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First published online May 30, 2007
doi: 10.1242/10.1242/dev.02857
1 Developmental Genetics Program and Department of Cell Biology, Skirball
Institute of Biomolecular Medicine, New York University School of Medicine,
New York, NY 10016, USA.
2 Department of Biology, Queens College, The City University of New York,
Flushing, NY 11367, USA.
3 Institute of Molecular and Cellular Biology, National Taiwan University,
Taipei, Taiwan.
* Author for correspondence (e-mail: yelon{at}saturn.med.nyu.edu)
Accepted 28 March 2007
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MATERIALS AND METHODS
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DISCUSSION
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Key words: Endocardium, Myocardium, Zebrafish, cloche, miles apart (edg5), Morphogenesis
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MATERIALS AND METHODS
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DISCUSSION
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;
Harvey, 2002;
Moorman and Christoffels,
2003). Once they reach the embryonic midline, the bilateral
populations merge through a process called cardiac fusion. Cardiac fusion
produces an intermediate structure, referred to as a cardiac crescent in
amniotes (Harvey, 2002;
Moorman and Christoffels,
2003) and a cardiac cone in zebrafish
(Glickman and Yelon, 2002),
that gradually transforms into a primitive heart tube. This simple cylinder
immediately becomes responsible for driving embryonic circulation; therefore,
the precise coordination of heart tube assembly has significant consequences
for embryonic physiology.
Previous studies of heart tube assembly in the zebrafish embryo have
provided an overview of this dynamic process. Cardiac fusion initiates with
contacts between posterior subsets of contralateral cells, followed by
interactions between anterior subsets of contralateral cells
(Glickman and Yelon, 2002;
Yelon et al., 1999)
(Fig. 1A). These connections
create a ring of cardiomyocytes surrounding the centrally located precursors
of the vascular endocardium (Stainier et
al., 1993; Trinh and Stainier,
2004) (Fig. 1A).
Next, the myocardial ring, which is also called the cardiac cone, converts
into a tube (Glickman and Yelon,
2002; Stainier et al.,
1993; Yelon et al.,
1999). Through gradual extension of the length of its axis, the
once shallow cone becomes an elongated cylinder, and the inner and outer
circumferences of the cone become the arterial and venous apertures of the
nascent heart tube (Glickman and Yelon,
2002; Stainier et al.,
1993; Yelon et al.,
1999). At the same time, the endocardial precursors spread out to
create the endothelial lining of the myocardial cylinder
(Stainier et al., 1993). Thus,
the cardiac cone provides an essential foundation for the dimensions and
composition of the zebrafish heart tube.
Despite the importance of creating a strong foundation for heart tube
formation, the morphogenetic mechanisms that regulate cardiomyocyte behavior
during this process remain relatively undefined. Work in multiple model
organisms has indicated that interactions between the myocardium and
neighboring gut endoderm are crucial for the recruitment of cardiomyocytes
toward the embryonic midline (e.g. Dickmeis
et al., 2001; Kikuchi et al.,
2001; Li et al.,
2004; Narita et al.,
1997). Medial recruitment also appears to require the organization
of cardiomyocytes into polarized epithelia. During cardiac fusion,
cardiomyocytes begin to form the junctional complexes typical of epithelia and
exhibit molecular characteristics of apicobasal polarity
(Trinh and Stainier, 2004).
Mutations that interfere with epithelialization, such as natter (also
known as fibronectin 1), prevent cardiomyocytes from moving properly
toward the midline (Trinh and Stainier,
2004). However, it is not clear whether the proximity of the
contralateral myocardial populations at the midline is sufficient to
coordinate their integration into a heart tube. Does tube construction simply
result from a continuation of the initial medial movement of cardiomyocytes,
or does it require a distinct mode of cell behavior subject to independent
regulation?
