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First published online April 24, 2009
doi: 10.1242/10.1242/dev.030924
1 Hubrecht Institute and University Medical Centre Utrecht, 3584 CT, Utrecht,
The Netherlands.
2 Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular
Medicine, New York University School of Medicine, New York, NY 10016,
USA.
3 Graduate Program in Areas of Basic and Applied Biology, Universidade do Porto,
4050-465 Porto, Portugal.
4 Interuniversity Cardiology Institute of the Netherlands, 3511 GC, Utrecht, The
Netherlands.
* Authors for correspondence (e-mails: yelon{at}saturn.med.nyu.edu; j.bakkers{at}niob.knaw.nl)
Accepted 4 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Fgf, Differentiation, Heart, Islet1, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Growth of the heart tube could be mediated either through cell
proliferation or by recruitment of new cardiomyocytes into the organ. Although
cardiac cells do proliferate, it is rare in the linear heart tube. In the
chick heart, myocardial proliferation only starts at stage 12, when the heart
tube has already looped and chambers start to emerge
(Soufan et al., 2006
). The
early work of de la Cruz in the chick embryo demonstrated that the heart
lengthens by the addition of cells to the arterial (outflow) pole of the heart
(de la Cruz et al., 1977).
Later studies in mouse and chick confirmed that, in these organisms, the
embryonic primitive heart tube grows and becomes structurally elaborate owing
to the significant addition of cells to the right ventricle and the outflow
tract (OFT) from the pharyngeal mesoderm, which is also referred to as the
secondary or anterior heart field (Kelly
et al., 2001; Mjaatvedt et
al., 2001; Waldo et al.,
2001). Later studies revealed an extension of this population of
progenitor cells, more posteriorly, that also contribute to the venous pole of
the heart (atria) (Cai et al.,
2003). The progenitor cells located within the pharyngeal mesoderm
that contribute cardiomyocytes to both the arterial pole and the venous pole
are referred to as the second heart field (SHF), as opposed to the first heart
field, which gives rise to the cells of the early cardiac tube
(Buckingham et al., 2005). A
lack of markers that are specific for the primary heart field has made it
difficult to determine its location. The recent observation that Islet1
protein, previously used as a marker for the SHF, is present in all
cardiomyocytes has initiated a discussion about significance of the different
heart fields (Prall et al.,
2007). This discussion has led to the alternative suggestion that
the heart tube grows by a continuous differentiation process
(Moorman et al., 2007).
However, the inability to visualize cardiomyocyte differentiation has made it
difficult to resolve these issues.
Here, we investigate the dynamics of cardiomyocyte differentiation during cardiac development, and by taking advantage of opportunities for high-resolution and live imaging of the zebrafish heart, we visualize this process for the first time. By using a developmental timing assay and a photoconvertible marker, we identify two phases of cardiomyocyte differentiation. These previously unrecognized phases of cardiomyocyte differentiation are separated in time, space and the molecular mechanisms by which they are controlled.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
Tg(–5.1myl7:nDsRed2)f2
(Mably et al., 2003) lines.
Founder fish with germline integration of Tg(cmlc2:kaede) were
generated by Tol2 transposase-mediated transgenesis
(Fisher et al., 2006) and were
outcrossed to produce embryos for photoconversion. The islet1 mutant
K88X (isl1SA0029) was isolated from an ENU-mutagenized
library, using target-selected mutagenesis, as described before
(Wienholds et al., 2002).
Cell counts and developmental timing assay
To count cardiomyocytes at various stages, we used the
Tg(cmlc2:dsred2-nuc) line stained using an 
-DsRed antibody
(Clontech). The embryos were grown under normal culture conditions
(Westerfield, 1995) up to the
desired stage and subsequently fixed (overnight at 4°C) in 2%
paraformaldehyde containing glycerol and washed with PBS containing 0.1% Tween
(PBST) the following day. The embryos were counterstained with DAPI [15
minutes at room temperature, 1:5000 DAPI (Boehringer Mannheim) in PBST]. The
embryos were flat-mounted and imaged ventrally in Vectashield containing DAPI
(Vector Laboratories).
Mounted embryos were imaged using a Leica TCS SPE confocal microscope with a 20x oil immersion lens. The images were zoomed in to 1.96x with the LAS-AF TCS SPE software and sequential confocal images were taken using the laser channels 405, 488 and 532 nm with a standardized step size of 0.642 µm in the z-direction. The pinhole was set to 1 airy unit and the scanning speed was 600 Hz.
3D reconstructions of confocal stacks were made using Volocity version 4.1 software (Improvision). Quantification of the GFPposDsRedpos and GFPposDsRedneg myocardial cells was also performed using Volocity 4.1 software. For each individual cell positive in the GFP channel, it was carefully determined whether it was DsRedpos or DsRedneg by opening and closing the different channels. All embryos were counted at least two times.
