RESEARCH ARTICLE 2389
Development 138, 2389-2398 (2011) doi:10.1242/dev.061473
© 2011. Published by The Company of Biologists Ltd
Zebrafish cardiac development requires a conserved
secondary heart field
Danyal Hami1,2, Adrian C. Grimes3, Huai-Jen Tsai4 and Margaret L. Kirby1,2,5,*
SUMMARY
The secondary heart field is a conserved developmental domain in avian and mammalian embryos that contributes myocardium
and smooth muscle to the definitive cardiac arterial pole. This field is part of the overall heart field and its myocardial component
has been fate mapped from the epiblast to the heart in both mammals and birds. In this study we show that the population that
gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field,
and contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. We also show
that Isl1, a transcription factor associated with undifferentiated cells in the secondary heart field in other species, is active in this
field. Furthermore, Bmp signaling promotes myocardial differentiation from the arterial pole progenitor population, whereas
inhibiting Smad1/5/8 phosphorylation leads to reduced myocardial differentiation with subsequent increased smooth muscle
differentiation. Molecular pathways required for secondary heart field development are conserved in teleosts, as we demonstrate
that the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish
arterial pole.
INTRODUCTION
In chick and mouse, cardiac progenitors are organized bilaterally
in the epiblast on either side of the primitive streak (Hatada and
Stern, 1994; Tam et al., 1997). Previous fate mapping in the chick
and mouse epiblast has established that the cranial-to-caudal
organization of cardiac progenitors before gastrulation corresponds
to the cranial-to-caudal organization of the heart (Rosenquist, 1970;
Yang et al., 2002). The cranially located arterial pole progenitors
ingress through the primitive streak earlier than the more caudal
venous pole progenitor cells (Chuai and Weijer, 2009; Cui et al.,
2005). After gastrulation, these progenitors reorganize into two
bilateral fields in the anterior lateral plate mesoderm (ALPM) prior
to cardiogenesis, with arterial pole progenitors located medially and
venous pole progenitors located laterally (Abu-Issa and Kirby,
2008; Yang et al., 2002). As cardiogenesis proceeds, these
mesodermal fields fuse at the ventral midline to form a heart tube
that lacks septation and chambers (Arguello et al., 1975). In chick,
the cardiomyocytes in the heart tube that have undergone early
differentiation typically do not proliferate while the heart tube
continues to elongate (van den Berg et al., 2009). Thus, the
continuous addition of cells from a progenitor population allows
the developing heart to elongate and loop (Buckingham et al.,
2005; de la Cruz et al., 1977).
The portion of the heart field that gives rise to the initial heart
tube has been called the first heart field, whereas the second heart
field is added later. In mouse, these two populations of cardiac
1
Department of Pediatrics, Duke University, Durham, NC 27710, USA. 2University
Program in Genetics and Genomics, Duke University, Durham, NC 27710, USA.
3
Department of Pediatrics, University of Wisconsin Madison, Madison, WI 53711,
USA. 4Institute of Molecular and Cellular Biology, National Taiwan University, Taipei,
Taiwan 106. 5Department of Cell Biology, Duke University, Durham, NC 27710, USA.
*Author for correspondence ([email protected])
Accepted 17 March 2011
progenitors are spatially and molecularly distinct (Kelly et al.,
2001; Cai et al., 2003). Second heart field progenitors differentiate
as they are added to the heart tube to give rise to the right ventricle,
the outflow tract and part of the atria (Buckingham et al., 2005).
The definitive arterial pole is formed from the most medial portion
of the second heart field, known as the secondary heart field (AbuIssa and Kirby, 2007; Dyer and Kirby, 2009b). In chick, this
progenitor population remains in the pericardial wall caudal to the
developing arterial pole until it is added to the heart tube to form
the most distal outflow myocardium and the smooth muscle at the
base of the great arteries (Mjaatvedt et al., 2001; Waldo et al., 2001;
Waldo et al., 2005).