To distinguish between these models, we have used time-lapse imaging to resolve the direction, rate and organization of individual cardiomyocyte movements during cardiac fusion in wild-type and mutant zebrafish embryos. Our data indicate that two morphologically and genetically separable phases of cardiomyocyte movement underlie cardiac fusion: a first phase that recruits cells toward the midline and a second phase that creates the morphology of the cardiac cone. Additionally, we find that cardiomyocyte movement during the second phase is regulated by the presence of the endocardium, thereby revealing a previously unappreciated role for myocardial-endocardial interactions during heart tube assembly.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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;
Qian et al., 2005) carrying
Tg(cmlc2:egfp) (Huang et al.,
2003) were mated to generate experimental embryos. Embryos were
screened for robust egfp expression using a Zeiss M2Bio microscope.
For mil mutants, the genotype was assessed prior to time-lapse
imaging: these mutants exhibit a clear cardia bifida phenotype at the
18-somite stage (Yelon et al.,
1999). Selected embryos were mounted in a drop of 3%
methylcellulose within a larger drop of 2% low-melting point agarose in embryo
medium. Mounting slides were constructed by placing a single layer of
electrical tape on a standard microscope slide and cutting a small square out
of the center to create a well. Embryos were oriented to permit a dorsal view
of the heart fields, using the tip of the notochord as a guide. Finally, a
drop of embryo medium with tricaine was placed on top of each agar-mounted
embryo.
Data collection and processing
Embryos were maintained at 28.5°C during data collection, using an
upright Zeiss LSM510 confocal microscope equipped with a heated stage, a
25x Plan-Neofluor multi-immersion objective and LSM510 software. In
order to generate 3D reconstructions, 30-40 focal planes were collected at
intervals of 1.5-2.5 µm, a distance sufficient to accommodate sample
movement during the course of the time-lapse. Images were collected at 2-3
minute intervals. Typically, time-lapses began around the 15- to 18-somite
stage and ended at the completion of fusion, around the 20- to 21-somite
stage. Time of initial imaging was often dependent upon GFP intensity, which
varies at early stages. After imaging, embryos were coaxed from their mounting
agar and transferred to fresh embryo medium. Embryonic stage was determined
both before and after mounting; manipulation and imaging did not delay
development. Additionally, embryonic health and genotype were assessed at 24
hours post-fertilization (hpf). Only data collected from embryos that appeared
completely normal at 24 hpf were analyzed.
For 4D analysis, LSM510 databases were imported into a Volocity (Improvision) library. An image sequence composed of a linear array of 3Drendered time points was then generated for each data set. As appropriate, image appearance (brightness, contrast, opacity) was modified to optimize visualization of cells of interest. Optimization of image appearance was a key step: GFP intensity varies widely among individual cardiomyocytes and increases over time, so resolution of each cell required different adjustments of image settings. Image settings shown in Figs 1-4 allow resolution of many, but not all, tracked cells. Images were exported as QTmov files for visualization of a time-lapse from a preset view.
Cell tracking and analysis
Examination of high-resolution confocal images indicated that the
myocardium is a relatively flat sheet of cells, approximately 8 µm thick,
from the 15- to 20-somite stages (N.G.H. and D.Y., unpublished). During these
stages, most cardiomyocyte movement is along the x (mediolateral) and
y (anteroposterior) axes, rather than along the z
(dorsoventral) axis. The z-axis displacement of tracked
cardiomyocytes did not exceed one cell diameter over the course of our
time-lapses. Therefore, when tracking cardiomyocyte movements, we treated the
myocardium as a 2D tissue and focused on tracking the x and
y coordinates of the center of each cell. At time points following
our window of analysis, we have observed significant dorsal cardiomyocyte
movements as the cardiac cone extends into a tube (N.G.H. and D.Y.,
unpublished). These dorsal movements are consistent with prior histological
observations (Stainier et al.,
1993; Trinh and Stainier,
2004), although we have not observed lateral displacement of
dorsal cardiomyocytes as suggested by Trinh and Stainier
(Trinh and Stainier,
2004).
