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 Developmental Biology, Biology I, University of Freiburg,
Freiburg, Germany
* Author for correspondence (e-mail: yelon{at}saturn.med.nyu.edu)
Accepted 22 March 2004
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SUMMARY |
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Key words: Zebrafish, Ventricle, Atrium, Myocardium, Endocardium, Fate map, Nodal
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Introduction |
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Prior to heart tube formation, distinct populations of ventricular and
atrial myocardial precursors reside within the lateral plate mesoderm (LPM).
In chick, LPM fate maps indicate a rostrocaudal organization of ventricular
and atrial precursors (DeHaan,
1963; DeHaan,
1965
; Hochgreb et al.,
2003
; Redkar et al.,
2001
; Stalsberg and DeHaan,
1969
), concurring with the regionalized expression patterns of
chamber-specific genes (Bisaha and Bader,
1991
; Yutzey et al.,
1994
). Similarly, in zebrafish and mouse, restricted gene
expression patterns suggest spatial organization of chamber precursors within
the LPM (Yelon et al., 1999
;
Berdougo et al., 2003
;
OBrien et al., 1993
;
Lyons et al., 1995
). Thus,
there exists a clear ventricular-atrial pattern within the LPM, but how is
this pattern established? Fate maps of the chick embryo suggest that
organization of ventricular and atrial lineages begins at early stages, with
ventricular progenitors tending to be located in rostral regions of the
cardiogenic portion of the primitive streak and atrial progenitors tending to
be located more caudally (Garcia-Martinez
and Schoenwolf, 1993
;
Rosenquist, 1970
). However, it
is not known whether these positional tendencies correlate with a distribution
of chamber specification signals within the primitive streak.
In zebrafish, previous lineage analysis has shown that labeling of a single
cardiogenic blastomere at the midblastula (2000 cell/high) stage results in
labeled progeny in either the ventricle or the atrium, but never in both
chambers (Stainier et al.,
1993). These data indicate an early separation of ventricular and
atrial lineages, but they do not reveal how this separation is regulated. One
possibility is that ventricular and atrial progenitors are spatially organized
at early stages and are thereby differentially exposed to key specification
signals. Alternatively, ventricular and atrial progenitors could be
intermingled within the blastula, with lineage separation mediated by
stochastic and/or lateral inhibition mechanisms.
To distinguish between these models of chamber specification, we have constructed a fate map of chamber progenitors in the zebrafish blastula, including analysis of myocardial and endocardial fates in both the ventricle and the atrium. The resolution of our fate map demonstrates that ventricular and atrial myocardial progenitors are spatially organized prior to gastrulation, such that ventricular progenitors are found closer to the embryonic margin and the dorsal midline than are atrial progenitors. By contrast, ventricular and atrial endocardial progenitors are intermixed at this stage. The relative proximity of ventricular myocardial progenitors to the embryonic margin indicates that Nodal signals, which emanate from the margin, could influence ventricular fate assignment. Consistent with this hypothesis, we find that antagonism of Nodal signaling alters the myocardial fate map, demonstrating an important role of the Nodal pathway during cardiac chamber specification.
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Materials and methods |
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At 40% epiboly [5 hours post fertilization (hpf)], we oriented embryos
laterally in 3% methylcellulose, using the expression of Tg(gsc:gfp)
to indicate the location of the dorsal margin
(Doitsidou et al., 2002
). To
activate caged fluorescein in individual blastomeres, we exposed cells to a 10
second pulse from a 375 nm pulsed nitrogen laser (MicroPoint Laser System,
Photonic Instruments) focused through a 40x water-immersion objective on
a Zeiss Axioplan 2 microscope, equipped with a cooled CCD camera (Pentamax,
Princeton Instruments) and automated shutters (Uniblitz) controlled by the
Metamorph 4.5 Imaging System (Universal Imaging). In each experiment, we
labeled a specific number of neighboring blastomeres within a single tier of
the deep cell layer (DEL) of the blastoderm. At 40% epiboly, the first four
tiers of the DEL are one to three cells thick
(Warga and Kimmel, 1990
). For
consistency between experiments, we always labeled cells in the most
superficial layer of the DEL. In several control experiments, we fixed embryos
immediately after initial photography (see below) and proceeded with detection
of activated fluorescein (see below); results confirmed that labeling was
restricted to the intended blastomeres (data not shown).