The organization of cardiac progenitors seen in zebrafish mirrors
that observed in chick and mouse. Atrial and ventricular precursors
are organized in partially overlapping zones in the lateral marginal
zone (LMZ) of the blastoderm before gastrulation (Keegan et al.,
2004). The atrial progenitors are located ventrally and farther from
the ingressing LMZ relative to ventricular progenitors, defining the
order of gastrulation. After gastrulation, the mesodermal cardiac
progenitors are organized into bilateral fields with ventricular
progenitors located medially relative to atrial progenitors
(Schoenebeck et al., 2007).
These mapping studies in zebrafish were terminated prior to
the addition of smooth muscle of the bulbus arteriosus, the most
distal component of the intrapericardial arterial pole immediately
cranial to the ventricular myocardium. The adult zebrafish
outflow tract, with the most proximal smooth muscle surrounded
by a sleeve of myocardium, is homologous to that observed in
animals with a divided arterial pole (Grimes et al., 2006; Grimes
et al., 2010). Zebrafish also display a low level of proliferation
in early differentiating cardiomyocytes, which would necessitate
the addition of cells to the heart tube after its formation (de Pater
et al., 2009) (K. A. Nembhard and M.L.K., unpublished).
Although such an addition has been shown (de Pater et al.,
2009), the location of arterial pole precursors has not been
DEVELOPMENT
KEY WORDS: Bmp signaling, Cardiac arterial pole, Cardiac progenitors, Hedgehog signaling, Second heart field, tbx1, Zebrafish
2390 RESEARCH ARTICLE
MATERIALS AND METHODS
Embryo collection, preparation and identification
Zebrafish (Danio rerio) were maintained following published protocols
(Westerfield, 1993; Nüsslein-Volhard and Dahm, 2002). Where indicated,
Tübingen wild-type fish (Tu), Tg(cmlc2::GFP) (kindly provided by Deborah
Yelon, UCSF), Tg(gsc::GFP) (kindly provided by Deborah Yelon), tbx1tu285/+
(AB) (kindly provided by Tatjana Piotrowski, University of Utah),
smob577/+(AB) (kindly provided by Stephen Devoto, Wesleyan University),
Tg(sox10::eGFP) (kindly provided by Thomas Schilling, UC Irvine),
Tg(cmlc2::GFP); tbx1tu285/+(AB) and Tg(cmlc2::GFP); smob577/+(AB) were
used to obtain embryos. Genotyping for tbx1tu285 (AB) histological sections
was performed as described (Piotrowski et al., 2003). All embryos were
anesthetized with 0.016% tricaine and fixed in 4% paraformaldehyde at the
desired stage, then left in methanol overnight at –20°C.
DiI labeling
DiI (2.5 mg/ml in DMSO) was injected into the branchial region through
the dorsal side of Tg(cmlc2::GFP) zebrafish embryos at 24-36 hours postfertilization (hpf). The embryos were anesthetized with 0.016% tricaine and
embedded in 3% methylcellulose for stability during injection, and then
placed in embryo medium until observation at 48 and 72 hpf. Embryos
were again anesthetized and placed in 3% methylcellulose during imaging.
Blastula fate mapping
Tg(gsc::GFP) embryos were injected at the 1-cell stage with ~0.2 nl of a
2.5% solution of lysine-fixable caged fluorescein:rhodamine dextran (1:1
v/v) (Molecular Probes) dissolved in 0.2 M KCl. The unfixable rhodamine
permitted visualization of the photolabeled cells independently of GFP,
whereas the fixable fluorescein permitted immunolabeling of the resulting
progeny. Embryos were kept in the dark until 40% epiboly, then placed in
2% agarose molds. Using gsc::GFP expression to determine orientation,
caged dye was photoactivated using a 368-nm pulsed nitrogen laser
focused through a 40⫻ water-immersion objective. Embryos were again
kept in the dark until 72 hpf.
ALPM fate mapping
For cell tracing at later stages, Tu, Tg(cmlc2::GFP) or Tg(sox10::eGFP)
embryos were injected at the 1-cell stage with either fluorescein dextran
only, or, in the case of Tg(sox10::eGFP) embryos, with the
fluorescein:rhodamine combination described above. These were kept in
the dark until the 6- to 8-somite stage, and then embedded in 1% low
melting point agarose (Lonza) in distilled water. Using the notochord and
neural plate as references, caged dye was photoactivated as described
above.