For each time-lapse, we examined all z-stack images and tracked as many cells as were resolvable in any focal plane throughout the entire period of imaging. Figs 1-4 show reconstructed projections of z-stacks and include a representative subset of tracks from the time-lapses depicted. To track a cell, we selected a pixel at its visual center at each time point, modifying the image intensity as necessary, and recorded each pixel's coordinates using Volocity. Each series of cell locations was then converted to a track using Volocity's `manual track' function. This function automatically generates a measurement file that includes the distance traveled, velocity and a series of xy positions for each cell. These files were exported into Microsoft Excel for further analysis, including calculation of net direction of movement. To visually depict cell tracks (as in Fig. 1E-I, Fig. 2C-E and Fig. 3C,D) we generated lines connecting each series of cell locations, added arrowheads to indicate the direction of movement, and overlaid these tracks on processed images of individual time points. As a control for accuracy of tracking, all of the cells in one wild-type embryo were tracked twice. The two sets of distances, velocities and directions of movement did not vary by more than 5%.
For quantitative analysis of the net direction of movement for each tracked
cell, we calculated the percentage of the y-axis displacement
relative to the x-axis displacement in radians, using the formula:
(
y/x)x100. This can also be expressed as
an angle (°) using the formula:
atan(y/x)x57.295. The resulting value
reflects the cell's degree of displacement along the anterior-posterior axis:
a value of 0° corresponds to direct medial movement, and a value of
90° corresponds to direct anterior or posterior movement. Examination of
the degree of displacement of wild-type cardiomyocytes during the first phase
of cardiac fusion demonstrated that more than 80% of these cells were
displaced by less then 30° (Fig.
1J). We therefore defined `medial movement' as a displacement of
less than 30° and `angular movement' as a displacement of greater than
30°. Some tracked cells exhibited little movement: we defined `no net
displacement' as movement of less than 10% of a cell diameter over the course
of a 20-minute period (as in Fig.
1I). Additionally, we defined `no net directed movement' as
failure to move beyond a cell diameter from the starting position over the
course of a time-lapse lasting at least 90 minutes (as in
Fig. 4C,D).
To compare regional differences in the patterns of cardiomyocyte movement, we divided the bilateral cardiomyocyte populations into three regions: anterior, central and posterior. In wild-type embryos, we defined regions retrospectively, based on the positions of individual medial cardiomyocytes at the completion of cardiac fusion (Fig. 1M). Cells located at the anterior or posterior interfaces between contralateral populations were considered to be in anterior or posterior regions, respectively. Cells located between these areas were considered to be in the central region. In clo, mil and mil;clo mutant embryos, we defined regions differently, as cardiac cone morphology is aberrant. In these cases, we divided populations into equivalent thirds along the anterior-posterior axis, based on the number of cells present at the medial edge of the population at the beginning of the time-lapse (Fig. 2G and Fig. 3F).
In situ hybridization
Single-color whole-mount in situ hybridization for cmlc2 and
flk1 was performed according to a standard protocol
(Liao et al., 1997;
Yelon et al., 1999). Two-color
fluorescent whole-mount in situ hybridization was conducted according to a
protocol that will be reported separately (J.J.S., B. R. Keegan and D.Y.,
unpublished). Riboprobes for cmlc2 and fli1a
(Thompson et al., 1998) were
labeled with dinitrophenol (Mirus) and digoxygenin (Roche) haptens and
detected by deposition of Cy3 and fluorescein tyramides (PerkinElmer),
respectively.
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DISCUSSION
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).
Tg(cmlc2:egfp) expression mimics endogenous cardiac myosin light
chain 2 (cmlc2; myl7 - ZFIN) expression, beginning
before cardiomyocytes approach the embryonic midline and continuing throughout
development (Huang et al.,
2003; Yelon et al.,
1999) (Fig. 1B-D).
We visualized cell movements over time by collecting confocal
z-stacks at 2-minute intervals and rendering a three-dimensional
time-lapse. To quantify patterns of cell behavior, we measured the
displacement of individual cells between time points, creating a track
representative of each cell's movement over time (detailed imaging and
tracking protocols are provided in the Materials and methods). Using this
strategy, we resolved the direction, rate and organization of individual
cardiomyocyte movements throughout the process of cardiac fusion. Although we
focused our analysis on cells at the medial edge of each cardiomyocyte
population, we also found that lateral cells exhibit comparable patterns of
movement (data not shown).
