© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Endoderm convergence controls subduction of the myocardial
precursors during heart-tube formation
ABSTRACT
Coordination between the endoderm and adjacent cardiac mesoderm is
crucial for heart development. We previously showed that myocardial
migration is promoted by convergent movement of the endoderm, which
itself is controlled by the S1pr2/Gα13 signaling pathway, but it remains
unclear how the movements of the two tissues is coordinated. Here, we
image live and fixed embryos to follow these movements, revealing
previously unappreciated details of strikingly complex and dynamic
associations between the endoderm and myocardial precursors. We
found that during segmentation the endoderm underwent three distinct
phases of movement relative to the midline: rapid convergence, little
convergence and slight expansion. During these periods, the myocardial
cells exhibited different stage-dependent migratory modes: co-migration
with the endoderm, movement from the dorsal to the ventral side of the
endoderm (subduction) and migration independent of endoderm
convergence. We also found that defects in S1pr2/Gα13-mediated
endodermal convergence affected all three modes of myocardial cell
migration, probably due to the disruption of fibronectin assembly around
the myocardial cells and consequent disorganization of the myocardial
epithelium. Moreover, we found that additional cell types within the
anterior lateral plate mesoderm (ALPM) also underwent subduction, and
that this movement likewise depended on endoderm convergence. Our
study delineates for the first time the details of the intricate interplay
between the endoderm and ALPM during embryogenesis, highlighting
why endoderm movement is essential for heart development, and thus
potential underpinnings of congenital heart disease.
KEY WORDS: Myocardial migration, Endoderm convergence,
Subduction, S1pr2/Gα13, In vivo imaging
INTRODUCTION
Interplay between adjacent tissues is crucial for organ formation.
During vertebrate development, the heart tube is formed by the
fusion of two populations of myocardial precursors, which migrate
from bilateral regions of embryos. This migration requires proper
proliferation, differentiation and epithelial organization of
myocardial cells, as well as an environment conducive to their
migration (Bakkers, 2011; Evans et al., 2010; Staudt and Stainier,
2012).
Among the environment factors crucial for myocardial migration
is the endoderm, a tissue adjacent to the cardiac mesoderm. Mouse
and zebrafish mutants with endodermal defects display cardia bifida
Department of Anatomy and Cell Biology, Carver College of Medicine, University of
Iowa, 1-400 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242-1109,
USA.
*Present address: State Key Laboratory of Freshwater Ecology and Biotechnology,
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
‡
These authors contributed equally to this work
§
Author for correspondence ([email protected])
Received 27 September 2014; Accepted 21 July 2015
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(David and Rosa, 2001; Narita et al., 1997; Reiter et al., 1999), and
in chick embryos, disruption of the endoderm perturbs myocardial
migration (Rosenquist, 1970). The endoderm has been reported to
influence heart formation by secreting growth factors required for
cardiac specification and differentiation (Andrée et al., 1998; Lough
and Sugi, 2000; Nascone and Mercola, 1995; Schultheiss et al.,
1995). However, this is not always the case; for example,
myocardial differentiation is unaffected in zebrafish mutants that
lack endoderm (Yelon et al., 1999). The endoderm is also thought to
provide a physical substrate over which cardiac precursors migrate,
because of the close anatomic proximity of these tissues and the
finding that in most animal models, myocardial migration fails in
the absence of endoderm (David and Rosa, 2001; Lough and Sugi,
2000). Recent work in chick, however, suggests that endodermal
movement directs migration of the heart field by providing
mechanical forces that pull cardiac mesoderm to the midline
(Varner and Taber, 2012), and that active migration by the cardiac
cells is minimal (Cui et al., 2009). Similarly, in mouse, invagination
of the endoderm-derived foregut is directly linked to migration of
the pericardial mesoderm (Madabhushi and Lacy, 2011; Maretto
et al., 2008). Recently, in Drosophila the neighboring ectoderm
regulates cardiac movement at certain stages (Haack et al., 2014).
Thus, the movement of tissues surrounding myocardial cells can
influence their migration.
In zebrafish, myocardial migration requires signaling by
sphingosine-1-phosphate (S1P) via its cognate G protein-coupled
receptor S1pr2. Zebrafish mutants that lack either the S1P-receptor
(S1pr2; miles apart) or the S1P transporter (Spns2; two of hearts)
develop cardia bifida as a result of defective myocardial migration
(Kawahara et al., 2009; Kupperman et al., 2000; Osborne et al.,
2008). We recently identified Gα13 as the downstream effector of
S1pr2 in regulating myocardial migration, and showed that S1pr2/
Gα13 signaling regulates convergent movement of the endoderm,
which in turn promotes myocardial migration (Ye and Lin, 2013).
However, exactly how S1pr2/Gα13-mediated endoderm movement
controls myocardial migration remains unclear.
Here, we employ transgenic lines that label specifically cardiac
precursors and the endoderm to assess directly developmental
interactions between these tissues. Our study delineates the highly
dynamic associations between the endoderm and myocardial cells,
as well as other cells of mesodermal origin, during segmentation,
thereby identifying a previously unappreciated movement of the
mesodermal cells from the dorsal to the ventral side of the endoderm.
RESULTS
S1pr2/Gα13-mediated convergent movement of the
endoderm regulates myocardial migration
To determine whether the endodermal defect is the root cause of the
impaired myocardial migration observed in S1pr2/Gα13-deficient
embryos, we generated a transgenic line (sox17:mCherry-2Agna13a) in which Gα13a [a morpholino (MO)-insensitive form] is
DEVELOPMENT
Ding Ye*,‡, Huaping Xie‡, Bo Hu‡ and Fang Lin§
RESEARCH ARTICLE
expressed specifically in the endoderm under control of the sox17
promoter (Woo et al., 2012). Embryos obtained from crossing these
animals with Tg(my7:EGFP) and Tg(sox17:EGFP) fish (Mizoguchi
et al., 2008) showed that the expression of mCherry (Gα13a) was
detected only in the EGFP-labeled endoderm (Fig. 1A-A″).
Immunofluorescence analysis revealed that in the transgenic
embryos injected with gna13a/b MO, Gα13 was expressed in the
endoderm but not in the adjacent mesodermal cells (supplementary
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
material Fig. S1), confirming that transgenic endodermal Gα13a
expression is MO-resistant.
