 7, A–D).
However, a signicant proportion of cells expressing DN
Rac1 was found within mesodermal tissues such as the so-
mites and notochord (arrows in Fig. 7 [A, C, E, and F]). The
percentage of cells residing in such nonendodermal positions
was signicantly higher among DN Rac1–expressing donor-
derived tissue than control (Fig. 7 G). Intriguingly, these cells
were still Tg(sox17:GFP) positive but exhibited the charac-
teristic cell shapes and expressed molecular markers of the
Figure 6. prex1 is a target of Nodal signaling, promotes Rac1 activ-
ity, and regulates endodermal cell motility. (A) Expression of prex1 was
measured by real-time quantitative PCR. Inhibition of Nodal signaling by
SB-505124 treatment (SB) down-regulated prex1 expression (normalized
to DMSO-treated controls), and overactivation of the Nodal pathway by
expression of taram-A* (TA*) increased prex1 expression (normalized
to control embryos expressing mCherry). The data shown are mean fold
changes from six independent experiments. (B) Section through an embryo
at 70% epiboly processed for prex1 in situ hybridization. prex1 appears to
be enriched within the endodermal layer (arrows). Bar, 25 µm. (C and D)
Representative ratiometric images of control (Ctrl; C) and Prex1 MO–
injected (D) cells expressing RFP-PBD and colabeled with fluorescent dex-
tran. Images are pseudocolored based on ratio value. Warmer colors indi-
cate enrichment of PBD relative to dextran. Bars, 10 µm. (E) Quantification
of the mean ratio of PBD to dextran indicates that Prex1 knockdown reduces
Rac1 activity. Control, n = 124 cells; MO, n = 70 cells. (F and G) Rep-
resentative migration tracks over a 1-h period from control (E) and Prex1
MO–injected (F) embryos. Dorsal is to the right. Bars, 25 µm. (H and I)
Quantification of migration persistence (H) and instantaneous velocity (I)
from control and Prex1 MO–injected embryos. Prex1 knockdown signifi-
cantly increased migration persistence and moderately reduced migration
velocity. Control, n = 80 cells; MO, n = 33 cells. (J and K) Overexpressing
Prex1 can rescue random migration (J) and partially rescue migration ve-
locity (K) in embryos treated with the Nodal inhibitor SB-505124. DMSO,
n = 44 cells; SB-505124, n = 34 cells; SB-505124 + Prex, n = 52 cells.
All error bars represent SEM. *, P < 0.05.
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Published September 3, 2012
947Regulation of endoderm migration by Nodal and Rac1 • Woo et al.
(boxed region in Fig. 7 K). These experiments suggest that
the migration behavior of endodermal cells during gastrula-
tion is important for maintaining endoderm identity.
In this study, we have shown that during gastrulation stages,
endodermal cells undergo developmentally regulated changes
in migration behavior, which are driven by corresponding
changes in actin cytoskeletal dynamics. We have also shown
that the increased actin dynamics and random motility of cells
during early gastrulation stages depend on Nodal signaling and
Rac1 activity. Furthermore, we showed that Nodal signaling in-
duces the expression of the Rac-specic GEF prex1 and that
Prex1 functions downstream of Nodal signaling to promote ran-
dom migration at early gastrulation stages. Together, these ob-
servations indicate that the early random migration of endodermal
cells is driven by Nodal-induced Rac1 activation.
Interestingly, our data also suggest that the transition to
directed migration during late gastrulation may not be simply a
result of down-regulation of Nodal and/or Rac1 signaling. First,
we observed that Rac1 activity increases rather than decreases
during late gastrulation (Fig. S4 I). This increase in Rac1 activity
may correlate with the onset of Cxcl12–Cxcr4 chemokine signal-
ing (Mizoguchi et al., 2008), which has been reported to signal
through Rac1 (Xu et al., 2012). Second, when we examined en-
dodermal cell migration during late gastrulation in Nodal- or
Rac1-inhibited embryos, we found that although cell migration
was not severely affected, directional persistence was slightly in-
creased (Fig. S4, C and G). This result suggests that Nodal-
dependent signals may still be operating to promote random
motility, but, at late stages, they are now superseded by direc-
tional cues provided by putative chemoattractants such as
Cxcl12. Therefore, we propose a model in which Nodal, via
Prex1, induces global Rac1 activation, which results in direction-
ally random cell migration during early gastrulation stages. Then,
as endodermal cells become responsive to directional cues during
late gastrulation, these cues may lead to strongly polarized Rac1
activation that overwhelms the Nodal-dependent global Rac1 ac-
tivation, leading to highly persistent, dorsal-directed migration.