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; Keegan et al.,
2004; Lyons et al.,
1995; O'Brien et al.,
1993; Stalsberg and DeHaan,
1969; Yelon et al.,
1999). In accordance with these previous studies, our time-lapse
examination of wild-type embryos demonstrated that cardiac fusion begins with
a phase of coherent medial movement that brings the cardiomyocytes into
position for the initiation of cardiac cone formation
(Fig. 1B,C,E,F and see Movie 1
in the supplementary material). All cardiomyocytes exhibited similar patterns
of behavior, following relatively parallel paths of similar length with
general maintenance of neighbor relationships
(Fig. 1E,F). Examination of
cell tracks revealed that most cardiomyocytes move directly toward the
midline, exhibiting an angular displacement of less than 30° along the
anterior-posterior axis (Fig.
1J and Table 1). We
classify this behavior as `medial movement' (red arrows/bars), in contrast to
`angular movement' (yellow arrows/bars), which we define as an angular
displacement of greater than 30° (Fig.
1E,F,J). Overall, the observed coherence of cardiomyocyte movement
is consistent with the status of the migrating myocardium as a maturing,
polarized epithelium (Trinh and Stainier,
2004).
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As a consequence of regional transitions to angular movement, the bilateral populations of cardiomyocytes formed mirror-image arcs, ultimately encircling the endocardial precursors and creating the cardiac cone (Fig. 1C,D,G,H). Regional differences in the duration of movement also contribute to arc formation. Although all cardiomyocytes moved at comparable rates during the second phase of cardiac fusion (0.36±0.03 µm/minute), cells in central regions tended to stop moving earlier than cells in anterior or posterior regions. Examination of cell movements during the final 20 minutes of cone formation (20 minutes before the 20-somite stage, Fig. 1I) revealed that most centrally located cells exhibit no net displacement during this interval [64% (35 of 55) of cells examined; white asterisks]. By contrast, most cells in anterior and posterior regions continued moving during this time interval, with only 33% (21 of 63) of these cells coming to a halt. Together, our data indicate that regionally restricted transitions in the direction and duration of cardiomyocyte movement facilitate cardiac cone formation.
No angular movement in the absence of endocardium
Our time-lapse analyses in wild-type embryos indicated that cardiac fusion
involves two morphologically distinct phases of cell behavior. We hypothesized
that these two phases could represent separately regulated morphogenetic
processes, with endoderm-derived cues driving the initial medial movement
(Alexander et al., 1999;
Dickmeis et al., 2001;
Kikuchi et al., 2001;
Kupperman et al., 2000) and
independent cues triggering the transition from medial to angular movement.
Previous studies have suggested that the endocardial precursors cluster at the
future center of the cardiac cone
(Stainier et al., 1993;
Trinh and Stainier, 2004). To
test whether the endocardium influences the redirection of anterior and
posterior cardiomyocytes toward a central point, we tracked cardiomyocyte
movements in cloche (clo) mutant embryos
(Stainier et al., 1995).
Although the identity of the clo gene has not yet been reported,
prior analyses have established that clo is required
cell-autonomously for formation of endothelial precursors, and clo
mutants therefore exhibit a general loss of endothelium, including the
endocardium (Liao et al.,
1997; Stainier et al.,
1995).