Our results indicate that endodermal expression of Gα13a does
not affect either endoderm convergence or myocardial migration, as
the endoderm appeared to be normal and a single heart tube formed
(Fig. 1D). As we reported previously (Ye and Lin, 2013), control
embryos injected with gna13a/b MO (morphants) exhibited a
widened endodermal sheet, cardia bifida, a lack of circulation, and
pericardia edema (80%, n=205) (Fig. 1C,F; supplementary material
Fig. S2B,B″; and data not shown). By contrast, Tg(sox17:mCherry2A-gna13a) embryos injected with the MO had a single heart,
normal circulation and normal endoderm morphology (99.2%,
n=328; Fig. 1E,F; supplementary material Fig. S2D,D″; and data
not shown), although they exhibited tail-fin blistering (47±7.5%,
P=0.25; Fig. 1F; supplementary material Fig. S2D,D′), which
is another characteristic phenotype in S1pr2/Gα13-deficient
embryos (54±3.3%, n=205; supplementary material Fig. S2B,B′)
(Kupperman et al., 2000; Ye and Lin, 2013). These findings indicate
that endodermal expression of Gα13a is sufficient to rescue the
endodermal defects and cardia bifida, and that the tail-fin defect is
independent of cardia bifida in these animals. Thus, endoderm
convergence is crucial for the migration of myocardial precursors.
Fig. 1. Endoderm-specific expression of Gα13 rescues defects in
migration of the endoderm and myocardial cells caused by global
Gα13 depletion. (A-A″) Epifluorescence images of anterior endoderm and
myocardial cells in embryos indicated. (B-E) Epifluorescence images of
anterior endoderm of control and gna13a/b MO-injected embryos indicated.
(F) Frequencies of cardia bifida and tail blistering in embryos indicated
(same as in B-E) at 2 dpf. Dorso-anterior view, with anterior up; yellow
dots, cardiomyocytes; white lines (equivalent length), width of the anterior
endodermal sheet. *P<0.001, #P=0.25. Data are mean±s.e.m.
To investigate how endoderm convergence influences myocardial
migration, we first examined the dynamic movements of the
endoderm. We performed time-lapse experiments on the anterior
region of the endoderm (in which myocardial precursors migrate)
of Tg(sox17:EGFP) embryos during the 7-20 somite stage(s)
(supplementary material Movie 1), as previously reported (Ye and
Lin, 2013). We found that the endoderm underwent three distinct
phases of movement. First, at 7-14 s, the endoderm converged
rapidly towards the midline, reaching its narrowest point by 14 s,
with a significant reduction (49%) in endoderm width (from 308 to
157 µm; Fig. 2A-D,O). Second, at 14-17 s, the convergence slowed
to a minimum, producing no significant change in endoderm width
(from 148 to 150 µm; Fig. 2D,E,O). Third, at 18-20 s, the endoderm
expanded slightly, increasing its width (from 150 to 164 µm;
Fig. 2F,G,O). Similarly, we determined the speed of convergence by
analyzing the displacement of the lateral limits of the endoderm at
various developmental stages, and found that it accelerated from 0.2
to 0.5 µm/min at 8-10 s, peaked at 1.2 µm/min at 12 s, and then
dramatically fell to 0.2 µm/min by 14 s (Fig. 2P); it averaged 0.01
and −0.13 µm/min during 14-17 s and 18-20 s, respectively, which
is consistent with the small change and the slight expansion of the
endodermal sheet during these two periods (Fig. 2O,P).
In s1pr2/mil mutants, at 8 s, the endodermal sheet was already
significantly wider than that in the control siblings (Fig. 2H versus
A), suggesting that endoderm convergence was already impaired by
this stage. In these embryos, the endodermal sheet remained wider
throughout segmentation, yet displayed convergence patterns
similar to those of their controls (Fig. 2A-O). Notably, in these
embryos, at 8-10 s the endoderm converged at speeds comparable to
those in controls; however, speed acceleration did not occur until
12 s and the peak speed was slower (0.68 versus 0.98 µm/min in
controls) (Fig. 2P). Thus, the reduction in endodermal width was
significantly smaller than that in controls (110 versus 151 µm).
Furthermore, at 15-17 s the endoderm continued to converge at a
speed of 0.21 µm/min, whereas the control endoderm had
completed convergence by 14 s (Fig. 2O,P). During 18-20 s, the
speed of expansion was also reduced relative to that in controls
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DEVELOPMENT
The endoderm displays three distinct phases of convergent
movement during segmentation
RESEARCH ARTICLE
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
(−0.04 versus −0.16 µm/min; Fig. 2P). Thus, in control embryos,
endoderm convergence occurs mainly before 14 s and is rapid from
10-12 s; however, in s1pr2/mil mutant embryos, the acceleration is
delayed and inefficient (Fig. 2P). The defects in rapid convergence
in s1pr2/mil mutants probably contribute to the widening of the
endodermal sheet throughout segmentation. Similar results were
found in gna13a/b morphants (supplementary material Fig. S3).
Overall, these data demonstrate that S1pr2/Gα13 signaling is
required for efficient endoderm convergence during segmentation,
a period during which myocardial migration occurs.
Myocardial cells associate with the endoderm during its
three phases of convergence
Our observations indicate that the endoderm completes convergence
at 14 s (Fig. 2), which is earlier than when myocardial precursors
have been reported to migrate to the midline (16-20 s) (Holtzman
et al., 2007). Moreover, our data indicate that the endoderm expands
slightly from 18-20 s (Fig. 2). Thus, it seems that endoderm
convergence and myocardial migration do not occur during the same
time window. To understand how endoderm convergence affects
myocardial migration, we characterized interactions between the two
cell populations during heart-tube formation.
In a first approach, we evaluated the relative positions of the
endoderm and myocardial precursors in fixed Tg(sox17:EGFP)
embryos at various developmental stages. We performed in situ
hybridization (ISH) using a probe for the cardiac transcription factor
nkx2.5, labeling myocardial precursors, and then conducted GFP
immunostaining to assess endoderm morphology. Our results
revealed intricate and dynamic changes in the anatomy of the two
tissues (Fig. 3). Before 12 s, the anterior endoderm narrowed, as
shown in Fig. 2, and the myocardial precursors migrated towards the
midline (the distance between the two populations became smaller)
(Fig. 3A-C); notably, migrating myocardial precursors populated,
and remained at, the lateral boundary of the endodermal sheet
(Fig. 3A,B). These data indicate that myocardial precursors and the
endodermal layer co-migrated and retained their relative positions.
At 12 s, however, the myocardial precursors were located outside
the lateral boundary of the endoderm (Fig. 3C), suggesting that the
endoderm was uncoupled from myocardial cells and had moved
further than the myocardial cells. During 16-24 s, endoderm width
changed very little, but two myocardial populations moved closer to
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the midline and merged to form the heart cone (Fig. 3D-F),
suggesting that these two tissues migrate independently during this
period.