Recording the location of labeled blastomeres
For each experiment, we recorded the latitude (distance from the margin)
and longitude (distance from the dorsal midline) of the labeled blastomeres.
Immediately after fluorescein activation, we captured DIC and fluorescent
images to record the latitude of the labeled cells; this position is expressed
in tiers, or cell diameters, from the margin, with tier 1 being the row of
blastomeres closest to the yolk (Fig.
1A-C). Before documenting the longitude of the labeled cells, we
incubated embryos for an hour at 20°C, allowing expression of
Tg(gsc:gfp) to become appropriately robust. We then recorded
longitude using animal views; this position is expressed in degrees around the
circumference of the embryo, with the center of Tg(gsc:gfp)
expression defined as 0° (Fig.
1D). To estimate the change in position introduced by waiting 1
hour to record longitude, we compared longitude positions immediately
following labeling and 1 hour after labeling. Convergence of labeled
blastomeres toward dorsal was always less than 5° (n=13; data not
shown).
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Counterstaining techniques enhanced visualization of the heart. In some
cases, we used the MF20 anti-myosin heavy chain monoclonal antibody
(Bader et al., 1982) to
recognize myocardium. We incubated embryos with a 1:10 dilution of MF20
hybridoma supernatant overnight at 4°C, followed by detection with a goat
anti-mouse Ig(H+L) secondary antibody conjugated to ß-galactosidase
(1:100 dilution, 2 hours at room temperature; Southern Biotechnology
Associates). After washing (four washes, 15 minutes each), we equilibrated
embryos in staining buffer (2 mM MgCl2, 0.2% NP-40, 1 mM
deoxycholic acid, 5 mM K3Fe(CN)6, 5 mM
K4Fe(CN)6) and stained with the ß-galactosidase
substrate 6-Chloro-3-Indoxy-beta-D-galactopyranoside
(salmon-gal, 0.5 mg/ml; Biosynth B-7200), generating a pink
precipitate (Fig.
1E,F,H,L).
In an alternate counterstaining method, we detected myocardium with a
cardiac myosin light chain 2 (cmlc2) riboprobe
(Yelon et al., 1999). In these
embryos, we performed whole-mount in situ hybridization before detecting
fluorescein. We used a standard protocol
(Yelon et al., 1999
), except
for the incorporation of 175 µg/ml 5-Bromo-6-Chloro-3-Indolyl Phosphate
(magenta-phos; Sigma B-5667) as the substrate for the alkaline
phosphatase reaction, generating a pink precipitate
(Fig. 1G,I-K;
Fig. 5L,M;
Fig. 6E,F).
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When possible, we assessed contributions to non-cardiac lineages, based on morphology and location of labeled cells (Fig. 4A,C,E,G). However, we were not able to score all lineages thoroughly: both pigmentation and MF20 staining in the somites obscured visualization of labeled cells, especially in the gut tube.
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Immunofluorescence
Whole-mount immunofluorescence was performed as described previously
(Yelon et al., 1999). MF20
(Bader et al., 1982
) and S46
(generous gift from F. Stockdale) monoclonal antibodies were detected with
goat anti-mouse IgG2b-TRITC and goat anti-mouse IgG1-FITC secondary antibodies
(Southern Biotechnology Associates), respectively.
RNA injection
We synthesized capped lefty1 mRNA
(Thisse and Thisse, 1999)
using the SP6 mMessage mMachine system (Ambion) and injected embryos with both
1 pg mRNA and caged fluorescein at the one- to two-cell stage.
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Results |
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A detailed description of our fate mapping strategy is provided in the Materials and methods. In brief, using laser-mediated activation of caged fluorescein, we labeled two or three neighboring blastomeres in each of 184 wild-type embryos. For each embryo, we recorded the latitude (Fig. 1A-C) and longitude (Fig. 1D) of the labeled cells. At 44 hours post fertilization (hpf), we assessed the contribution of labeled cells to the myocardium and endocardium of the ventricle and atrium (Fig. 1E-L). Thus, by correlating cardiac contributions with initial positions of labeled blastomeres, we generated a fate map of chamber progenitors at 40% epiboly (Tables 1, 2; Figs 2, 3).