Dorsomorphin application
Dorsomorphin (Sigma-Aldrich), a selective small molecule inhibitor of
Bmp signaling, was dissolved in DMSO at 5 mM and diluted to either 20
M or 40 M in embryo medium at the time of application. Embryos were
dechorionated and kept in the dark during application until the desired
stage.
General immunolabeling
Once rehydrated in PBST (1⫻ PBS containing 0.1% Tween 20), embryos
were permeabilized by proteinase K treatment (10 mg/ml) and heating at
65°C for 10 minutes, placed in blocking solution [1% BSA, 0.5% Triton
X-100, 5% normal goat serum (NGS) in 1⫻ PBS] for 2 hours and then
incubated with primary antibodies MF20 [Myosin, sarcomere;
Developmental Studies Hybridoma Bank (DSHB), University of Iowa, IA,
USA; 1:10], S46 (Slow myosin heavy chain 1, 2, 3; DSHB; 1:10) and antiEln2 (Tropoelastin 2 or Elastin b; kindly provided by Fred W. Keeley,
University of Toronto; 1:1000) in blocking solution overnight at 4°C. For
39.4D5 (Islet1 homeobox; DSHB) and anti-phosphorylated (p) Smad1/5/8
(Cell Signaling) labeling, embryos were rehydrated in PBST and blocked
using blocking solution containing 0.3% Triton X-100 for 2 hours and then
incubated with primary antibodies MF20, CT3 (Cardiac troponin T; DSHB;
1:10), 39.4D5 (1:100) or anti-pSmad1/5/8 (1:100) in blocking solution
overnight at 4°C. Fluorescent secondary antibodies (Invitrogen; 1:200)
were used for detection as appropriate.
For fluorescein labeling, fixed embryos were serially rehydrated in
PBST and permeabilized as described above. Blocking was performed with
5% sheep serum and 2 mg/ml BSA in PBST, after which embryos were
incubated overnight with alkaline phosphatase-conjugated anti-fluorescein
antibody (Roche; 1:5000). The labeled cells were visualized using
NBT/BCIP (Roche) or Fast Red (Roche) staining.
GFP signal amplification
For 14-somite stage Tg(cmlc2::GFP) embryos, photoactivation of cardiac
progenitors was conducted as described above for the 7-somite stage.
Embryos were dehydrated in methanol after fixation and kept at –20°C
overnight, then serially rehydrated in PBST. Embryos were permeabilized
as described above, and then blocked in 10% NGS and 2% BSA in PBST,
followed by incubation in rabbit anti-GFP (Invitrogen; 1:1000) overnight
at 4°C. After washing in PBST, embryos were incubated in anti-rabbit IgG
HRP (Invitrogen; 1:1000) for 1.5 hours at room temperature. After further
washing with PBST, the TSA-Plus Tetramethylrhodamine System (Perkin
Elmer) was applied according to the manufacturer’s instructions with the
fluorophore-conjugated tyramide at 1:1000 in the reaction buffer.
Histology
Labeled embryos were dehydrated in methanol, transferred into Histoclear
(National Diagnostics) for 20 minutes and embedded in paraffin at 56°C.
The cooled blocks were then sectioned as described (Grimes et al., 2006).
In situ analysis
After rehydration into PBST, embryos were permeabilized as described
above, then hybridized with a digoxygenin-conjugated riboprobe for hand2
at 67°C overnight. The hand2 construct was kindly provided by Deborah
Yelon (UCSF) (Schoenebeck et al., 2007). Following hybridization,
embryos were rinsed and transferred into PBST, then incubated in anti-
DEVELOPMENT
investigated. Furthermore, the incorporation of such cardiac
progenitors into the smooth muscle of the bulbus arteriosus has
not been explored.