Additionally, we observed that the morphology of the myocardial
population changed during this period: before 12 s, the cell clusters
had a broad spindle-like shape (Fig. 3A,B); at 12 s, they became
more compact and condensed into narrow strips (Fig. 3C); by 16 s,
they were significantly expanded and had migrated towards each
other (Fig. 3D); and by 24 s the clusters had formed a heart cone
(Fig. 3E,F). Transverse sections of these embryos revealed an
intriguing and unexpected phenomenon (Fig. 3A′-F′): before 9 s,
the myocardial precursors laid dorsal to the endoderm; by 12 s, they
had moved ventral to the endoderm, and this migration was similar
to the subduction that had been described in Caudata and Xenopus,
i.e. the movement of cells from a superficial to a deeper location
(Shook et al., 2002, 2004); by 16 s, the myocardial precursors were
all ventral to the endoderm.
In a second approach, we employed in vivo imaging to follow
the migration of the endoderm and myocardial precursors. We
utilized Tg(nkx2.5:ZsYellow) (Zhou et al., 2011), in which
ZsYellow can be detected in myocardial precursors as early as
9-10 s, and our newly generated line Tg(sox17:memCherry),
which expresses membrane-localized mCherry in the endoderm
(Fig. 4A). Consistent with our findings in fixed embryos (Fig. 3),
during 9-12 s the ZsYellow-expressing myocardial populations
were located at the lateral boundaries of the endoderm, and
migrated with the endoderm towards the midline (Fig. 4A-C;
supplementary material Movie 2). Further measurement showed
that the width of the endoderm and the distance between two
myocardial populations were very similar (Fig. 4O), confirming
that the endoderm and myocardial cells migrated together and
at similar speed. However, at 12-13 s these tissues became
uncoupled (Fig. 4O, arrow), with the endoderm moving minimally
but myocardial precursors continuing their migration towards
the midline until they merged and formed the heart cone
(Fig. 4D-G,O). At 17-20 s, in vivo imaging on Tg(nkx2.5:
lifeact-GFP) embryos revealed that myocardial cells at the
leading region extended active protrusions towards the midline
(supplementary material Movie 3 and Fig. S4C-F).
Collectively, these data indicate that myocardial precursors
undergo migration in three major stages that might involve
DEVELOPMENT
Fig. 2. S1pr2 is required for efficient endoderm
convergence during segmentation.
Epifluorescence time-lapse experiments were
performed on control and mil mutant
embryos (supplementary material Movie 1).
(A-N) Snapshots of the anterior endoderm from the
movies, at the stages indicated. Dorso-anterior
views; yellow lines (equivalent length for embryos
at the same stage), width of the anterior
endodermal sheet, showing that the endodermal
sheet was wider in mutant. (O,P) The average
endoderm width (O) and convergence speed (P).
Red, cyan and pink lines represent the periods of
rapid convergence, little convergence and
expansion, respectively. Arrows and arrowheads
mark completion of convergence. *P<0.05;
#P<0.01 versus control. Data are mean±s.e.m.
RESEARCH ARTICLE
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
Fig. 3. S1pr2 is required for the
subduction and active migration of
myocardial precursors. (A-L) Overlays
of GFP-labeled endoderm (as revealed
by immunofluorescence) and nxk2.5
expression (as revealed by whole-mount
in situ hybridization), in embryos at the
stages indicated. (A′-L′) Transverse
sections of the embryos shown in A-L.
Red dashed lines indicate lateral
boundaries of the endodermal sheet;
yellow lines denote width of the area
covered by the myocardial populations;
arrowheads indicate myocardial cells;
arrows indicate that myocardial cells are
located above (yellow) and below (red)
the endoderm. Scale bars: 100 µm.
the endoderm; and (3) after 14 s, they engage in migration
towards the midline, independent of convergent movement of the
endoderm.
Fig. 4. Gα13 is required for all stages of
myocardial migration. Epifluorescence
time-lapse experiments performed on
the embryos indicated (supplementary
material Movie 2). (A-N) Snapshots of
the anterior endoderm and myocardial
cells from the movies at the stages
indicated. Dorso-anterior views. White
arrowheads indicate myocardial cells
(MCs). (O) Endoderm width (red) and the
distance between the two populations of
myocardial cells (green) at the stages
indicated. Black arrow and arrowhead
denote the timepoints at which myocardial
precursors were dissociated from the
endoderm in the control and gna13a/b
MO-injected embryos, respectively. Data
are mean±s.e.m.
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DEVELOPMENT
different mechanisms (Fig. 6E, model): (1) before 12 s, they depend
on the endoderm to migrate towards the midline; (2) at 12-14 s, they
undergo subduction, moving from the dorsal to the ventral side of
RESEARCH ARTICLE
S1pr2/Gα13 signaling is required for all stages of myocardial
migration
Next, we investigated which stage(s) of myocardial migration is
affected in S1pr2/Gα13-deficient embryos. We found that in mil
mutant embryos, myocardial precursors populated at the lateral
boundaries of the widened anterior endodermal sheet as in their
control counterparts, but that they remained in these positions for a
much longer period (6-16 s versus 6-12 s) (Fig. 3G-J versus A-C).
Similar results were found in gna13a/b morphants using transgenic
lines in which the myocardial and endodermal cells are labeled
(supplementary material Fig. S3F-I versus A,B). Live imaging
revealed that myocardial precursors and the endodermal layer
remained at constant relative positions and migrated together for a
much longer period, until 16 s (Fig. 4H-M versus A-C,O;
supplementary material Movie 2). These findings indicate that in
S1pr2/Gα13-deficient embryos, the first phase of myocardial
migration was impaired because of the defect in endoderm
convergence. Furthermore, in embryos globally deficient for
Gα13, endodermal expression of Gα13 rescued defects in both
endoderm convergence and myocardial migration (supplementary
material Movie 4). These data support our hypothesis that, prior to
subduction, myocardial precursors depend on the endoderm to
migrate.