Examination of clo mutants demonstrated that cardiac fusion begins normally in the absence of endocardium. The medial direction of movement exhibited by clo mutant cardiomyocytes (Fig. 2A-D and see Movie 2 in the supplementary material) resembled the initial behavior of wild-type cardiomyocytes (Fig. 1E,F). Additionally, the mean rate of clo mutant cardiomyocyte movement (0.62±0.12 µm/minute) was similar to the mean rate observed during the first phase of wild-type cardiac fusion (0.68±0.04 µm/minute). However, in contrast to wild-type behavior, clo mutant cardiomyocytes failed to make a transition to angular movement (Fig. 2C,D,F,G,H and Table 1). Instead, anterior and posterior clo mutant cardiomyocytes continued to move medially throughout cardiac fusion. Additionally, central cardiomyocytes in clo mutants remained in motion over a longer duration than their wild-type counterparts (compare Fig. 2E with Fig. 1I). During the last 20 minutes of cone formation, only 31% (10 of 26) of the centrally located cells tracked in clo mutant embryos came to a halt. Together, these abnormal movement patterns result in the formation of a dysmorphic cardiac cone (Fig. 2B). Thus, the clo mutant phenotype suggests that the endocardium exerts significant influence on the direction and duration of cardiomyocyte movement during the second phase of cardiac fusion. Subsequently, heart tube extension proceeds slowly and aberrantly in clo mutants, such that the misshapen cardiac cone transforms into an abnormally short and wide tube (Fig. 2I,J). The aberrant dimensions of the clo mutant cardiac cone might be directly responsible for the dimensions of the clo mutant heart tube. However, it is also possible that the morphology of the clo mutant heart tube reflects a separate role of the endocardium in regulating heart tube extension.
Angular movement in the absence of prior medial movement
The aberrant patterns of cell behavior in clo mutants suggest that
the endocardium might actively guide the direction of cardiomyocyte movement.
Alternatively, the endocardium might play a more passive role in shaping the
cardiac cone, perhaps by creating an obstacle for the moving cardiomyocytes.
In this scenario, the central location of the endocardium, together with the
intracellular junctions connecting the myocardial epithelium
(Trinh and Stainier, 2004),
could create physical constraints for the medially migrating cardiomyocytes,
resulting in the conversion of medial movement into angular movement at the
anterior and posterior ends of each heart field. To test whether the
transition to angular movement requires an initial phase of medial movement,
we tracked cardiomyocyte movement in mutants exhibiting cardia bifida, a
phenotype in which the heart fields do not move toward the midline and instead
form two lateral hearts. Zebrafish mutations that disrupt endoderm
specification or morphogenesis, such as casanova (cas;
sox32 - ZFIN) and miles apart (mil; edg5 -
ZFIN), result in cardia bifida. The cas locus encodes a Sox-related
transcription factor that is required cell-autonomously for endoderm
specification (Alexander et al.,
1999; Dickmeis et al.,
2001; Kikuchi et al.,
2001). The mil locus encodes a sphingosine-1-phosphate
receptor that plays a cell non-autonomous role in recruiting cardiomyocytes to
the midline (Kupperman et al.,
2000). Endoderm morphogenesis is disrupted in mil mutants
(Kupperman et al., 2000),
possibly as a consequence of defects in endoderm-extracellular matrix
interactions (Matsui et al.,
2007), and the mil myocardial defects are presumed to be
a result of the observed endoderm defects. We tracked cardiomyocytes in both
cas mutants and mil mutants. As we observed comparable
phenotypes in both cases, we focus here on our mil mutant data.
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). Thus,
the mil mutant phenotype indicates that the angular movements that
establish the cardiac cone can occur even in the absence of initial medial
movement.
Endocardium actively induces cardiomyocyte movement
Our data indicate that cone formation in cardia bifida mutants involves
angular cell movements reminiscent of those observed during wild-type cone
formation (Fig. 3C-G and
Fig. 1G,H,L,M), suggesting that
lateral cone formation might also depend on the presence of endocardium. In
mil mutants, the presumed endocardial precursors do not move to the
midline (Fig. 3H-K). Instead,
these cells remained clustered laterally, adjacent to the central regions of
the mil mutant heart fields (Fig.
3H-K). Thus, angular cardiomyocyte movements are directed toward
centrally located endocardial cells in both wild-type and mil mutant
embryos. To test whether endocardium is required for the angular movements
observed in mil mutants, we tracked cardiomyocyte movements in
mil;clo double mutants (Fig.