Photoconversion of Kaede
Photoconversion of Kaede fluorescence from green to red was achieved by
exposing transgenic embryos to UV light on a Zeiss Axioplan microscope
equipped with a DAPI filter set, as previously described
(Hatta et al., 2006). Confocal
z-stacks were obtained using a Zeiss LSM 510 laser-scanning
microscope and analyzed with Zeiss LSM and Volocity software.
Histological methods
BrdU labeling was performed by soaking the embryos in embryo medium
containing 5 mg/ml BrdU (Roche) for 24 hours. -BrdU (Roche) and
-phospho-His (Upstate) antibody labeling was performed on 6- and
10-µm thick paraffin sections, respectively, which were then stained with
3,3'-Diaminobenzidine (DAB). Other antibodies used in this study were:
-DsRed (Clontech), -eGFP (Chemokine) and -Isl (Hybridoma
bank clone 39.4D5). Immunofluorescence was performed according to Smith et al.
(Smith et al., 2008).
SU5402 treatment
Embryos were treated with either DMSO or SU5402 at a concentration of 12.5
µM in embryo medium from 24 hpf until 48 hpf in glass vials in the dark at
normal culture temperatures.
Morpholinos
Antisense morpholinos targeting fgf8
(Draper et al., 2001) or
isl1 (Hutchinson and Eisen,
2006) were injected at the one-cell stage. Uninjected and control
MO (Gene Tools) injected embryos from the same egg lay were used as controls
for all experiments.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
identify all cardiomyocytes expressing DsRed, Tg(cmlc2:dsred2-nuc)
embryos were fixed for immunofluorescence staining using an -DsRed
antibody (Fig. 1A-F). At 24
hours post-fertilization (hpf), the heart tube has formed from the cardiac
disk and was found to contain 151±12 (mean±s.e.m., n=5)
cardiomyocytes. Over the next 24 hours, a significant increase in the number
of cardiomyocytes was observed (to 311±10, n=5, at 48 hpf;
Fig. 1E; see also Table S1 in
the supplementary material).
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To quantify the total number of cardiomyocytes that had undergone at least
one round of DNA replication during heart looping, embryos were soaked in a
solution containing BrdU from 24 hpf until 48 hpf. When sectioned and stained
by an -BrdU antibody, only 16±2 (n=6) BrdU-positive
cardiomyocytes per embryo were observed
(Fig. 1I,J; see also Figs S1,
S2 in the supplementary material). Surprisingly, 54% of the BrdU-positive
cells found in the myocardium were located near the two cardiac poles (within
four tiers of cells at the end the myocardial tube), adjacent to the highly
proliferating mesenchyme (Fig.
1J). From the BrdU incorporation analysis and the phospho-His
staining, we conclude that the low rate of proliferation within the myocardium
cannot account for the substantial increase in cardiomyocyte number that we
observed in the heart between 24 and 48 hpf.
Distinct phases of cardiomyocyte differentiation at the venous and arterial poles
Our finding that a large fraction of BrdU-positive cells was located near
the poles suggested either a local zone of proliferation or the addition and
differentiation of cells that originate from the adjacent proliferating
mesenchyme to the poles of the heart tube. To investigate cardiomyocyte
differentiation, we used a developmental timing assay by examining double
transgenic animals expressing both eGFP and DsRed in differentiating
cardiomyocytes from the cardiac myosin light chain 2 (cmlc2)
promoter [Tg(cmlc2:eGFP)/Tg(cmlc2:dsred2-nuc)]. This approach takes
advantage of the observation that the DsRed protein requires more time
(approximately 24 hours) to mature and fluoresce than does eGFP, which matures
and fluoresces very rapidly (Lepilina et
al., 2006; Verkhusha et al.,
2001). If cardiomyocyte differentiation occurs at different time
points, one would expect to find two populations of cells within the heart
myocardium: eGFPposDsRedpos cells, which have initiated
differentiation early, and eGFPposDsRedneg cells, which
have initiated differentiation at a later stage. If cardiomyocyte
differentiation occurs at the same time in all cells, one would expect to find
a single population of eGFPposDsRedpos cells.