A secondary heart field in zebrafish would require molecular
regulation prior to addition to the heart tube, as is the case in
mammals and birds. In the latter models, the transcription factor
Isl1 has been detected in cardiac progenitors prior to differentiation
(Ma et al., 2008; Yuan and Schoenwolf, 2000), whereas Bmp
signaling is needed for myocardial differentiation (Prall et al.,
2007; Waldo et al., 2001). Another transcription factor, TBX1, has
been implicated in defects of the arterial pole in human (Chieffo et
al., 1997; Merscher et al., 2001). The Sonic hedgehog (Shh)
pathway is an important regulator of Tbx1 (Garg et al., 2001;
Yamagishi et al., 2003), and defects in this pathway mirror those
observed in Tbx1 mutants (Washington Smoak et al., 2005). Shh
signaling and Tbx1 are both important for the normal proliferation
of secondary heart field progenitors (Dyer and Kirby, 2009a;
Kochilas et al., 2002; Xu et al., 2004).
We hypothesized that secondary heart field progenitors in
zebrafish would be organized in a similar pattern to that seen in
chick and mouse, and that the pathways controlling the
development of these progenitors would be conserved. We show
that the zebrafish has a subset of cardiac progenitors within the
mesodermal heart fields that contribute to both the myocardium of
the ventricle and the smooth muscle of the bulbus arteriosus. This
population of cells is located in the most medial portion of the
mesodermal heart fields in zebrafish and is present in the
pericardial wall adjacent to the arterial pole after early heart tube
formation. The molecular mechanisms involved in the formation
of the zebrafish arterial pole are also broadly conserved. These
conserved features of arterial pole development make zebrafish an
ideal organism in which to study preseptation development of the
arterial pole.
Development 138 (11)
digoxygenin antibody (Roche; 1:5000) in 2% sheep serum and 2 mg/ml
BSA overnight at 4°C. The hand2 expression domain was visualized using
NBT/BCIP staining.
RESULTS
Zebrafish arterial pole precursors are clustered
dorsally among ventricular progenitors prior to
gastrulation
To establish the location of arterial pole progenitors in the zebrafish
blastoderm we took advantage of the Tg(gsc::GFP) line, which
expresses GFP in the Goosecoid homeobox transcription factor
domain, allowing clear identification of the dorsal axis prior to
gastrulation (Doitsidou et al., 2002). Adopting similar fate-mapping
strategies to those of Keegan et al. (Keegan et al., 2004), caged
fluorophores were used as lineage tracers to identify cells of the
blastula that contribute to the most cranial myocardium of the
ventricle and the smooth muscle of the bulbus arteriosus. By
activating caged fluorophores with laser photolysis in no more than
one or two neighboring blastomeres and recording their position
within the blastula, the contribution of these labeled cells to the
arterial pole was assessed at 72 hpf. As previously published
(Keegan et al., 2004), atrial and ventricular progenitors were found
in distinct populations of cells in the first four tiers of cells in the
LMZ at 40% epiboly. Ventricular myocardial progenitors were
found to reside between 55° and 140° from dorsal in tiers one
through three, with atrial precursors between 75° and 160° from
dorsal in tiers two through four (Fig. 1). In addition to the
identification of atrial and ventricular precursors, we identified cells
that populated the myocardium at the distal outflow tract of the
ventricle, restricted to the first and second tiers between 55° and
105° from dorsal (Fig. 1). No labeled cells were detected in the
smooth muscle of the bulbus arteriosus at 72 hpf. As excessive
proliferation might lead to dilution of the fluorescein in
photolabeled cells, we looked for smooth muscle progenitors at a
later stage in development.
Arterial pole precursors are located
mediocranially in the zebrafish heart fields
As we were unable to identify smooth muscle progenitors in the
zebrafish blastula, we next looked for arterial pole progenitors in
the ALPM. We labeled cells at the 7-somite stage, for which a fate
map of myocardial precursors has already been generated
(Schoenebeck et al., 2007). This map is based on lineage tracing in
the expression domains of nkx2.5 and hand2 in the ALPM, with
hand2 being the most reliable marker of cardiogenic precursors in
zebrafish, and shows a spatial separation of ventricular and atrial
precursors, with ventricular precursors located medially
(Schoenebeck et al., 2007). Based on this work and our previous
data at 40% epiboly (Fig. 1), we photoactivated dye in four-cell
blocks primarily in the medial portion of the cardiogenic field. We
RESEARCH ARTICLE 2391
confirmed the photoactivation of cells within the hand2 domain by
immunolabeling fluorescein in photoactivated cells (Fig. 2A,B).