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The resolution of our fate map reveals a previously unrecognized spatial organization of ventricular and atrial myocardial progenitors. Ventricular progenitors tend to be found closer to the margin than do atrial progenitors (Table 1, Fig. 2B). Specifically, we find ventricular progenitors primarily in tiers 1 (11/48 experiments in tier 1) and 2 (7/57 experiments), rarely in tier 3 (1/53 experiments), and never in tier 4 (0/26 experiments). By contrast, atrial progenitors never originate in tier 1 (0/48 experiments) but are found in tiers 2 (4/57 experiments), 3 (4/53 experiments) and 4 (3/26 experiments). Furthermore, within tiers 2 and 3, ventricular progenitors tend to be located closer to the dorsal midline than are atrial progenitors: ventricular progenitors are found 60-125° from dorsal, and atrial progenitors are found 90-140° from dorsal (Fig. 2B).
In addition to establishing the spatial relationships of ventricular and
atrial progenitors, our data provide an estimate of the density of myocardial
progenitors in the blastula. Labeling 2 neighboring blastomeres within the
LMZs produces myocardial progeny in 20% (23/115) of experiments,
suggesting a relatively sparse distribution of individual myocardial
progenitors (Fig. 2B). In these
experiments, the number of labeled myocardial cells ranges from two to six,
with an average of 4.3 labeled myocardial cells per embryo
(Table 1), suggesting that an
average myocardial progenitor produces four myocardial progeny by 44 hpf. In
this regard, it is interesting to note that experiments labeling three
blastomeres and experiments labeling five blastomeres also yield an average of
4.3 labeled myocardial cells, just as in experiments labeling 2 blastomeres
(Table 1). The consistency of
the average myocardial output, regardless of the initial number of labeled
blastomeres, lends further support to the notion that individual myocardial
progenitors are spread at a low density, rather than tightly clustered, within
the LMZs.
Intermingling of ventricular and atrial endocardial progenitors
As in previous work (Warga and
Nüsslein-Volhard, 1999), we find endocardial progenitors in
the same LMZs as myocardial progenitors
(Table 2,
Fig. 3). However, in contrast
to myocardial progenitors, ventricular and atrial endocardial progenitors are
found throughout the LMZs without apparent organization. Both ventricular and
atrial endocardial progenitors are found in all four tiers and over the entire
60-140° range (Table 2, Fig. 3). Furthermore, and also
in contrast to our data for myocardial progenitors, in many (17/37)
experiments, labeled endocardial cells are found in both cardiac chambers
(Table 2, Fig. 3). In most (34/37)
experiments, labeled endocardial cells are grouped together within the heart
tube (Fig. 1G,K,L). Embryos
with labeled cells in both ventricular and atrial endocardium are especially
interesting: in most (14/17) of these cases, a chain of labeled endocardial
cells extends across the atrioventricular boundary
(Fig. 1L).
As with myocardial progenitors, our data indicate the frequency of labeling
endocardial progenitors at 40% epiboly. When labeling two neighboring
blastomeres in the LMZs, 28% (32/115) of embryos contain labeled
endocardial cells (Fig. 3).
Owing to the difficulty of discerning endocardial cell boundaries, especially
in contiguous chains of cells, it is challenging to determine the number of
labeled endocardial cells reliably; in scoreable cases, the number of labeled
endocardial cells per embryo ranged from two to 20.
Multiple lineages arise from the lateral marginal zone
Supporting and extending previous fate maps, our data indicate that cardiac
chamber progenitors are intermingled with progenitors of other lineages in the
LMZs (Kimmel et al., 1990;
Warga and Nüsslein-Volhard,
1999
). These other lineages include endoderm, endothelium,
pectoral fin mesenchyme, blood, head muscle and pharyngeal tissue
(Fig. 4)
(Warga and Nüsslein-Volhard,
1999
). Although our protocol is not optimal for analysis of all
organs (see Materials and methods), we were often able to score contribution
of labeled cells to head vessels, circulating blood, pharyngeal pouches and
pectoral fin mesenchyme (Fig.
4). We find progenitors of head vessels, pharyngeal pouches and
fin mesenchyme in locations compatible with and in addition to those noted in
other studies (Fig. 4B,E,H)
(Warga and Nüsslein-Volhard,
1999
). We also find a previously unreported population of blood
progenitors in tiers 1 and 2, 45-100° from dorsal
(Fig. 4D). Some blastomeres in
this area give rise to circulating cells in cephalic regions; based on their
location and morphology, these progeny are probably myeloid cells, most likely
macrophages known to reside in a region of LPM anterior to the myocardial
precursors (Herbomel et al.,
1999
; Herbomel et al.,
2001
; Lieschke et al.,
2002
).