We could confirm the long maturation time for DsRed protein in the double transgenic embryos [Tg(cmlc2:eGFP)/Tg(cmlc2:dsred2-nuc)]. At 24 hpf, the heart tube has formed, and strong eGFP fluorescence was detected in the cardiomyocytes (Fig. 2A). The DsRed fluorescence was still not detectable (Fig. 2B,C), even though antibody staining demonstrated that the DsRed protein was abundantly present at that time (see Fig. S3 in the supplementary material). The DsRed fluorescence was detectable by confocal microscopy only from 36 hpf onwards. Intriguingly, we found two pools of cardiomyocytes, eGFPposDsRedpos and eGFPposDsRedneg cells (Fig. 2D-F), suggesting that cardiomyocyte differentiation had indeed occurred in different phases. The eGFPposDsRedpos cells, which had initiated differentiation early, were located in the ventricle and in a specific region of the atrium (the inner curvature). The eGFPposDsRedneg cells, which had initiated differentiation at a later time point, were consistently found in the atrium (mainly in the outer curvature) and at the arterial pole (Fig. 2D-F). This is consistent with our observation that the number of cmlc2-expressing cells in the lateral plate mesoderm increases gradually over time, with expression in ventricular precursors preceding expression in atrial precursors (see Fig. S4 in the supplementary material). At 48-55 hpf we still observed eGFPposDsRedneg cells located at both the venous and the arterial poles (Fig. 2G-I). In a single z-scan, at the level of the ventricle/outflow region, the different intensities of the eGFP versus the DsRed signal could be appreciated, and is suggestive of the addition of newly differentiating cardiomyocytes at the arterial pole (Fig. 2J-L).
To examine the timing of cardiomyocyte addition in more detail, we employed
a transgene [Tg(cmlc2:kaede)] in which expression of the red-to-green
photoconvertible fluorescent protein Kaede
(Ando et al., 2002) is driven
by the cmlc2 promoter. In Tg(cmlc2:kaede) embryos,
photoconversion of Kaede can mark the differentiated cardiomyocytes present at
a specific timepoint: the green form of Kaede in all cmlc2-expressing
cells converts into the red form, labeling these cells with red fluorescence
even as they continue to produce additional green Kaede (see Fig. S5 in the
supplementary material). By contrast, newly differentiating cells that begin
expressing cmlc2 after the time of photoconversion will fluoresce
green, but not red (Fig. S5 in the supplementary material).
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Islet1 mutants have reduced cardiomyocyte differentiation at the venous pole
Next we wanted to identify the signals that regulate these two waves of
cardiomyocyte differentiation. Islet 1 (Isl1) deficient mouse embryos
lack part of the atria and most of the outflow tract and right ventricle,
suggesting that Isl1-expressing cells contribute to both the venous
and the arterial poles of the mouse heart
(Cai et al., 2003). More recent
studies demonstrated that Isl1 is expressed throughout the heart
(Prall et al., 2007), and is
also required for heart morphogenesis and early cardiomyocyte specification in
Drosophila and Xenopus, respectively
(Brade et al., 2007;
Tao et al., 2007). To identify
a possible isl1-positive cardiac progenitor cell population in the
zebrafish, we analyzed expression at various stages. Using an antibody
recognizing both Isl1 and Isl2 proteins
(Hutchinson and Eisen, 2006;
Wan et al., 2006), we observed
a strong nuclear signal in the trigeminal sensory ganglia, the vascular
endothelium, and the endoderm, which lays dorsal to the cardiac field
(Trinh Le and Stainier, 2004)
(Fig. 3B,C). We also observed a
nuclear signal in cells located at the periphery of the cardiac field where
future atrial cells are located (Fig.
3A-F; see Fig. S6 in the supplementary material). Frequently the
Isl-positive cells also expressed Tg(cmlc2:eGFP), indicating that
cardiomyocyte differentiation had been initiated in the Isl-positive
cells.
To address whether zebrafish Isl1 is required for normal heart development,
we screened for mutant alleles in an ENU-mutagenized library
(Wienholds et al., 2002). We
identified one allele that results in a premature stop codon in the third
exon, removing part of the LIM-domain and the entire homeobox
(Fig. 4A,B). At 24 hpf, the
isl1 mutant embryos look morphologically normal but are immotile,
which can be explained by the function of Isl1 described in primary
motoneurons (Hutchinson and Eisen,
2006) (see Fig. S7A-D in the supplementary material; data not
shown). In addition to the motility defect, the heart of isl1 mutant
embryos contracts irregularly and with a reduced frequency (bradycardia;
Fig. 4C). Whereas the heart
frequency in wild-type siblings will rise from 80±1 beats/minute at 24
hpf to 163±4 beats/minute at 48 hpf, the heart frequency of
isl1 mutants remains low (95±3 beats/minute at 48 hpf,
58±3 beats/minute at 72 hpf; see Table S2 in the supplementary
material). In addition, isl1 mutant hearts showed frequent pauses in
the cardiac contraction, resulting in reduced blood circulation in the trunk
and tail (see Movie 1 in the supplementary material). Subsequently,
isl1 mutant larvae die between 5 and 7 dpf.