We found a contribution to the arterial pole from the cranial and
medial portion of the area mapped, with a consistent contribution
to the arterial pole from grid zone F (Fig. 2C,D, Table 1). These
precursors can give rise to both the smooth muscle and
myocardium of the arterial pole at 72 hpf (Fig. 2E,F). We
confirmed the colocalization of the fluorescein-labeled cells with
the myocardium and smooth muscle of the arterial pole in sagittal
sections of 72 hpf hearts (Fig. 2G-K). Labeled cells were not
observed in the endocardium, and labeled cells that did not enter
the heart were typically observed elsewhere in the branchial region
of the larvae (data not shown).
Contribution to the arterial pole is delayed in
early heart tube formation
To assess the timing of secondary heart field contribution to the
zebrafish heart, we examined the location of the arterial pole
progenitor population during the early differentiation of cardiac
cells at the 14-somite stage in Tg(cmlc2::GFP) fish (cmlc2 is also
known as myl7 – Zebrafish Information Network), which express
GFP in the myocardium (Huang et al., 2003). GFP becomes
detectable in differentiating cardiac cells at the 14-somite stage
through immunohistochemical amplification of the signal. Cells
photoactivated in grid zone F at the 7-somite stage, which
contributed exclusively to the arterial pole and the ventricle, were
not GFP positive, suggesting that they remained undifferentiated.
These cells were dorsal to the population of GFP-positive cardiac
cells in Tg(cmlc2::GFP) zebrafish at the 14-somite stage (Fig. 3AC), as the GFP-positive cardiomyocytes moved towards the
midline to form the cardiac cone. Cells labeled in grid zones B and
H, which contributed to both the atrium and the ventricle, could be
found both within and outside the GFP-positive domain at the 14somite stage (Fig. 3D,E).
Given that Isl1 has been observed in differentiated cardiac tissue
in chick (Yuan and Schoenwolf, 2000), we examined Isl1
expression in the cells that form the heart in zebrafish. As the
cardiogenic fields approached the dorsal midline at the 14-somite
stage, we observed Isl1 in an area ventral and lateral to the
trigeminal placode as is the case in the cardiac tissue in the GFPpositive domain in Tg(cmlc2::GFP) fish (Fig. 3F-H). Isl1 is
robustly expressed in the trigeminal placode also, making it a
reliable landmark at the 14-somite stage (Thisse et al., 2004). In
transverse sections, we observed Isl1 in the layer of differentiated
cardiomyocytes and in the mesodermal cells dorsal to them (Fig.
3I,J), where labeled cells that contribute to the arterial pole were
visible at this stage (Fig. 3A-E), with no observable coelom in
between. At 30 hpf, cells from grid zone F were within the
branchial region, typically in the pericardial wall, but had not been
incorporated into the heart tube (Fig. 3K).
Fig. 1. Arterial pole progenitors at 40% epiboly. Fate
map of arterial pole progenitors in the zebrafish embryo
at 40% epiboly. Shown are the first four tiers of the
lateral marginal zone (LMZ) for the left and right sides of
the embryo, dorsal to the right (0°). The equatorial
circumference of the embryo has been divided into 3°
segments, each segment corresponding approximately to
a single cell diameter. The fate map is presented with the
embryo flattened for simplicity. Dots represent
experiments in which caged fluorophores were activated
in single cells and colors indicate the position of their
progeny at 72 hpf.