4A-D and see Movie 4 in the supplementary material).
mil;clo double mutants displayed the expected lateral heart fields;
however, unlike mil mutant cardiomyocytes, mil;clo
double-mutant cardiomyocytes did not display any directed movement
(Fig. 4C,D). Although cells
appeared to jostle relative to their neighbors, they did not exhibit net
medial or angular movement. As a consequence, the mil;clo double
mutants did not form cardiac cones or tubes; instead, each heart field
remained as a sheet of cells. Thus, the mil;clo double-mutant
phenotype, like the clo mutant phenotype, suggests that the
endocardium is required for angular cardiomyocyte movement during cone
formation. Moreover, the requirement for endocardium in a cardia bifida
scenario suggests that the endocardium is not merely an obstacle that diverts
the medial progress of cardiomyocyte movement. Rather, the endocardium appears
to play an active role in inducing the angularly directed cell movements that
configure the cardiac cone.
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MATERIALS AND METHODS
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DISCUSSION
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Endocardium influences the induction, direction and duration of cardiomyocyte movement
Our data suggest a new function for the endocardium in regulating
myocardial behavior: the endocardium actively provokes a critical transition
in cardiomyocyte movement, inducing a phase of movement that is necessary to
shape the cardiac cone. The endocardium may also actively dictate the angular
direction of cardiomyocyte movement during the second phase of cardiac fusion.
However, our data cannot rule out a more passive mechanism in which the
physical position of the endocardial cells influences the angle of
cardiomyocyte movement. In addition to its role in triggering cardiomyocyte
movement, the endocardium also appears important for bringing cardiomyocytes
to a halt: it is likely that the duration of central cardiomyocyte movement is
limited by the physical impediment of the centrally located endocardial
precursors.
The molecular mechanisms by which the endocardium stimulates cardiomyocyte
movement remain to be explored. The endocardium might communicate with the
myocardium via direct cell-cell contacts or via secreted cues. Previous
studies have demonstrated multiple requirements for myocardial-endocardial
crosstalk during later phases of heart development. For example,
myocardial-endocardial communication mediated by Tgf-ß, Wnt, Notch,
Calcineurin and Vegf signaling is crucial during atrioventricular valve
formation (e.g. Armstrong and Bischoff,
2004; Beis et al.,
2005; Chang et al.,
2004; Hurlstone et al.,
2003; Lee et al.,
2006; Timmerman et al.,
2004). Additionally, Neuregulin and Plexin-Semaphorin signaling
are crucial during trabecular outgrowth
(Meyer et al., 1997;
Toyofuku et al., 2004),
Neuregulin and Notch signaling are crucial during specification of the cardiac
conduction system (Milan et al.,
2006; Rentschler et al.,
2002), and Heart of glass signaling is crucial during chamber wall
thickening (Mably et al.,
2003). In future work, it will be interesting to evaluate whether
any of these signaling pathways are crucial for the myocardial-endocardial
interactions that coordinate heart tube assembly. Alternatively, endocardium
could play a less direct role, perhaps contributing to the local deposition of
extracellular matrix components (Trinh and
Stainier, 2004) in a way that affects the available routes for
cardiomyocyte movement.
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;
Harvey, 2002;
Moorman and Christoffels,
2003). In this regard, it is interesting to compare our findings
with data regarding cardiac morphogenesis in Ciona intestinalis
(Davidson et al., 2005). The
Ciona embryo contains a pair of cardiac progenitor cells that exhibit
two separate phases of directed movement as they travel from their origin in
the tail toward the ventral midline. First, the cardiac progenitor cells
migrate anteriorly in close association with the endoderm. Then, the cells
switch direction and move ventrally to facilitate cardiac fusion at the
midline. Combining data from zebrafish and Ciona, we suggest that a
two-phase model of cardiac fusion might be applicable throughout chordate
phyla, with an initial phase of cardiomyocyte recruitment relying on
interactions with the endoderm, and a second phase of cardiomyocyte
integration relying on independent factors such as those supplied by the
endocardium during zebrafish cardiac cone formation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2379/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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), the
percentage of cells moving angularly is significantly reduced
(t-test, P<0.01) compared with that observed during the
second phase of wild-type fusion (see 