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To address whether the observed cardiovascular defects in isl1
mutant embryos could result from a defect in cardiomyocyte differentiation, we
used the above-described developmental timing assay. To do so, we crossed the
isl1 mutation into Tg(cmlc2:eGFP)/Tg(cmlc2:dsred2-nuc)
double transgenic embryos and quantified the number of
eGFPposDsRedpos cardiomyocytes and
eGFPposDsRedneg cardiomyocytes in confocal images after
3D reconstruction (Fig. 5A-F).
We observed no significant difference in the number of
eGFPposDsRedpos cells (siblings, 200±6; mutants,
203±7) nor in the number of eGFPposDsRedneg cells
at the arterial pole (sibs, 28±1; mutants, 32±2;
Fig. 5G-K; see also Table S3 in
the supplementary material), demonstrating that cardiomyocyte differentiation
still occurs. We did, however, observe a significant reduction in the number
of eGFPposDsRedneg cells at the venous pole of
isl1 mutants (25±3 in sibling hearts and 10±3 in mutant
hearts, P<0.01; Fig.
5D-F,K). To confirm that the observed defects in cardiomyocyte
differentiation resulted from the loss of Isl1 function, we used antisense
morpholinos (MO) against isl1, which affect splicing of the
isl1 mRNA and thereby prevent Isl1 protein production
(Hutchinson and Eisen, 2006)
(see Fig. S8 in the supplementary material). Using the developmental timing
assay, we again observed a significant reduction in the number of
eGFPposDsRedneg cells at the venous pole, while the
number of eGFPposDsRedneg cells at the arterial pole
remained unaffected. The stronger effects on cardiac morphology and the number
of eGFPposDsRedpos cells observed after isl1 MO
injection were probably due to an additional toxic effect of the MO
injection.
Because isl1 mutant embryos display cardiac dysfunction, we
addressed whether cardiac dysfunction by itself can affect cardiomyocyte
addition. To interfere with cardiac function, we injected tnnt2 MOs
(Sehnert et al., 2002).
Although cardiac contraction was abolished in tnnt2 Mo-injected
embryos, the addition of new cardiomyocytes to both poles of the heart tube
was not significantly altered (see Fig. S9 in the supplementary material).
In conclusion, the loss of bmp4 expression in the SV combined with the reduced number of eGFPposDsRedneg cardiomyocytes at the venous pole demonstrate that, in zebrafish, Isl1 is specifically required to complete cardiomyocyte differentiation at the venous pole and not at the arterial pole. This suggests that other, Isl1-independent pathways regulate the addition of cardiomyocytes to the arterial pole (see Discussion).
Fgf signaling is required for cardiomyocyte addition at the arterial pole
Two previous studies using conditional and hypomophic Fgf8 alleles
in mouse have demonstrated that Fgf signaling is crucial for the recruitment
of SHF cells to the arterial pole of the heart
(Ilagan et al., 2006;
Park et al., 2006). Consistent
with the suggested role for fgf8 in arterial pole formation, the
zebrafish fgf8 mutation acerebellar (ace) is known
to diminish the size of the ventricle
(Reifers et al., 2000).
Furthermore, inhibition of Fgf signaling between 24 and 48 hpf reduces the
number of ventricular cardiomyocytes in the zebrafish heart, but the cellular
mechanism responsible for this deficiency has not yet been elucidated
(Marques et al., 2008). To
address whether Fgf signaling is activated at the arterial pole, we analyzed
sprouty4 (spry4) expression at stages between 24 and 48 hpf,
because Sprouty proteins are Fgf antagonists that are induced by Fgf signaling
(Furthauer et al., 2001;
Hacohen et al., 1998). At 24
hpf, we observed very strong spry4 expression at the
midbrain-hindbrain boundary and much weaker expression near the arterial pole
of the linear heart tube (Fig.
6A,B). At 36 hpf, spry4 expression was observed in the
heart tube and at the position where the arterial pole connects to the head
mesoderm (Fig. 6C). Speculating
that Fgf8 might regulate the addition of cells to the arterial pole of the
zebrafish heart tube, we injected the eGFP/DsRed double transgenic embryos
with an antisense MO targeting fgf8 and reproducing the
ace/fgf8 mutant phenotypes (see Fig. S10 in the supplementary
material) (Draper et al.,
2001). In agreement with the reported role of Fgf signaling in
cardiomyocyte specification, the hearts of fgf8 MO-injected embryos
were much smaller than those of wild type, and we found that the total number
of myocardial cells was significantly decreased (control, 305±23,
n=3; fgf8 MO, 196±6, n=4;
P<0.05; Fig.
6E-G,N). In addition, a significant reduction in the number of
eGFPposDsRedneg cells at the arterial pole was observed
(control, 29±1, n=3; fgf8 MO, 17±4,
n=4; P<0.05), while the number of
eGFPposDsRedneg cells at the venous pole did not change
significantly (control, 32±4, n=3; fgf8 MO,
26±2, n=4; Fig.