DEVELOPMENT
Zebrafish arterial pole development
2392 RESEARCH ARTICLE
Development 138 (11)
Fig. 2. Arterial pole progenitors at the 7-somite stage. (A)Dorsal view of a 7-somite zebrafish embryo, cranial to the top. Photoactivated
fluorescein, labeled immunohistochemically (red, arrow), can be identified relative to the hand2 expression field (blue/black). (B)A magnification of
the image in A with a grid superimposed on the region targeted for uncaging to show experimental division of the heart field in the right anterior
lateral plate mesoderm (ALPM). Gray circles represent ~4-cell clusters that were labeled in the mesodermal heart field. In this specimen, cells were
labeled in grid zone F (red). The dashed line highlights the cranial tip of the notochord. (C)Dorsal view of a 7-somite embryo with a map showing
the cardiogenic field (red) and arterial pole progenitors within that field (blue), based on Table 1. (D)Overlay of the grid from B and map from C.
(E)Diagram of the ventral view of a zebrafish embryo at 72 hpf, with cranial indicated by the eyes (pink) and caudal indicated by the yolk (yellow).
The branchial muscles (light blue) are cranial to the bulbus arteriosus (green), ventricle (orange) and atrium (red). (F)Zebrafish in similar orientation
as in E. Progeny of cells labeled in grid zone F (black) at the 7-somite stage are shown relative to the bulbus arteriosus (anti-Eln2, green), and
striated muscle (MF20, red). (G,H,J,K) Sagittal sections of 72 hpf zebrafish, with cranial to the left, showing progeny of the photoactivated
population at grid zone F in the arterial pole (arrows). (I)Diagram showing the various parts of the heart seen in sagittal sections. Caudal is
indicated by the yolk (yellow) and cranial by the bulbus arteriosus (green). A, atrium; V, ventricle; Y, Yolk. Scale bars: 10m.
Progenitors progressively move into the arterial
pole after heart tube formation
Cells that contribute to the arterial pole did not differentiate during
initial heart tube formation and remained in the branchial region
during looping, which is reminiscent of secondary heart field
progenitors in chick embryos (Waldo et al., 2005). We therefore
predicted that we could label cells in the branchial region and
observe them in the myocardium from 48 hpf, when myocardial
addition nears completion. We labeled cells in the dorsal pericardial
wall adjacent to the arterial pole with the lipophilic dye DiI at 24,
30 and 36 hpf and observed incorporation into the ventricle at both
48 and 72 hpf. In these experiments, we observed cells in the
myocardium in 82% (19/23) of embryos injected with DiI at 24
hpf, 62% (18/29) of embryos injected at 30 hpf, and 55% (11/20)
of embryos injected at 36 hpf. Cells labeled at 30 and 36 hpf were
generally restricted to the most cranial portion of the ventricle at
the developing outflow tract, whereas cells labeled earlier migrated
further into the ventricle (Fig. 4; data not shown). The presence of
these DiI-labeled cells in the myocardium was confirmed by
confocal imaging of the hearts (Fig. 4C,F).
Grid zone
A
B
C
D
E
F
G
H
I
J
K
OFT (BA+M)*,‡
1
2
4
2
15
1
OFT (M only)†
Ventricle
Atrium
A/V
1
1
4
1
1
2
1
9
4
2
3
2
1
1
In total, 111 cells were mapped.
A/V, atrioventricular canal; OFT, outflow tract.
*Bulbus arteriosus (BA) and myocardium (M) at the arterial pole.
†
Only the myocardium of the arterial pole.
‡
Labeling of smooth muscle only was never observed.