When myocardial cells engaged in subduction at ∼12 s, the shape
of the myocardial population changed from spindle- to stripe-like
(Fig. 3B versus C), and the cells migrated from the dorsal to the
ventral side of the endoderm (Fig. 3B′ versus C′). Thus, this stripe
reflects a morphological change in the myocardial population
during subduction. Notably, in mil mutant embryos and gna13a/b
morphants, appearance of the stripe was delayed until around 16 s
(Fig. 3J; supplementary material Fig. S3I), with myocardial cells
first emerging on the ventral side of the endoderm at this time
(Fig. 3J′; supplementary material Fig. S3I′). To monitor subduction,
we performed confocal in toto imaging on Tg(sox17:memCherry)/
Tg(nkx2.5:Kaede) embryos (Fig. 5). We analyzed the XZ views to
determine the spatial organization of memCherry-expressing
endoderm and Kaede-expressing myocardial cells. Consistent
with the findings in Fig. 3, in control embryos myocardial
precursors located dorsal to the endodermal layer at 10 s
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
(Fig. 5A′), but as the endodermal sheet converged at 12-14 s, they
slid underneath the endoderm (Fig. 5B′-C′), indicating that
myocardial subduction is a relatively quick process, lasting about
30 min (11-12 s, Fig. 5A′-B′; supplementary material Movie 5). An
earlier study had shown that myocardial populations displayed twolayered structures from 16 s (Trinh and Stainier, 2004). Here, we
found that the myocardial populations appeared to be a single
monolayer before subduction, and were converted to two layers
during the subduction period (Fig. 5A′ versus B′-C′). Notably, cell
tracking revealed that the lateralmost cells (3 embryos, 3-5 cells per
embryo) migrated towards the midline (a phenomenon that could
contribute to formation of a second layer of the myocardial cells),
whereas the dorsalmost cells stayed in the same relative position
(supplementary material Movie 5, control, cell #1 versus cell #2).
By contrast, in S1pr2/Gα13-deficient embryos, subduction of the
myocardial cells initiated at 13-14 s (Fig. 5E-F′) and lasted more
than 60 min (13-16 s; Fig. 3I-L, Fig. 5F′; supplementary material
Fig. S3H-I″, Movie 5). Thus, subduction of myocardial precursors
occurred in the context of the S1pr2/Gα13 deficiency but was
significantly delayed and inefficient.
Finally, in control embryos, after subduction (12 s) the
myocardial cells were positioned ventral to the endoderm, and
from 18-20 s they migrated towards the embryonic midline to form
the heart cone (Fig. 3D,E and Fig. 4E-G). In S1pr2/Gα13-deficient
embryos, the myocardial cells exhibited little medial migration,
even after they had moved beneath the endoderm (Fig. 3K,L,
Fig. 4M,N; supplementary material Fig. S3J, Movie 2); consistent
with this finding, these cells formed protrusions only briefly
(supplementary material Fig. S4G-K). These data indicate that
S1pr2/Gα13 signaling is required for the migration of myocardial
precursors at all stages.
Endoderm convergence controls the initial migration and
subduction of myocardial cells
The observation that myocardial migration (during 17-20 s) is
impaired in the context of the S1pr2/Gα13 deficiency seems to
contradict the findings that myocardial migration is independent of
endoderm convergence at this stage (Figs 3 and 4). To identify
precisely which stage(s) of myocardial migration requires S1pr2/
DEVELOPMENT
Fig. 5. Subduction of myocardial precursors is
impaired in Gα13 morphants. (A-F′) Snapshots
from in toto confocal time-lapse movies of control
(A-C′) or gna13a/b MO-injected (D-F′) Tg(sox17:
memCherry)/(nkx2.5:Kaede) embryos at 10-14 s
(supplementary material Movie 5). XY view (A-F),
XZ view (A′-F′). Dashed lines denote the midline.
D, dorsal; V, ventral. Scale bars: 20 µm.
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Fig. 6. Gα13 expression prior to myocardial subduction is crucial for
myocardial migration. (A-C) Classes of heart morphology phenotypes at
26 hpf, in embryos heat shocked at various stages: normal heart (A), two fused
hearts (B) and two separated hearts (C). Arrowheads denote hearts.
(D) Distribution of heart phenotype classes in embryos heat shocked at the
indicated stages of development. The total number of embryos is indicated.
*P<0.001; #P=0.8, percentage of normal-like embryos in indicated groups
versus non-heat shocked group. (E) Model of how the endoderm and
myocardial cells interact during heart-tube formation. The relative positions
and velocities of migration of the endodermal sheet (green) and myocardial
precursors (red dots) in control and S1pr2/Gα13-defective embryos at the
indicated stages of myocardial-cell migration, with the mode of migration
indicated. Arrow direction denotes direction of migration; arrow thickness
indicates speed of migration.
Gα13-mediated endoderm convergence, we manipulated Gα13a
expression temporally by utilizing a transgenic Tg(hsp70:gna13a)
line that expresses Gα13a under the control of the heat shock
promoter hsp70 (Kwan et al., 2007). Embryos derived from crosses
between Tg(hsp70:gna13a) and Tg(sox17:memCherry) animals
were injected with the gna13a/b MO.
At the following stages (during which myocardial precursors
undergo different modes of migration), the injected embryos were
placed at 37°C for 30 min to induce the expression of Gα13a: the
tail-bud (TB) stage, 6 s, 10 s and 16 s. The expression of targeted
proteins can be detected after 45 min of heat shock treatment (Shoji
and Sato-Maeda, 2008). Thus, heat shock (HS) at the TB stage is
expected to cause Gα13 expression at 2-3 s, before myocardial
subduction is initiated; HS at 6 s will induce Gα13 expression at
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
8-9 s, just before myocardial subduction occurs; HS at 10 s and 16 s
will induce Gα13 expression at 12-13 s and 18-19 s, when
myocardial cells undergo and complete subduction, respectively.
Indeed, immunostaining assay revealed that in control embryos
Gα13 expression was present in both endodermal and nonendodermal cells (supplementary material Fig. S5A,B), but in
gna13a/b morphants it was present at 13 s only if HS was applied at
TB (supplementary material Fig. S5C versus D). Both control and
HS embryos were evaluated for heart morphology at 26 hpf, by
which time control myocardial cells would have fused and formed a
single heart (Fig. 6A), and embryos with defects in myocardial
migration typically have two hearts, which can either be separated
completely (cardia bifida, Fig. 6C) or fused (Fig. 6B).