Location prior to gastrulation correlates with location in lateral plate mesoderm
Despite the intermingling of multiple mesendodermal lineages in the LMZs,
our fate map suggests that ventricular and atrial myocardial progenitors might
remain relatively organized during and following gastrulation. To explore this
model, we examined whether blastomeres from distinct regions of the LMZs
migrate to particular regions of the LPM. Specifically, we compared the
destinations of LMZ blastomeres from a dorsal portion of tier 1 with those
from a ventral region of tier 3 (Fig.
5A-H). At the end of gastrulation (tailbud stage, 10 hpf), we
found that blastomeres from 60-100° of tier 1 contribute to a rostral and
medial portion of the LPM (n=4;
Fig. 5A,B,E,F). By contrast,
blastomeres from 100-140° of tier 3 contribute to a relatively caudal and
lateral portion of the LPM (n=5;
Fig. 5C,D,G,H).
Extending this analysis further, we assessed positions of labeled cells as
the heart tube begins to form (20-22 somites, 19-20 hpf). At this stage, the
LPM has migrated towards the embryonic midline, allowing bilateral populations
of myocardial cells to meet and form a shallow cone
(Yelon et al., 1999).
Expression patterns of chamber-specific genes suggest that the central region
of the cone, expressing ventricular myosin heavy chain
(vmhc), will form the ventricle; and that the peripheral portion of
the cone, expressing atrial myosin heavy chain (amhc), will
form the atrium (Fig. 5I-K)
(Berdougo et al., 2003
;
Yelon et al., 1999
).
Correspondingly, myocardial progenitors from tier 1 contribute to the central
portion of the cone (n=5; Fig.
5L). Furthermore, myocardial progenitors from a ventral portion of
tier 3 contribute to the peripheral portion of the cone (n=2;
Fig. 5M).
Together, these results demonstrate that ventricular and atrial myocardial progenitors retain their relative organization from blastula stages through heart tube formation. In the blastula, ventricular and atrial progenitors occupy different regions of the LMZs, with ventricular progenitors located more marginally and dorsally than are atrial progenitors (Fig. 2B). As gastrulation proceeds, blastomeres from different regions of the LMZs form different regions of the LPM, with presumed ventricular progenitors located more rostrally and medially than presumed atrial progenitors (Fig. 5A-H). Finally, relative orientation within the LPM remains consistent as heart tube assembly proceeds, with medial cells contributing to the future ventricle and lateral cells contributing to the future atrium (Fig. 5I-M).
Nodal signaling promotes ventricular specification
Our fate map demonstrates that myocardial progenitors are spatially
organized prior to gastrulation; however, it does not indicate when chamber
fates are specified. Nevertheless, the relative positions of ventricular and
atrial progenitors suggest that differential reception of secreted signals
could influence chamber fate. For example, ventricular progenitors would have
preferential exposure to signals that are found at the margin or in a
vegetal-animal gradient. In the zebrafish blastula, Cyclops and Squint,
members of the Nodal family of TGFß-related ligands, are generated in
marginal blastomeres and the yolk syncytial layer
(Erter et al., 1998;
Feldman et al., 1998
;
Rebagliati et al., 1998a
;
Rebagliati et al., 1998b
;
Sampath et al., 1998
).
Furthermore, zebrafish Nodal signals have been shown to influence cell fate
relative to distance from the margin, particularly in the dorsal mesoderm
(Chen and Schier, 2001
;
Dougan et al., 2003
;
Gritsman et al., 2000
).
Therefore, we hypothesized that Nodal signaling could regulate chamber fate
specification.
Prior studies have established that complete inhibition of Nodal signaling
in the zebrafish embryo blocks specification of all mesendodermal lineages at
the blastula margin, resulting in the elimination of cardiac mesoderm
(Carmany-Rampey and Schier,
2001; Chen and Schier,
2001
; Feldman et al.,
1998
; Gritsman et al.,
1999
; Thisse and Thisse,
1999
). Partial reduction of Nodal signaling, as in zebrafish
mutants lacking zygotic supplies of the EGF-CFC co-receptor One-eyed pinhead
(Oep), causes a variable loss of myocardium, with the ventricle tending to be
more significantly affected than the atrium
(Reiter et al., 2001
). Nodal
signaling can also be inhibited by the extracellular antagonist Lefty1
(Chen and Schier, 2002
;
Cheng et al., 2004
;
Thisse and Thisse, 1999
).