Thus, we speculate that by promoting global Rac1 activation, the
function of Nodal/Prex1 during early gastrulation stages is to
generate noise in the subcellular distribution of activated Rac1,
ensuring that endodermal cells do not inappropriately respond to
weak directional cues that may be present at these stages (perhaps
guiding mesodermal cell migration). Our observations that loss
of Nodal or Rac1 signaling during early gastrulation stages leads
to increased directional persistence could be a result of the un-
masking of these weak polarization signals that would normally
be overwhelmed by the global Rac1 activity induced by high
Nodal signaling at these early stages. This model is also consis-
tent with our cell transplantation results in which precociously
inducing persistent migration by DN Rac1 expression results in
the mistargeting of endodermal cells to mesodermal tissues.
Notably, our observations differ from cell culture studies in
which decreasing Rac1 activity was sufcient to switch cells
from random to persistent migration (Pankov et al., 2005).
tissues in which they resided (Fig. 7, C–F). To better under-
stand how Rac1-decient cells became mislocalized to the
mesoderm, we performed time-lapse imaging soon after trans-
plantation (Fig. 7 [H–K] and Video 7). We observed that at
75% epiboly, control cells were spread out along the dorsal
ventral and animal–vegetal axes. In contrast, DN Rac1–
expressing cells appeared dispersed along the animal–vegetal
axis only (Fig. 7 I). As a result, during the switch to dorsally
oriented migration beginning at 90% epiboly, the Rac1-decient
cells reached the dorsal end of the embryo rst, whereas control
cells were still relatively spread out dorsoventrally (Fig. 7 J).
Subsequently, we observed some of the dorsal-most Rac1-
decient cells extruding away from their neighbors and taking
on an elongated cell shape reminiscent of notochord cells
Figure 7. Cells expressing DN Rac1 are less likely to contribute to en-
dodermal tissues. (A–D) Lateral view of an embryo 22 h after fertilization
containing donor endodermal cells from Tg(sox17:GFP) embryos express-
ing DN Rac1 as well as donor control cells from Tg(sox17:dsRed) embryos.
Host embryo is labeled with phalloidin. Images in C and D are taken from
the boxed regions in A and B, respectively. Arrows point to DN Rac1–
expressing cells that appear to aberrantly reside in the somites. Bars, 100 µm.
(E and F) In situ hybridization analyses of myoD (E) or ntla (F) expression
show that some DN Rac1–expressing donor cells (arrows) express markers
for muscle (E) or notochord (F), respectively, despite also being labeled
with the Tg(sox17:GFP) transgene (green). Bars, 25 µm. (G) Quantification
of donor cell contribution to endodermal or nonendodermal tissues. Expres-
sion of DN Rac1 significantly increased the likelihood of cells contributing
to nonendodermal tissues. n = 23 embryos. *, P < 0.05 by
test. (H–K)
Frames from a time-lapse video (Video 7) showing the relative movements
of control (red) and DN Rac1–expressing cells (green) from mid-gastrulation
to early somitogenesis. Numbers indicate hours elapsed. Bars, 100 µm.
(inset) Enlarged region of the boxed area showing DN Rac1–expressing
cells that have migrated into the notochord. Bar, 25 µm.
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Published September 3, 2012
JCB • VOLUME 198 • NUMBER 5 • 2012 948
(Xu et al., 2012), making it very likely that Prex1 lies directly in
this signaling pathway. However, in terms of endoderm develop-
ment, several questions remain about the role of Prex1. First, to
what extent are both PIP
and G necessary for Prex1 function
in vivo? Mizoguchi et al. (2008) suggested that phosphoinositide
signaling is not highly active in migrating endodermal cells, and
it may be possible to activate Prex1 with G alone, espe-
cially under conditions of low PIP
concentrations (Welch et al.,
2002). If PIP
and/or G are required for full Prex1 activity, are
they generated downstream of receptors such as Cxcr4, and, if
so, how do those signaling pathways interact with Nodal signal-
ing? Given that most studies of Prex1 to date have used neutro-
phils in culture, the developing zebrash endoderm may represent
a useful system to probe important questions about Prex1 func-
tion in vivo.