We found that Gα13 expression at different stages rescued
myocardial migration defects to varying degrees. Consistent with
our previous findings (Ye and Lin, 2013), among morphants that
were not subjected to HS, 64.4±7.8% displayed cardia bifida,
27.9±5.9% exhibited fused hearts and only 7.7±3.3% had a single
heart (Fig. 6D). Strikingly, when subjected to HS at the TB stage,
100% morphants had a single heart (Fig. 6D, P=5.3E-07 versus
non-HS), suggesting that myocardial migration defects in gna13a/b
morphants were completely rescued by Gα13 expression at this
stage. When subjected to HS at 6 s, 16.8±12.8% of morphants had
fused hearts, and the rest (83.2±12.8%, P=7E-06 versus non-HS)
had single hearts, suggesting that Gα13 expression just prior to
myocardial subduction rescues myocardial migration significantly,
although not fully. HS at 10 s (induces Gα13 expression at 12-13 s,
prior to or during myocardial subduction in gna13a/b morphants)
led to 6.9±4.3% of embryos exhibiting cardia bifida, 33.1±3.8%
fused hearts and 65±3.4% (P=3.3E-6 versus non-HS) normal hearts
(Fig. 6D), suggesting that Gα13 expression during myocardial
subduction rescued the myocardial migration defects only partially.
Finally, in embryos subjected to HS at 16 s, when myocardial
subduction is complete, the incidence of fused hearts and cardia
bifida was similar to that of non-HS controls (94.2% versus 92.3%,
respectively; P=0.8), although they more often displayed fused
hearts (48.9±5.2% versus 27.9±5.9% in non-HS control) than
cardia bifida (45.3±4.7% versus 64.4±7.8% in non-HS controls)
(Fig. 6D). This suggests that the induction of Gα13 expression at
16 s has only a minor effect on myocardial migration.
Overall, our rescue experiments indicate that: (1) the earlier in
segmentation Gα13 is expressed in the context of a Gα13 deficiency,
the better the chances that defects in myocardial migration will be
rescued; (2) endodermal Gα13 expression (and thus normal
endoderm convergence) at 12-14 s is essential for myocardial
migration and subduction; and (3) Gα13 expression after myocardial
subduction is insufficient to rescue defects in myocardial migration
(Fig. 6E, model).
Organization of the myocardial epithelium is disrupted in
Gα13 morphants
The epithelial organization of myocardial cells matures from
16-20 s, and this step is crucial for their migration (GaravitoAguilar et al., 2010; Trinh and Stainier, 2004). To explore the
cellular mechanisms that underlie myocardial defects in the
context of the Gα13 deficiency, we first assessed expression of the
polarity marker ZO-1 in myocardial cells. First, we performed
whole-mount immunostaining in Tg(nkx2.5:Kaede) embryos at
12 s and 16 s, the times at which myocardial cells undergo and
complete subduction, respectively, and analyzed Z-projections of
XY views of Kaede-labeled cells (Fig. 7A-E). In control
myocardial cells, ZO-1 expression was observed at the cell
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DEVELOPMENT
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Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
edges, with enrichment at the vertices (where three or more cells
meet; Fig. 7A′,C′, circles), and faint punctate structures were
presented in some areas (Fig. 7A′,C′, arrowheads). In Gα13deficient myocardial cells, the punctate ZO-1 staining at the cell
edges was much more prominent (Fig. 7B′,D′, arrowheads), and
the ZO-1 expression was significantly increased at both the edges
and vertices (Fig. 7A-E, circles and arrowheads).
To examine the epithelial organization of myocardial precursors
in greater depth, we performed immunostaining on vibratomesectioned embryos. In control myocardial cells, punctate ZO-1
expression was present in the lateral membrane between cells
starting at 12 s, and became restricted to the basal domain of the
lateral membrane by 20 s (Fig. 7F,H,J, white arrowheads),
indicating that the epithelial organization matured during
myocardial migration, consistent with a previous report (Trinh
and Stainier, 2004). Notably, the timing of ZO-1 expression at 12 s
(Fig. 7F,F′) coincided with that of the initiation of subduction,
suggesting that in addition to contributing to myocardial migration
at later stages, ZO-1 expression might be important for subduction.
Consistent with our findings from Z-projections, in Gα13-deficient
2934
embryos ZO-1 expression was diffuse and its domain was expanded,
with some localized in ectopic regions such as the apical membrane
(Fig. 7G,I,K, yellow arrowheads). In addition, two other polarity
markers, αPKC and β-catenin, are localized apicolaterally and
basolaterally, respectively, in control myocardial cells (Trinh and
Stainier, 2004). In the mil mutant and Gα13 morphants, these
proteins were mislocalized, concomitant with the aberrant ZO-1
expression (supplementary material Fig. S6). Collectively, our
findings indicate that in S1pr2/Gα13-deficient embryos the epithelial
organization of the myocardial population is disrupted – a feature
that might lead to impairment of their subduction and their medial
migration at later stages.
Fibronectin (Fn) assembly surrounding myocardial cells is
disrupted in Gα13 morphants
Proper expression of Fn in the microenvironment is crucial for
establishing the apicobasal organization that myocardial cells need
for their medial migration (Arrington and Yost, 2009; GaravitoAguilar et al., 2010; Sakaguchi et al., 2006; Trinh and Stainier,
2004; Trinh et al., 2005). Furthermore, S1pr2/Mil has been shown
DEVELOPMENT
Fig. 7. Epithelial organization of myocardial cells is disrupted in Gα13 morphants. (A-E) Whole-mount ZO-1 immunostaining was performed in embryos
indicated at 12 s and 16 s. (A-D) Projections of confocal z-stacks of the right lateral mesoderm showing ZO-1 expression (gray) in the nkx2.5:Kaede-expressing
(myocardial) cells (green). (A′-D′) Higher-magnification views of areas shown in boxes in A-D. The vertices were defined as regions where membranes from
three adjacent cells come into contact, and the edges as the cell periphery excluding the vertices. Yellow dashed line, midline; circles, vertices; arrowheads,
edges. (E) Intensity of ZO-1 expression in myocardial cells at the vertices and edges in control embryos (54 cells from 7 embryos at 12 s and 25 cells from 4
embryos at 16 s) and gna13a/b MO-injected embryos (51 cells from 8 embryos at 12 s and 20 cells from 4 embryos). *P<0.001 versus control. Data are
mean±s.e.m. (F-K) Transverse vibratome sections immunostained for Kaede (magenta), ZO-1 (green), actin (Rhodamine-Phalloidin, red) and nuclei (DAPI,
blue). Yellow dashed line indicates midline; white dashed lines outline myocardial cells; white and yellow arrowheads indicate normal and ectopic ZO-1
expression, respectively. Scale bars: 20 µm.
to interact genetically with Fn to promote myocardial migration
(Matsui et al., 2007). Similarly, we found that Gα13 interacted
genetically with Fn in regulating endoderm convergence and
myocardial migration (not shown). To determine whether Gα13
affects Fn expression, we examined its patterns in whole-mount
embryos at various stages.