Fittingly, we find that embryos ectopically expressing low levels of
lefty1 exhibit variable reductions of myocardium, with preferential
loss of ventricular myocardium (Fig.
6A-D); the range of cardiac phenotypes observed mimics that found
in zygotic oep mutants (Reiter et
al., 2001
). These phenotypes implicate Nodal signaling in
ventricular development, but they fail to establish its precise role. Nodal
signaling may promote ventricular fate specification; alternatively, Nodal
signaling could play a later role in promoting ventricular differentiation,
growth, or survival.
To clarify the role of Nodal signaling in ventricle formation, we examined alterations to the myocardial fate map in embryos injected with lefty1 mRNA. To detect a shift in the vegetal-animal distribution of fates, we focused our analysis on tier 1, where wild-type myocardial progenitors are exclusively ventricular (Fig. 2B). In these experiments, we typically labeled multiple adjacent blastomeres, in order to facilitate a rapid survey of resident progenitors. We detected myocardial progeny in 24% (7/29) of the lefty1-injected embryos in which we labeled tier 1 LMZ blastomeres (Fig. 6G). In a striking contrast to our wild-type fate map, we find both atrial and ventricular progenitors in tier 1 of lefty1-injected embryos (Fig. 6E-G), indicating a fate transformation of tier 1 blastomeres when Nodal signaling is antagonized. Therefore, Nodal signaling contributes to the vegetal-animal patterning of myocardial progenitors, promoting the assignment of ventricular fate in blastomeres near the embryonic margin.
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Discussion |
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Strikingly, our data show that myocardial and endocardial progenitor populations are organized considerably differently. Myocardial chamber progenitors are spatially organized at 40% epiboly; by contrast, endocardial chamber progenitors are distributed without apparent organization. In general, ventricular myocardial progenitors are located more marginally and dorsally than are atrial myocardial progenitors, although there is a zone of overlap in which both ventricular and atrial myocardial progenitors can be found (Fig. 7A). Nevertheless, none of our experiments in wild-type embryos yielded myocardial progeny in both the ventricle and atrium. Thus, ventricular and atrial myocardial lineages are clearly separated by 40% epiboly. This lineage separation could be the consequence of early patterning events that initiate chamber specification. Alternatively, lineage separation could be facilitated by orderly migration of LMZ blastomeres, preserving the relative orientation of ventricular and atrial progenitors until chamber specification occurs. Our data suggest that both scenarios orderly migration and early patterning apply to the myocardial progenitors.
|
When gastrulation begins, the blastomeres closest to the margin involute
first, followed by the higher tiers (Fig.
7B,C) (Warga and Kimmel,
1990). Thus, in the hypoblast, ventricular progenitors would tend
to be slightly more rostral than are atrial progenitors. Additionally, by
virtue of their initial dorsal positions, ventricular progenitors would be
closer to the embryonic midline than are atrial progenitors. Subsequently,
through convergence and extension, the wide zones containing myocardial
progenitors become more narrow and extend rostrally
(Fig. 7D-F)
(Myers et al., 2002
;
Sepich et al., 2000
). After
gastrulation, myocardial chamber progenitors occupy discrete regions of the
LPM (Fig. 7F,G). During
somitogenesis stages, these bilateral populations migrate medially and
eventually fuse to form the ventricular and atrial regions of the heart tube
(Fig. 7G-I)
(Yelon et al., 1999
). Thus,
our data are consistent with a generally orderly process of myocardial
morphogenesis. In this regard, our findings are comparable to fate maps of
other zebrafish germ layers: in particular, zebrafish neural and endodermal
fate maps suggest early organization and orderly migration of tissue
progenitors during gastrulation (Kozlowski
et al., 1997
; Warga and
Nüsslein-Volhard, 1999
;
Woo et al., 1995
).