6N,O; see also Table S3 in the supplementary material).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Cardiomyocyte differentiation at the venous pole
By using two different assays, developmental timing and Kaede
photoconversion, we demonstrated that cardiomyocyte differentiation is
initiated in the ventricle. Subsequently, atrial cells are added at the venous
pole by a continuation of cardiomyocyte differentiation. These results are in
agreement with the previous findings in both chick and zebrafish that the
induction of atrium-specific myosin gene expression occurs after the induction
of ventricle-specific myosin expression
(Berdougo et al., 2003;
Yutzey et al., 1994). Our
Kaede photoconversion experiments demonstrate that the differentiation at the
venous pole is completed by 34 hpf at the latest. Finally, our results
demonstrate that Isl1 is required to complete the cardiomyocyte
differentiation process at the venous pole. Interestingly, we observed
bradycardia and frequent pauses in cardiac contraction in the zebrafish
isl1 mutant embryos. This was not reported for the mouse
Isl1 mutants, which could have been because of the severe heart
failure observed in these embryos (Cai et
al., 2003). Earlier experiments in chick demonstrated that
intrinsic heart beat frequency increases over the anteroposterior axis and
that a single pacemaker area becomes established at the venous pole
(Moorman et al., 1998).
Problems in pacemaker activity result in a so-called sick sinus syndrome,
which is characterized by arrhythmias, such as sinus bradycardia and sinus
pauses, or arrests (Dobrzynski et al.,
2007). Therefore, the reduced heart frequency and the pauses in
contraction that we observed in isl1 mutant embryos could be
explained by a failure of cardiomyocyte differentiation at the venous pole.
Indeed, we observed a specific loss of bmp4 expression and a
significant reduction in the number of eGFPposDsRedneg
cells at the venous pole, which demonstrated reduced cardiomyocyte
differentiation at the venous pole. One explanation for the residual
cardiomyocyte differentiation observed in isl1 mutant embryos could
be that Isl1 is redundant with other Islet factors during cardiomyocyte
differentiation. Because the -Isl antibody we used can recognize both
Isl1 and Isl2 proteins, and because almost all signal is lost in the
isl1 mutant (see Fig. S7 in the supplementary material), we do not
believe this to be a likely explanation. An alternative explanation would be
that Isl1 is required for the differentiation of a specific subset of
cardiomyocytes located at the venous pole. As Isl1 is expressed in various
cell types near and in the differentiating cardiomyocytes
(Fig. 3), the autonomy of Isl1
during cardiomyocyte addition at the venous pole remains an open and
interesting question.
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), we did
not observe any effect on the expression of early cardiac markers (e.g.
nkx2.5) in isl1 mutant or isl1 MO-injected embryos
(E.d.P. and J.B., unpublished observations).
From our observations in zebrafish, Mouse Isl1 seems to have a much broader
function than does zebrafish Isl1. Mouse Isl1 is required for the recruitment
of cells to both the venous pole (both atria) and the arterial pole (right
ventricle and OFT) (Cai et al.,
2003). Possible explanations for these differences could be that
at the arterial pole zebrafish Isl1 is redundant with other Islet factors,
which, from the arguments described above, we do not believe to be likely. An
alternative and more likely explanation would be that these differences
reflect evolutionary differences between teleosts and amniotes, differences
that are responsible for the recruitment of the extra cardiomyocytes required
to form additional chambers (see also below).
Cardiomyocyte differentiation at the arterial pole
The Kaede photoconversion experiments demonstrate that there is
discontinuity between the initial phase of differentiation, which gives rise
to cardiomyocytes that form the ventricle and atrium, and the later addition
of cells to the arterial pole. At 19-26 hpf, when cardiomyocytes are still
added to the venous pole, addition of new cardiomyocytes to the arterial pole
was hardly observed. Addition of new cardiomyocytes at the arterial pole was
apparent only at later stages of cardiac development (34-48 hpf).
Interestingly, BrdU labeling in chick has revealed a population of labeled
cardiomyocytes at the venous pole at stage 10, when no such cells were evident
at the arterial pole (Soufan et al.,
2006). It was suggested that these venous pole cells had
incorporated BrdU in the splanchnic mesoderm before being added to the venous
pole. Together with the observation that cells are not added to the arterial
pole until after stage 12 (de la Cruz et
al., 1977; Mjaatvedt et al.,
2001), this would suggest that cardiomyocyte differentiation at
the venous pole is occurring much earlier than differentiation at the arterial
pole in chick embryos. However, the visualization of cardiomyocyte
differentiation in mouse or chick embryos would be required to allow a direct
comparison of the dynamics.