In control embryos, Fn fibrils were assembled in striking patterns
that were previously unappreciated. At 12 s, when myocardial cells
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
underwent subduction, Fn fibrils were enriched between the
endoderm and the myocardial populations, and were aligned
along the anterior-posterior axis (Fig. 8A,A′, white arrowheads);
at 14 and 16 s, during which myocardial cells complete subduction
and migrate towards the midline, the direction of Fn fibril
assembly was re-aligned in the putative direction of migration
of the myocardial cells (Fig. 8C,C′,E,E′, white arrowheads).
Furthermore, transverse XZ sections revealed that Fn fibrils
Fig. 8. Fn assembly patterns are disrupted in Gα13 morphants. Whole-mount Fn immunostaining was performed in the embryos indicated. (A-F) Projections of
XY views of confocal z-stacks spanning the myocardial cells (magenta), showing Fn assembly (green). (A′-F′) Magnification images of areas shown in boxes in AF. White and yellow arrowheads indicate leading regions of myocardial populations in control and gna13a/b MO-injected embryos, respectively. (A″-F″) Images of
XZ transverse sections of the regions indicated by white lines in A-F. White and yellow arrows indicate Fn assembly in the leading front of myocardial cells
in control and gna13a/b MO-injected embryos, respectively. Yellow dashed line, midline; white dashed lines outline myocardial cells; D, dorsal; V, ventral.
Scale bars: 20 µm.
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surrounded the myocardial population and were highly enriched at
the leading edge of the myocardial population during subduction
and medial migration (Fig. 8A″,C″,E″, white arrows). These
findings suggest that the assembly of Fn fibrils is crucial for the
subduction and medial migration of myocardial cells.
In Gα13 morphants, overall Fn expression level is not
significantly different (Fig. 8). However, Fn assembly exhibited
completely different patterns: at 12 s, myocardial cells had not
initiated subduction, and Fn fibrils appeared to be smaller and
displayed no obvious directionality, although Fn assemblies
surrounded the myocardial cells (Fig. 8B-B″); at 14 and 16 s, Fn
fibrils were aligned along the anterior-posterior axis, and
condensed in the lateral region of the myocardial population
(Fig. 8D′,F′, yellow arrowheads); cross-sectional views revealed
that Fn fibrils were not enriched, but rather expressed broadly, in
the leading edge of the myocardial population (Fig. 8D″,F″, yellow
arrows). Considering the crucial role that Fn assembly plays in
epithelial organization of the myocardial cells, we postulate that
abnormal Fn assembly might contribute to defects in their
epithelial polarity, and thus to defects in their subduction and
medial migration. On the other hand, the disrupted patterns of Fn
assembly might reflect impairment of myocardial subduction and
migration.
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
Cells in the ALPM engage in subduction that depends on
endoderm convergence
The myocardial cells are components of the ALPM, and we
tested whether other cells within this structure engage in
similar subduction. The basic helix-loop-helix (bHLH) family
member hand2 is expressed in the ALPM (Trinh et al., 2005; Yelon
et al., 2000), and we used Tg(hand2:GFP)/Tg(sox17:memCherry)
transgenic lines to monitor movement of the ALPM and endoderm.
We found that two GFP+ ALPM populations, including myocardial
cells (Fig. 9), flanked the endodermal layer at 10 s, at which point
GFP can be detected (Fig. 9A, yellow arrowheads), and then
migrated medially and merged at the midline to form a single sheet
(Fig. 9A-F, supplementary material Movie 6). As shown in Fig. 3,
during subduction the myocardial population formed a stripe at
10-12 s. Notably, cells other than myocardial cells located in the
dorsalmost regions of the GFP+ domain exhibited a similar
morphology (Fig. 9A,B, red arrowheads), suggesting that these
ALPM cells also underwent subduction. Indeed, transverse sections
of three-dimensional (3D) confocal z-stacks revealed that, like
myocardial cells, the Hand2-GFP+ cells were dorsal to the
endodermal layer at 8 s, but gradually slid underneath and were
ventral to the endoderm at 10 s and 14 s (supplementary material
Fig. S7A-C′).
DEVELOPMENT
Fig. 9. The ALPM engages in
endoderm-dependent subduction.
Snapshots from epifluorescence timelapse movies of control and gna13a/b MOinjected Tg(sox17:memCherry)/(hand2:
EGFP) embryos at 10-20 s
(supplementary material Movie 6). Dorsoanterior views. (A-L) Overlays of anterior
memCherry-labeled endoderm (red) and
Hand2:GFP-labeled ALPM (green).
(A′-L′) Hand2:GFP-labeled ALPM.
(A″-L″) memCherry-labeled endoderm.
Yellow arrowheads indicate myocardial
cells (MCs); red arrowheads indicate
dorsalmost region of LPM cells; yellow
and red lines indicate gap between the
two myocardial populations and the
dorsalmost region of LPM cells. Scale
bars: 100 µm.
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In Gα13-deficient embryos, the ALPM cells flanked the widened
endodermal layer, and those in the dorsalmost regions, including the
myocardial cells, failed to migrate to the midline by 22 s, leaving a
gap (Fig. 9L, yellow and red lines; supplementary material Fig.
S7E-E″, Movie 6). Moreover, ALPM cells other than the
myocardial cells formed a stripe at 14-16 s, and these cells did not
slide underneath the endoderm until 16 s (Fig. 9H-J; data not
shown), indicating that subduction of these cells was delayed and
impaired. Thus, in the dorsalmost regions of Gα13-deficient
embryos, all ALPM cells are impaired with respect to both
subduction and medial migration, suggesting that medial
migration of this tissue depends on proper convergence of the
endoderm.
To test further whether the migration of ALPM cells relies on
endoderm convergence, we injected a MO targeting sox32, a
transcription factor required for endoderm development (Wong
et al., 2012; Yelon et al., 1999). In such morphants, which lack
endoderm, the Kaede-expressing myocardial precursors failed to
migrate towards the midline, remaining in the same position from
12-18 s (supplementary material Fig. S7I-K versus F-H, Movie 7).
In addition, unlike in control embryos, myocardial cells in the
morphants failed to form the stripe that is characteristic of
myocardial subduction (supplementary material Fig. S7I versus
F). These findings indicate that the endoderm is crucial for the
subduction and medial migration of ALPM cells.