It is interesting to compare the organization and morphogenesis of
myocardial progenitors in zebrafish and chick. The initial organization of
zebrafish myocardial progenitors places the ventricular progenitors closer to
both the margin and the dorsal organizer; similarly, avian fate maps indicate
a tendency of ventricular progenitors to involute earlier and closer to the
node than atrial progenitors
(Garcia-Martinez and Schoenwolf,
1993; Rosenquist,
1970
). However, although their initial organization seems
comparable, the orderly movements of zebrafish myocardial progenitors may not
resemble the migration patterns of chamber progenitors in chick. Although some
early cell labeling studies in chick have suggested relatively coherent
movements of groups of progenitors
(Rosenquist, 1966
;
Stalsberg and DeHaan, 1969
),
other analyses suggest that chamber progenitors can move and intermix
relatively freely until cardiac crescent formation is complete
(DeHaan, 1963
;
Redkar et al., 2001
).
In contrast to the orderly morphogenesis of myocardial progenitors,
zebrafish endocardial progenitors are likely to intermix substantially during
their migration into the heart tube. During somitogenesis stages, endocardial
cells are thought to aggregate near the embryonic midline, at the future
center of the myocardial cone (Stainier et
al., 1993). Once the cone forms, the endocardial cells are then in
position to generate the lining of the heart tube. This model, which is
consistent with our data, suggests that endocardial chamber progenitors do not
become spatially organized until they leave the midline to occupy a cardiac
chamber; furthermore, it is possible that endocardial chamber lineages are not
specified until chambers form. The high frequency of labeling both ventricular
and atrial endocardial cells in our fate map suggests that these lineages are
not separate at 40% epiboly (Fig.
3). Additionally, the observed chains of endocardial cells
extending into both chambers (Fig.
1L) suggest clonal relationships of labeled cells in these
experiments.
Nodal signaling patterns myocardial chamber progenitors
The vegetal-animal organization of myocardial chamber progenitors at 40%
epiboly suggests that vegetal-animal signaling gradients could influence
chamber specification. We demonstrate that ectopic expression of
lefty1 influences myocardial fate assignments in tier 1 blastomeres.
Given that Lefty antagonizes the function of EGF-CFC-co-receptor-dependent
TGFß molecules (Chen and Schier,
2002; Cheng et al.,
2004
), it is likely that our data reflect endogenous roles of the
Nodal ligands Squint and/or Cyclops in the zebrafish blastula
(Erter et al., 1998
;
Feldman et al., 1998
;
Rebagliati et al., 1998a
;
Rebagliati et al., 1998b
;
Sampath et al., 1998
).
Previous studies have shown that elimination of Nodal signaling alters the
fate map of the blastula margin, such that marginal fates, including
myocardium, are completely absent; and ectodermal fates, which are normally
found in higher tiers, are found in marginal locations
(Carmany-Rampey and Schier,
2001
; Dougan et al.,
2003
; Feldman et al.,
2000
). Our data indicate that reduction, rather than elimination,
of Nodal signaling creates a finer vegetal-animal adjustment in fate
assignments within the tiers of the LMZs, causing the distribution of
ventricular and atrial myocardial progenitors in tier 1 to resemble the
dorsoventral organization observed in tier 2 or 3 of the wild-type fate map.
These data are reminiscent of the prior observation that reduced Nodal
signaling causes fate transformations at the dorsal margin, such that
notochord progenitors, normally located in tier 3 or higher, are found in
tiers 1 and 2 (Gritsman et al.,
2000
). Although our results do not indicate how directly Nodal
signaling influences myocardial patterning, our data are congruent with a
general model in which reduction of Nodal signaling influences the entire
blastula margin, shifting relatively animal fate assignments to more vegetal
positions (Carmany-Rampey and Schier,
2001
; Dougan et al.,
2003
).
Our data clearly demonstrate the impact of Nodal signaling on chamber
specification; however, the Nodal pathway is unlikely to be the sole regulator
of this process. In particular, our fate map suggests that chamber fate
assignment could be accomplished through an intersection of vegetal-animal and
dorsal-ventral patterning. In the zebrafish blastula, Bmp, Wnt and Fgf signals
all regulate aspects of dorsal-ventral patterning
(Schier, 2001). Future studies
examining the influences of these signaling pathways on the myocardial fate
map are therefore likely to provide additional insight regarding the
integrated network of signals that pattern the myocardial progenitors.
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ACKNOWLEDGMENTS |
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Footnotes |
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