In the double transplantation experiments, we observed
that some cells in which random migration was suppressed by
DN Rac1 expression seemed unable to maintain endodermal
identity and instead contributed to mesodermal tissues. Al-
though we interpret these results as being a result of the sup-
pression of random migration during early gastrulation, it is
also possible that DN Rac1 impairs cell movements before
gastrulation, such as epiboly and ingression, which could
aberrantly place cells in the mesodermal layer. However,
although we did observe some endodermal cells that appar-
ently failed to ingress in Prex1 MO–injected embryos, we did
not see a similar effect with the low-level DN Rac expression
used throughout this study, suggesting that pregastrulation
movements are relatively unaffected. Thus, based on our time-
lapse analyses, we propose that DN Rac1 expression preco-
ciously induces persistent migration, causing cells to more
efciently reach the dorsal side of the embryo. Once there,
they may inappropriately interact with mesodermal cells or
mesoderm differentiation signals. It is also possible that Rac1
is required for later aspects of endoderm morphogenesis, such
as cell–cell adhesion during endodermal sheet formation,
which may also affect the ability of Rac-decient cells to
remain within the endoderm.
The ability of cells to switch their migratory behavior has
been observed in many different cell types and model systems
(Bak and Fraser, 2003; Wolf et al., 2003; Pankov et al., 2005;
Pézeron et al., 2008; Sanz-Moreno et al., 2008). In general, it is
thought that random migration plays either a dispersive or ex-
ploratory role, whereas persistent migration promotes rapid and
efcient translocation. The need for multiple modes of migra-
tion may be crucial not only during development but in the adult
as well. Most notably, processes such as wound healing and
axon regeneration require cells to switch from a stationary state
to a migratory one. Additionally, different types of invasive
tumor cells are characterized by different migratory behaviors
(Madsen and Sahai, 2010); some cells are even able to switch
between multiple migration modes (Sanz-Moreno et al., 2008),
which can impact the efcacy of drugs meant to block metasta-
sis (Wolf et al., 2003; Micuda et al., 2010). Therefore, the nd-
ings presented in this study have clear implications beyond
developmental processes.
lthough such a simple signaling mechanism may indeed be suf-
cient to regulate migratory behaviors under basic cell culture
conditions, our results illustrate the complexity of regulating cell
migration in the dynamic environment of the developing embryo.
The best-characterized role for Nodal signaling during
endoderm development has been the induction of endoderm-
specic transcription factor genes. Although it has been
previously suggested that Nodal may regulate cell movement
(Yokota et al., 2003; Pézeron et al., 2008), the mechanisms by
which Nodal could affect cell motility were unknown. Here, we
have shown that inhibition of Nodal signaling not only slowed
cell migration velocity and increased migration persistence but
also suppressed actin dynamics and Rac1 activity. We have
further identied the Rac-GEF Prex1 as a downstream target
of Nodal signaling. Rac1 is a well-known regulator of actin
polymerization and cell migration both in vitro (Gardiner et al.,
2002; Srinivasan et al., 2003; Pankov et al., 2005; Woo and
Gomez, 2006) and in vivo (Li et al., 2002; Kardash et al., 2010;
Yoo et al., 2010), and it has also recently been shown to be cru-
cial for the cell movements underlying gastrulation in mouse
(Migeotte et al., 2011). Although our results suggest that the
Nodal-dependent Rac1 activity we observed is a result of in-
creased expression of Prex1, Rac1 may be activated via a
transcription-independent pathway as well. We observed that
acute SB-505124 treatment lasting as little as 15 min was sufcient
to alter cell migration behavior (Fig. S5). Indeed, other TGF-
ligands have been to shown to induce both rapid Rho GTPase
activation that is Smad independent as well as sustained in-
creases in Rho activity that involve gene transcription (Kardassis
et al., 2009). It is also very likely that other cytoskeletal regula-
tory proteins besides Rac1 are involved in endoderm morpho-
genesis. Indeed, in our microarray analysis, we identied several
genes associated with cell migration and cytoskeletal dynamics
as potential targets of Nodal signaling (Fig. S3 A). In addition,
a study using a proteomics-based approach identied at least
four cytoskeleton-associated proteins that are differentially
regulated between mesendodermal and ectodermal cells (Link
et al., 2006); one of these proteins, Ezrin, was demonstrated
to function during the migration of prechordal plate progeni-
tor cells by regulating membrane protrusion (Diz-Muñoz
et al., 2010). Future studies will no doubt identify additional
cytoskeletal regulators important for tissue morphogenesis and
organ development.
In this study, we provide evidence that prex1 is transcrip-
tionally regulated by Nodal signaling. However, GEFs are also
subject to posttranscriptional regulation. Although most GEFs
are regulated by phosphorylation (Rossman et al., 2005), Prex1
is synergistically activated by PIP
and G (Welch et al., 2002;
Barber et al., 2007; Zhao et al., 2007). In neutrophils, Prex1 is
thought to act as a coincidence detector that allows for high
levels of Rac activation when both second messengers are
generated (Weiner, 2002), as occurs when G-protein–coupled
chemokine receptors are activated (Stephens et al., 1997).