1
Non-cardiac
Total
5
8
5
5
2
9
6
3
4
2
3
6
11
11
11
2
37
11
10
5
4
3
DEVELOPMENT
Table 1. Summary of mapped cells in the mesodermal heart fields
Zebrafish arterial pole development
RESEARCH ARTICLE 2393
Fig. 3. Cells that contribute to the arterial pole remain outside the early heart tube. (A)Diagram of a transverse section of a 14-somite
embryo, with dorsal to the top, showing the neural tube (NT, light blue) with differentiating myocardium (red) in the ALPM overlying the yolk (Y,
yellow). (B)Whole-mount 14-somite embryo in caudal view, dorsal to top. Labeled cells from grid zone F (black) were observed in the splanchnic
mesoderm of Tg(cmlc2::GFP) zebrafish that expresses GFP in differentiated cardiomyocytes (orange). (C)Transverse section oriented as in A showing
labeled cells (black) relative to differentiating myocardium (red). (D,E)Cells from grid zone B (black) can be seen in the differentiated myocardium
(red) at 14 somites in whole mount (D) and in section (E). (F)Diagram of a 14-somite embryo from the left side, dorsal to top, showing the position
of the cardiac progenitors (red), the eye (E, pink), the trigeminal placode (Tg, light blue) and the yolk (yellow). (G,H)A 14-somite embryo oriented as
in F, showing Isl1 (H, green) and cardiomyocytes (G, orange). The trigeminal placode and the non-differentiating portion of the heart field are Isl1
positive. Arrows indicate the anterior and posterior extent of cmlc2::GFP or Isl1 signal in G and H, respectively. (I,J) Transverse sections of embryos
at the 14-somite stage at the level of the trigeminal placode. Isl1 (green) can be barely detected in the nuclei (blue) of differentiating myocardium
(arrowhead) but more brightly in the nuclei of cells in the adjacent splanchnic mesoderm (arrow). (K)Dorsal view of a 30 hpf zebrafish. Cells
uncaged from grid zone F at the 7-somite stage (black, arrows) can be seen at some distance from the heart tube (orange). A, atrium; V, ventricle.
Scale bars: 10m.
Bmp promotes myocardial differentiation from
arterial pole progenitors
To determine the role of Bmp signaling we knocked down Bmpdriven phosphorylation by treating embryos with dorsomorphin,
which specifically blocks Bmp-mediated phosphorylation of
Smad1/5/8 in zebrafish (Yu et al., 2007). Embryos were treated
with 40 or 20 M dorsomorphin from 24 to 72 hpf, or with DMSO
as a control. Exposure of the embryos to 40 M dorsomorphin
resulted in a dramatic truncation of the ventricle, with subsequent
expansion of the smooth muscle of the bulbus arteriosus (Fig. 7AC). Development of the head muscles was also significantly
delayed after dorsomorphin treatment and appeared comparable to
the head musculature normally observed at 52 hpf (Fig. 7C,D). As
smooth muscle appears at 52 hpf, we also treated embryos with
dorsomorphin from 24 hpf to 48 hpf to observe any effects on the
ventricle exclusively. The ventricle appeared to be smaller and
looping was perturbed in the absence of Bmp signaling (Fig. 7E,F),
but the effect was milder than that previously observed at 72 hpf.
We verified the dorsomorphin-induced inhibition of Bmp signaling
by treating embryos with 40 M dorsomorphin or DMSO from 24
to 30 hpf and subsequently probing with anti-pSmad1/5/8. As
expected, Smad1/5/8 phosphorylation at the arterial pole was
significantly reduced at 30 hpf (Fig. 7G,H).
Arterial pole progenitors in zebrafish are distinct
from the neural crest population
Cardiac neural crest contributes to the septation of the arterial pole
in animals with a divided systemic and pulmonary circulation
(Kirby et al., 1983; Kirby et al., 1985), as well as to the innervation
of the heart (Kirby and Stewart, 1983), but not the myocardium,
during embryonic development. In zebrafish, two studies have
found a significant contribution of neural crest to the myocardium
(Li et al., 2003; Sato and Yost, 2003). To determine whether our
tracing studies had inadvertently labeled migrating neural crest
cells, we utilized Tg(sox10::eGFP) fish (Carney et al., 2006).
Sox10 is active in all neural crest cells during the early stages of
migration from the neural tube in zebrafish (Antonellis et al.,
2008). In Tg(sox10::eGFP) fish, GFP expression was restricted to
DEVELOPMENT
Progenitors adjacent to the arterial pole express
second heart field markers
In mouse, the transcription factor Isl1 is expressed in the mesoderm
of the pericardial wall after differentiation of the first heart field has
begun, in a population that continues to proliferate and remains
contiguous with the heart tube (Cai et al., 2003). Isl1 was detected
in the zebrafish branchial region at 24, 36 and 48 hpf, in cells
adjacent to the arterial pole (Fig. 5A-C⬘). These cells extended
from the pericardial wall into the developing ventricle. Isl1 was
largely absent at the arterial pole by 72 hpf (Fig. 5D,D⬘).