Our data demonstrate that cardiomyocyte differentiation at the arterial
pole is independent of Isl1 but requires Fgf8 signaling. The small ventricle
previously observed in ace/fgf8 mutant fish and the small ventricle
generated by blocking Fgf signaling at the linear heart tube stage
(Marques et al., 2008;
Reifers et al., 2000) can now
be explained by diminished cardiomyocyte differentiation at the arterial pole.
The differences that we observe after fgf8-MO injection or SU5402
treatment can be explained by a difference in timing when Fgf signaling is
blocked by these different treatments
(Marques et al., 2008). In the
case in which Fgf8 signaling is inhibited by the MO injection, Fgf8 signaling
is inhibited already at very early stages during cardiomyocyte specification,
which provides an explanation for the reduction in the number of
eGFPposDsRedpos cells we observed. In the case of SU5402
treatment, we added the inhibitor only at the tube stage (24 hpf), and
therefore this treatment affects only later cardiomyocyte addition. Whether
these two phases of Fgf requirement during cardiomyocyte differentiation also
exist in other vertebrates has been difficult to address owing to the early
lethality of mouse mutants with reduced Fgf signaling during gastrulation
(Dono et al., 1998;
Feldman et al., 1995;
Meyers et al., 1998). However,
by using different conditional Fgf8 mutant alleles, Park et al.
showed that the early loss of Fgf8 affects both ventricle and atrium
formation, whereas the late and specific deletion of Fgf8 in the
anterior heart field specifically affects the OFT
(Park et al., 2006).
Surprisingly, cardiomyocyte differentiation in zebrafish at the arterial pole does not require Isl1 (also discussed above). This demonstrates that not only is the temporal regulation of cardiomyocyte differentiation at both poles different, but also the mechanism and signals involved in regulating cardiomyocyte differentiation are different at each pole of the zebrafish heart.
Evolutionary adaptation
Our observation that cardiomyocyte differentiation at the arterial pole is
temporally separated from and regulated differently to the continuous wave of
differentiation in the rest of the heart tube may also help to explain the
different phenotypes (broad heart defects versus OFT defects) observed in
several mouse mutants. Mice deficient for Isl1 display broad cardiac
defects that affect the formation of ventricles as well as atria
(Cai et al., 2003). We now
suggest that the role for Isl1, which is required to complete the continuous
differentiation process in zebrafish, has been expanded in amniotes to
increase the relative size of the heart. Interestingly, during growth of the
heart tube in amniotes, both the venous and the arterial pole remain connected
to the dorsal mesocardium, where Isl1 is expressed. In zebrafish, however, the
Isl1-positive cells located laterally in the cardiac field are physically
separated from the future ventricle cells that are located medially in the
cardiac field.
Taken together, our results demonstrate that two distinct phases of cardiomyocyte differentiation contribute to the observed growth of the embryonic heart. This is the first time that the temporal regulation of cardiomyocyte differentiation has been visualized in any vertebrate organism. We believe that this new concept of two separate phases of cardiomyocyte differentiation that require different molecular mechanisms for their completion provides new insight into how the vertebrate heart grows and could have expanded during vertebrate evolution.
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/10/1633/DC1
We thank Dr Cuppen (Hubrecht Laboratory) and Dr Stemple (Welcome Trust Sanger Institute) for providing the isl1k88x zebrafish mutant, which was generated as part of the ZF-MODELS Integrated Project in the 6th Framework Programme (Contract No. LSHG-CT-2003-503496) funded by the European Commission. We also thank R. Kelly for discussions and suggestions when this work was in progress, K. Poss for providing the Tg(cmlc2:dsred2-nuc) fish, A. Moorman and K. Smith for critical reading of the manuscript and members of the Bakkers laboratory for stimulating discussions. Work in J.B.'s laboratory was supported by the Royal Dutch Academy of Arts and Sciences. Work in D.Y.'s laboratory was supported by the National Institutes of Health. E.d.P. was supported by EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart. S.M. was supported by the GABBA program and the Portuguese Foundation for Science and Technology (POCI 2010-FSE). Deposited in PMC for release after 12 months.
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. and Miyawaki,
A. (2002). An optical marker based on the UV-induced
green-to-red photoconversion of a fluorescent protein. Proc. Natl.
Acad. Sci. USA 99,12651
-12656.
Berdougo, E., Coleman, H., Lee, D. H., Stainier, D. Y. and
Yelon, D. (2003). Mutation of weak atrium/atrial myosin heavy
chain disrupts atrial function and influences ventricular morphogenesis in
zebrafish. Development
130,6121
-6129.
Brade, T., Gessert, S., Kuhl, M. and Pandur, P.
(2007). The amphibian second heart field: Xenopus islet-1 is
required for cardiovascular development. Dev. Biol.