DISCUSSION
Although the endoderm is crucial for myocardial migration, it is
unclear precisely how these two tissues interact. The current study
extends our previous findings that endoderm convergence is directly
linked to myocardial migration. Employing newly developed
transgenic lines, we have identified previously unknown features
of the movements of these tissues, as well as their intricate and
dynamic anatomic relationship during segmentation (Fig. 6E,
model). We show that the endoderm undergoes three distinct
phases of movement: rapid convergence before 12 s, little
convergence at 12-16 s and slight expansion after 16 s; and that
myocardial cells concomitantly undergo three distinct modes of
migration: initial migration that depends on endoderm convergence
(before 12 s), subduction from the dorsal to the ventral side of the
endoderm (12-14 s) and medial migration that appears to be
independent of endoderm convergence (after 14 s). These findings
will probably be fundamental to comprehending the role of the
endoderm in cardiac morphogenesis.
Myocardial cells depend on the endoderm for their migration
at early stages
The dependence of myocardial migration on the converging
endoderm was revealed by our imaging of live and fixed
embryos, which showed that before 12 s, when the endoderm
actively converges towards the midline and its width is reduced, two
populations of myocardial cells flank and remain at the lateral
boundaries of the endodermal sheet. Thus, at this stage myocardial
precursors depend on the endoderm and co-migrate with it towards
the midline (Figs 3,4; supplementary material Fig. S3, Movie 2).
This notion is supported by the findings that in the context of
S1pr2/Gα13 deficiency, myocardial precursors remained at the
lateral boundary of the widened endodermal sheet; thus, migration
of myocardial cells during this period was impaired because of
defects in endoderm convergence (Figs 3,4; supplementary material
Fig. S3, Movie 2). Moreover, endodermal expression of Gα13 before
12 s almost fully rescued the defects of endoderm convergence and
Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
myocardial migration caused by global inhibition of Gα13, whereas
such expression after 12 s had less effect on either endoderm
convergence or myocardial migration (Fig. 6; supplementary
material Movie 4). Our data provide direct evidence that prior to
12 s myocardial cells rely on endoderm convergence to move to the
midline. This finding is consistent with observations in other animal
model systems, showing that the migration of myocardial cells is
influenced by the movements of adjacent tissues [e.g. the endoderm
in chick and mouse (Madabhushi and Lacy, 2011; Maretto et al.,
2008; Varner and Taber, 2012), and the ectoderm in Drosophila
(Haack et al., 2014)].
Myocardial cells engage in subduction during their medial
migration
It was a surprise to find that during their medial migration,
myocardial cells not only migrate by distinct modes at different
stages, but also change their anatomic relationship with the
endoderm (Figs 3-5). The revelation that myocardial precursors
undergo such a process was possible because of high-resolution
in toto imaging (Fig. 5; supplementary material Movie 5).
Furthermore, our discovery that myocardial subduction was
delayed and ineffective in the context of an S1pr2/Gα13
deficiency (Figs 3-5; supplementary material Fig. S3) suggests
that endoderm convergence regulates the timing and efficiency of
myocardial subduction. This idea is supported by the finding that
myocardial subduction never occurred in the absence of endoderm
(supplementary material Fig. S7). In addition, we found that not
only cardiac mesodermal cells, but also other cells of the ALPM,
undergo subduction (Fig. 9; supplementary material Fig. S7),
suggesting that subduction is not specific to myocardial cells.
Notably, we also found that the myocardial population forms a twolayer structure during subduction (Fig. 5; supplementary material
Movie 5). Future experiments involving new transgenic lines that
label the nuclei and membranes of myocardial cells are expected to
shed light on the changes in cell shape and the migratory behavior of
myocardial cells at this stage. Although the mechanisms that promote
myocardial subduction are unknown, we found that Fn fibrils
displayed specific patterns of assembly around the myocardial cells,
and the area between them and the endoderm and that S1pr2/Gα13dependent endoderm convergence is crucial for this Fn assembly
(Fig. 8). Thus, Fn assembly might be crucial only for establishing
epithelial polarity, but perhaps also for facilitating myocardial
subduction and migration. Indeed, we found that by the onset of
myocardial subduction, the epithelial organization of the myocardial
cells had already initiated (Fig. 7). This is much earlier than that was
previously reported (Trinh and Stainier, 2004), suggesting that
epithelial polarity is necessary for myocardial subduction. In addition,
it is possible that Fn assembly facilitates mechanical contractility
between the endoderm and myocardial precursors, a phenomenon that
has been shown to promote tissue interactions (Lecuit et al., 2011;
Miller and Davidson, 2013). In Gα13 morphants, Fn fibrils failed to
assemble in the correct directions and were not enriched at the leading
regions of myocardial populations (Fig. 8), and the epithelial polarity
of myocardial cells was disrupted (Fig. 7; supplementary material
Fig. S6). These outcomes could affect the subduction of myocardial
cells and their subsequent medial migration.
Myocardial cells migrate independently of endoderm
convergence during the final period of their medial migration
Notably, from 14 s, when the endoderm completes convergence,
myocardial precursors continue to migrate medially towards the
midline to form a single cell population, seemingly independent of
2937
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RESEARCH ARTICLE
convergent movement of the endoderm (Figs 3,4; supplementary
material Movie 2). However, our experiments have not ruled out
other potential roles of the endoderm in myocardial migration
during this period. In particular, myocardial populations surrounded
by Fn retain contact with endodermal sheet (Fig. 8). During this
stage, the myocardial precursors are located between the
endodermal sheet and the yolk syncytial layer (YSL) (Trinh and
Stainier, 2004). Our data indicate that the endoderm stops migrating
medially, and there is no evidence that the YSL moves towards the
midline, although progenitor cell-mediated movement of internal
yolk syncytial nuclei (iYSN) has been reported (Carvalho et al.,
2009). Thus, there are no other cells/tissues adjacent to myocardial
cells that migrate medially and could provide the pulling forces
necessary to promote myocardial medial migration. Furthermore,
our live imaging shows that myocardial cells display migratory
protrusions at this stage (supplementary material Fig. S4C-F,
Movie 3). Additionally, in addition to undergoing medial migration,
myocardial cells were found to change their direction, angling
towards endocardial precursors (Holtzman et al., 2007). This
probably resulted from their intrinsic ability to migrate. Thus, the
subduction and medial migration of myocardial cells after 14 s is
probably due to their innate migration potential. Earlier studies in
chick suggested that myocardial cells have little ability to migrate,
and that endoderm movement drives their migration (Cui et al.,
2009; Varner and Taber, 2012). However, a new study showed that
chick myocardial cells instead exhibit active displacement and
autonomous ventral movement, and that these changes are
independent of the endoderm (Aleksandrova et al., 2015). It is
thus possible that the ventral movement of these cells in chick is
similar to the myocardial subduction we observed in zebrafish.