Zebrash endodermal cells also express chemokine receptors,
primarily Cxcr4a (Mizoguchi et al., 2008; Nair and Schilling,
2008). SDF-1–Cxcr4 signaling in primordial germ cells was
recently shown to activate Rac1 in a G-dependent manner
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Published September 3, 2012
Regulation of endoderm migration by Nodal and Rac1 • Woo et al.
prex1 in situ hybridization
To generate the prex1 in situ probe, the prex1 ORF was cloned into pCR-
Blunt II-TOPO (Invitrogen). For probe synthesis, pCR-Blunt II-TOPO-prex1
was digested by SpeI and in vitro transcribed with T7. For in situ hybridiza-
tion, embryos at 70% epiboly were dechorionated and fixed in 4% PFA
overnight at 4°C. Embryos were sunk in 30% sucrose, embedded in opti-
mal cutting temperature medium, and cryosectioned (12 µm thick). After
drying, sections were fixed in 4% PFA for 10 min at room temperature.
Sections were then acetylated with 0.1 M triethanolamine, 2.1 mM HCl,
and 0.25% acetic anhydride for 10 min at room temperature. Sections
were permeabilized with 1% Triton X-100 in PBS for 30 min at room
temperature. Nonspecific binding was blocked by incubating sections in
hybridization buffer (50% formamide, 5× SSC, 0.1% Tween 20, 50 mg/ml
heparin, and 500 mg/ml tRNA, pH 6.0) for 4 h at room temperature in a
humidified chamber. The prex1 probe was diluted to 200 ng/ml in hybrid-
ization buffer, and sections were incubated overnight at 65°C. Sections
were then washed once with SSC at 65°C, twice with 0.2× SSC at
65°C, and then transferred to room-temperature TBS (100 mM Tris HCl, pH
7.5, and 150 mM NaCl). Sections were blocked for 1 h at room tempera-
ture in 2% blocking reagent (Roche). Antidigoxigenin antibody (Roche)
was diluted 1:5,000 in 2% blocking reagent, and sections were incubated
overnight at room temperature. Sections were washed every 30 min for 4 h
with TBS and then equilibrated for 5 min in NTM buffer (100 mM Tris HCl,
pH 9.5, 100 mM NaCl, and 5 mM MgCl
). Sections were stained with
NBT/BCIP solution (1:50 in NTM buffer; Roche) overnight at room temper-
ature. After brief fixation with 4% PFA, sections were air dried overnight at
room temperature and then washed twice with xylene. Sections were
mounted in Permount (Thermo Fisher Scientific) and imaged on a micro-
scope (Axioplan; Carl Zeiss) with a 20×/0.75 NA objective lens.
Rac1 activity assay
pCS2-TagRFP-PBD was generated by replacing the GFP coding sequence of
pCS2-GFP-PBD (Miller and Bement, 2009) with TagRFP (Evrogen). Mosaic ex-
pression of TagRFP-PBD was accomplished using established cell transplanta-
tion techniques (Stafford et al., 2006; Chung and Stainier, 2008). Tg(sox17:
donor embryos were injected with 200 pg TagRFP-PBD mRNA,
300 pg sox32 mRNA, and 2 µg A647-dextran (10,000 molecular weight;
Invitrogen). At sphere stage, donor cells were transplanted to the marginal
zone of isochronic unlabeled host embryos. At 30% epiboly, embryos were
treated with 0.5% DMSO or 50 µM SB-505124 (Sigma-Aldrich). At shield
stage, embryos were embedded in 1% low-melting agarose and imaged
by spinning-disk confocal microscopy using a 20×/0.75 NA objective with
1. zoom. Z stacks were acquired at 4-µm intervals. Image processing and
analysis were performed using ImageJ software. The GFP channel was used
as a reference to ensure that only endodermal cells were included for analysis.
For the TagRFP-PBD and A647-dextran channels, maximum projections were
made, background was set to NaN (not a number), and images were normal-
ized to their own median value. Then, the TagRFP-PBD image was divided by
the A647-dextran image to generate a ratiometric image. The mean PBD/dex-
tran ratio was calculated by drawing user-defined regions of interest (ROIs)
around cells in the ratiometric images and measuring the mean gray value.
Using the same ROIs, we determined the cell area with ratio >1.0 by thresh-
olding the ratiometric images to include pixel values >1.0 and measuring the
area occupied by thresholded pixels within each cell.
Cell transplantations
Cell transplantations were performed as previously described (Stafford et al.,
2006; Chung and Stainier, 2008). For double transplantation experiments
(Fig. 7), control endodermal donor cells were generated by injecting Tg(sox17:
embryos with 300 pg sox32 mRNA. Rac1-deficient donor cells
were generated by injecting Tg(sox17:GFP)