311,297
-310.[CrossRef][Medline]
Buckingham, M. E., Meilhac, S. and Zaffran, S.
(2005). Building the mammalian heart from two sources of
myocardial cells. Nat. Rev. Genet.
6, 826-835.[CrossRef][Medline]
Cai, C. L., Liang, X., Shi, Y., Chu, P. H., Pfaff, S. L., Chen,
J. and Evans, S. (2003). Isl1 identifies a cardiac progenitor
population that proliferates prior to differentiation and contributes a
majority of cells to the heart. Dev. Cell
5, 877-889.[CrossRef][Medline]
de la Cruz, M. V., Sanchez, G. C., Arteaga, M. M. and Arguello,
C. (1977). Experimental study of the development of the
truncus and the conus in the chick embryo. J. Anat.
123,661
-686.[Medline]
Dobrzynski, H., Boyett, M. R. and Anderson, R. H.
(2007). New insights into pacemaker activity: promoting
understanding of sick sinus syndrome. Circulation
115,1921
-1932.
Dono, R., Texido, G., Dussel, R., Ehmke, H. and Zeller, R.
(1998). Impaired cerebral cortex development and blood pressure
regulation in Fgf2 deficient mice. EMBO J.
17,4213
-4225.[CrossRef][Medline]
Draper, B. W., Morcos, P. A. and Kimmel, C. B.
(2001). Inhibition of zebrafish fgf8 pre-mRNA splicing with
morpholino oligos: a quantifiable method for gene knockdown.
Genesis 30,154
-156.[CrossRef][Medline]
Feldman, B., Poueymirou, W., Papaioannou, V. E., DeChiara, T. M.
and Goldfarb, M. (1995). Requirement for Fgf-4 for
postimplantation mouse development. Science
267,246
-249.
Fisher, S., Grice, E. A., Vinton, R. M., Bessling, S. L.,
Urasaki, A., Kawakami, K. and McCalion, A. S. (2006).
Evaluating the biological relevance of putative enhancers using Tol2
transposon-mediated transgenesis in zebrafish. Nat.
Protoc. 1,1297
-1305.[CrossRef][Medline]
Furthauer, M., Reifers, F., Brand, M., Thisse, B. and Thisse,
C. (2001). sprouty4 acts in vivo as a feedback-induced
antagonist of FGF signaling in zebrafish. Development
128,2175
-2186.
Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. and Krasnow,
M. A. (1998). sprouty encodes a novel antagonist of FGF
signaling that patterns apical branching of the Drosophila airways.
Cell 92,253
-263.[CrossRef][Medline]
Hatta, K., Tsujii, H. and Omura, T. (2006).
Cell tracking using a photoconvertible fluorescent protein. Nat.
Protoc. 1,960
-967.[CrossRef][Medline]
Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J. and Tsai, H.
J. (2003). Germ-line transmission of a myocardium-specific
GFP transgene reveals critical regulatory elements in the cardiac myosin light
chain 2 promoter of zebrafish. Dev. Dyn.
228, 30-40.[CrossRef][Medline]
Hutchinson, S. A. and Eisen, J. S. (2006).
Islet1 and Islet2 have equivalent abilities to promote motoneuron formation
and to specify motoneuron subtype identity.
Development 133,2137
-2147.
Ilagan, R., Abu-Issa, R., Brown, D., Yang, Y. P., Jiao, K.,
Schwartz, R. J., Klingensmith, J. and Meyers, E. N. (2006).
Fgf8 is required for anterior heart field development.
Development 133,2435
-2445.
Kelly, R. G., Brown, N. A. and Buckingham, M. E.
(2001). The arterial pole of the mouse heart forms from
Fgf10-expressing cells in pharyngeal mesoderm. Dev.
Cell 1,435
-440.[CrossRef][Medline]
Lepilina, A., Coon, A. N., Kikuchi, K., Holdway, J. E., Roberts,
R. W., Burns, C. G. and Poss, K. D. (2006). A dynamic
epicardial injury response supports progenitor cell activity during zebrafish
heart regeneration. Cell
127,607
-619.[CrossRef][Medline]
Mably, J. D., Mohideen, M. A., Burns, C. G., Chen, J. N. and
Fishman, M. C. (2003). heart of glass regulates the
concentric growth of the heart in zebrafish. Curr.
Biol. 13,2138
-2147.[CrossRef][Medline]
Marques, S. R., Lee, Y., Poss, K. D. and Yelon, D.
(2008). Reiterative roles for FGF signaling in the establishment
of size and proportion of the zebrafish heart. Dev.
Biol. 321,397
-406.[CrossRef][Medline]
Meyers, E. N., Lewandorski, M. and Martin, G. R.
(1998). An Fgf8 mutant allelic series generated by Cre- and
Flp-mediated recombination. Nat. Genet.