Of note, evidence also indicates that displacement of the
extracellular matrix in the context of global tissue morphogenesis
accounts for the movement of certain tissues (Bénazéraf et al., 2010;
Cui et al., 2009; Zamir et al., 2008); this argues against cellautonomous migration of the myocardial cells. Interestingly, our
data show that the direction of Fn assembly aligns with that of
myocardial-cell migration (Fig. 8), suggesting that these two events
are linked. However, establishing whether Fn assembly resulted
from or directs myocardial migration will require in vivo labeling
and imaging of Fn. This is not feasible with the technologies
currently available for zebrafish.
In addition, we found that, although myocardial cells in S1pr2/
Gα13-deficient embryos eventually migrated to the ventral side of
the endoderm, they inevitably failed to migrate towards the midline
(Figs 3,4; supplementary material Fig. S3). This could be due
to defects in epithelial polarity of myocardial cells in context of
S1pr2/Gα13-deficiency (Fig. 7). However, we cannot exclude the
involvement of other factors, such as endocardial cells. Similar to
myocardial cells, two populations of endocardial precursors migrate
towards, and merge at, the midline, and then migrate towards
the myocardial cells to form the heart cone (Bussmann et al., 2007).
It would be interesting to assess whether the medial migration
of endocardial cells depends on endoderm convergence, whether
endocardial cells also undergo subduction and what the
consequences for myocardial migration are when endocardial
migration is defective in S1pr2/Gα13-deficient embryos.
In summary, our work shows that during heart-tube formation,
myocardial cells exhibit three distinct migratory modes, and their
migration depends on endoderm convergence during early stage,
but not during the late stage. Similarly, a recent study in Drosophila
showed that cardiac cells initially depend on the adjacent ectoderm
to migrate, but exhibit autonomous and active migration at later
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Development (2015) 142, 2928-2940 doi:10.1242/dev.113944
stages (Haack et al., 2014). Our results have significant implications
for the interplay of tissues in organ development. In particular,
many congenital heart defects, such as Cornelia De Lange and
DiGeorge syndromes (Arnold et al., 2006; Muto et al., 2011), also
display endoderm defects. Understanding the interactions between
the endoderm and the cardiac mesoderm might shed light on the
causes of such disease.
MATERIALS AND METHODS
Zebrafish strains and maintenance
AB*/Tuebingen, transgenic Tg(myl7:EGFP) (Huang et al., 2003), Tg
(sox17:EGFP) (Mizoguchi et al., 2008), Tg(hand2:GFP)pd24 (Yin et al.,
2010), Tg(nkx2.5:ZsYellow) (Zhou et al., 2011), Tg(nkx2.5:Kaede) (GunerAtaman et al., 2013) and milm93 zebrafish strains (Kupperman et al., 2000)
were used. Embryos were obtained by natural mating, maintained at 28.5°C
and, unless specified otherwise, were staged according to morphology or
hours post fertilization (hpf ), as described previously (Kimmel et al., 1995).
Generation of transgenic lines
The transgenic lines Tg(sox17:memCherry), Tg(sox17:mCherry-2Agna13a), Tg(hsp70:gna13a), Tg(myl7:memGFP) and Tg(nkx2.5:
lifeactGFP) were generated using a Tol2-based Multi-Site Gateway
system (Life Technologies) (Kwan et al., 2007; Villefranc et al., 2007).
All transgenic constructs were assembled using entry clones kindly
provided by Chi-Bin Chien and Kristen Kwan (University of Utah, USA),
unless specified otherwise. A 5′-entry clone containing a sox17
mini-promoter (Woo et al., 2012) was used to express genes specifically
in the endoderm. A 5′-entry clone containing an nkx2.5 promoter (Choe
et al., 2013) was used to express genes specifically in myocardial cells. A
middle clone encoding mCherry, and a 3′-entry clone encoding the 2A
peptide, a zebrafish gna13a-coding region (morpholino-insensitive), and
SV40, was created to express Gα13a (mCherry is used to identify lines
expressing Gα13a). Middle-entry clones encoding the zebrafish gna13a and
lifeact-GFP coding region were created using the pENTR Directional TOPO
Cloning Kit (Life Technologies). Tg(hsp70:gna13a) line was generated
using a destination vector that contains a myl7:GFP cassette (GFP
expression in the heart was used to identify embryos that contain the
gna13a transgene). Transgenic lines were established using standard
techniques as described previously (Xu et al., 2014). The founders were
bred to generate multiple stable lines.
Whole-mount in situ hybridization (WISH) and
immunofluorescence (IF)
Standard methods were used for WISH and IF. Please see supplementary
materials methods for further details.
Microscopy, time-lapse imaging and image analysis
Time-lapse imaging was performed as described previously (Ye and Lin,
2013). Please see supplementary materials methods for a detailed
description of microscopy, imaging and image analysis.
Morpholino (MO) injection
The previously validated MOs targeting the following genes were injected
into embryos at 1-cell stage: gna13a and gna13b (2-3 ng each) (Lin et al.,
2005), s1pr2/mil (15 ng) (Kawahara et al., 2009) and sox32 (4 ng)
(Wong et al., 2012).
Statistical analysis
Data were compiled from two to three independent experiments and are
presented as mean±s.e.m. Statistical analyses were performed using
unpaired Student’s two-tailed t-test and unequal variance, and P<0.05
was considered significant.
Acknowledgements
We are grateful to Caroline Burns and Geoffrey Burns (Harvard Medical School) for
providing the Tg(nkx2.5:ZsYellow) and Tg(nkx2.5:Kaede) lines; Sean Megason and
DEVELOPMENT
RESEARCH ARTICLE
Fengzhu Xiong (Harvard Medical School) for the mounting mould; Stephanie Woo
(University of California, San Francisco) for the sox17 promoter 5′ entry clone; Gage
Crump (University of Southern California) for the nkx2.5 promoter 5′ entry clone;
Carl-Philipp Heisenberg for lifeact-GFP entry clone (IST Austria); and Hui Xu and
Baosheng Xie (University of Iowa) for generating the Tg(hsp70:gna13a) line and
the Tg(nkx2.5:lifeact-GFP) construct, respectively.
Competing interests
The authors declare no competing or financial interests.
Author contributions
F.L. and D.Y. conceived the ideas and designed experiments; D.Y., H.X. and B.H.
performed the experiments; F.L. wrote the manuscript.
Funding
This work was supported by grants from the American Heart Association
[12GRNT11670009, 2012]; March of Dimes Foundation Research [2013]; and the
National Science Foundation [IOS-1354457] (all to F.L.).
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113944/-/DC1
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