18,136
-142.[CrossRef][Medline]
Mjaatvedt, C. H., Nakaoka, T., Moreno-Rodriguez, R., Norris, R.
A., Kern, M. J., Eisenberg, C. A., Turner, D. and Markwald, R. R.
(2001). The outflow tract of the heart is recruited from a novel
heart-forming field. Dev. Biol.
238,97
-109.[CrossRef][Medline]
Moorman, A. F., Christoffels, V. M., Anderson, R. H. and van den
Hoff, M. J. (2007). The heart-forming fields: one or
multiple? Philos. Trans. R. Soc. Lond. B Biol. Sci.
362,1257
-1265.
Moorman, A. F., de Jong, F., Denyn, M. M. and Lamers, W. H.
(1998). Development of the cardiac conduction system.
Circ. Res. 82,629
-644.
Park, E. J., Ogden, L. A., Talbot, A., Evans, S., Cai, C. L.,
Black, B. L., Frank, D. U. and Moon, A. M. (2006). Required,
tissue-specific roles for Fgf8 in outflow tract formation and remodeling.
Development 133,2419
-2433.
Prall, O. W., Menon, M. K., Solloway, M. J., Watanabe, Y.,
Zaffran, S., Bajolle, F., Biben, C., McBride, J. J., Robertson, B. R.,
Chaulet, H. et al. (2007). An Nkx2-5/Bmp2/Smad1 negative
feedback loop controls heart progenitor specification and proliferation.
Cell 128,947
-959.[CrossRef][Medline]
Reifers, F., Walsh, E. C., Leger, S., Stainier, D. Y. and Brand,
M. (2000). Induction and differentiation of the zebrafish
heart requires fibroblast growth factor 8 (fgf8/acerebellar).
Development 127,225
-235.[Abstract]
Sehnert, A. J., Huq, A., Weinstein, B. M., Walker, C., Fishman,
M. and Stainier, D. Y. (2002). Cardiac troponin T is
essential in sarcomere assembly and cardiac contractility. Nat.
Genet. 31,106
-110.[CrossRef][Medline]
Smith, K. A., Chocron, S., von der Hardt, S., de Pater, E.,
Soufan, A., Bussmann, J., Schulte-Merker, S., Hammerschmidt, M. and Bakkers,
J. (2008). Rotation and asymmetric development of the
zebrafish heart requires directed migration of cardiac progenitor cells.
Dev. Cell 14,287
-297.[CrossRef][Medline]
Soufan, A., van den Berg, G., Ruijter, J. M., de Boer, P. A. J.,
van den Hoff, M. J. and Moorman, A. F. (2006). Regionalized
sequence of myocardial cell growth and proliferation characterizes early
chamber formation. Circ. Res.
99,545
-552.
Tao, Y., Wang, J., Tokusumi, T., Gajewski, K. and Schulz, R.
A. (2007). Requirement of the LIM homeodomain transcription
factor tailup for normal heart and hematopoietic organ formation in Drosophila
melanogaster. Mol. Cell. Biol.
27,3962
-3969.
Trinh Le, A. and Stainier, D. Y. (2004).
Fibronectin regulates epithelial organization during myocardial migration in
zebrafish. Dev. Cell 6,371
-382.[CrossRef][Medline]
Verkhusha, V. V., Otsuna, H., Awasaki, T., Oda, H., Tsukita, S.
and Ito, K. (2001). An enhanced mutant of red fluorescent
protein DsRed for double labeling and developmental timer of neural fiber
bundle formation. J. Biol. Chem.
276,29621
-29624.
Waldo, K. L., Kumiski, D. H., Wallis, K. T., Stadt, H. A.,
Hutson, M. R., Platt, D. H. and Kirby, M. L. (2001).
Conotruncal myocardium arises from a secondary heart field.
Development 128,3179
-3188.
Wan, H., Korzh, S., Li, Z., Mudumana, S. P., Korzh, V., Jiang,
Y. J., Lin, S. and Gong, Z. (2006). Analyses of pancreas
development by generation of gfp transgenic zebrafish using an exocrine
pancreas-specific elastaseA gene promoter. Exp. Cell
Res. 312,1526
-1539.[CrossRef][Medline]
Westerfield, M. (1995). The
Zebrafish Book. Oregon: University of Oregon Press.
Wienholds, E., Schulte-Merker, S., Walderich, B. and Plasterk,
R. H. A. (2002). Target-selected inactivation of the
zebrafish rag1 gene. Science
297,99
-102.
Yutzey, K. E., Rhee, J. T. and Brader, D.
(1994). Expression of the atrial-specific myosin heavy chain
AMHC1 and the establishment of anteroposterior polarity in the developing
chicken heart. Development
120,871
-883.[Abstract]
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