Bmp signaling in the secondary heart field has been linked to the
differentiation of cardiac progenitors that are being added to the
arterial pole in mammals and birds (Prall et al., 2007; Dyer et al.,
2010). We observed Bmp signaling activity at the arterial pole of
zebrafish embryos using an antibody for phosphorylated (p)
Smad1/5/8, which are intracellular effectors of the Bmp pathway
that are phosphorylated when Bmp signaling is active (Faure et al.,
2000). pSmad1/5/8 staining was strong in cells adjacent to the
arterial pole between 24 and 48 hpf (Fig. 6A-C⬘), but appeared to
be restricted to a subset of Isl1-expressing cells closest to the
arterial pole myocardium up until 48 hpf (Fig. 6D). Bmp signaling
was also evident in the most proximal cells of the bulbus arteriosus
at 72 hpf (Fig. 6E,E⬘).
2394 RESEARCH ARTICLE
Development 138 (11)
Fig. 4. Cells in the pericardial wall are incorporated into the
developing heart tube. (A,E)Cells were labeled with DiI
(pseudocolored green) adjacent to the arterial pole of the heart tube
(pseudocolored red) in Tg(cmlc2::GFP) zebrafish at (A) 24 hpf and (E) 30
hpf. Pseudocoloring improved the visibility of DiI-labeled cells. Zebrafish
are shown from the left side, cranial to the top. (B,C,F,G) Labeled cells
were observed in the GFP-positive myocardium at (B,F) 48 hpf and
(C,G) 72 hpf. Zebrafish are shown from the right side, cranial to the
top. (D,H)Optical sections of 72 hpf zebrafish hearts from the right
side, cranial to the top, confirming that DiI-labeled cells were
incorporated into the myocardium. (I)Diagram showing the
approximate area of labeled cells (green) in the pericardial wall (black
line) when viewed dorsally at 30 hpf. The heart tube is shown (red).
Dashed lines indicate eyes (E). A, atrium; V, ventricle; Y, yolk. Scale bars:
10m.
the neural tube at the 7-somite stage (see Fig. S1A-C in the
supplementary material). Cells in the cranial neural crest dispersed
out towards the ALPM in three migrating streams by the 14-somite
stage (see Fig. S1D-F in the supplementary material). Arterial pole
progenitors in grid zone F (Fig. 2B) were photoactivated at the 7somite stage in the Tg(sox10::eGFP) zebrafish and, in five embryos
analyzed, the labeled arterial pole progenitors were distinct from
the GFP-positive cells that expressed sox10 (see Fig. S1C,F in the
supplementary material).
The addition of myocardial cells from the
branchial region to the heart tube is reduced in
mutants with arterial pole defects
Both the transcription factor Tbx1 and the Shh signaling pathway
have been established as important components of normal arterial
pole development in animals with systemic and pulmonary
circulations (Dyer and Kirby, 2009a; Kochilas et al., 2002; Xu et
al., 2004; Washington Smoak et al., 2005). To determine whether
Shh signaling and Tbx1 play similar roles in zebrafish, we studied
arterial pole development in strains with a hypomorphic mutation
in smoothened (smo) (Varga et al., 2001), an obligatory member of
the Hedgehog signaling pathway, and a nonsense mutation in tbx1
(van gogh) (Piotrowski et al., 2003).
All the components of the arterial pole are visible and well
defined by 72 hpf (Fig. 8A-C). Anti-Eln2 effectively labels the
bulbus arteriosus during embryonic development in zebrafish
(Miao et al., 2007) and provides a strong signal at this time. In smo
mutants, looping did not occur and the ventricle was significantly
reduced in size at 36 and 48 hpf. The smooth muscle of the bulbus
arteriosus did not typically appear in these mutants by 72 hpf (Fig.
8D-F). At 96 hpf, smooth muscle was visible in only five of 16 smo
mutants in section, with only two possessing a bulbus arteriosuslike structure (data not shown). In tbx1 mutants, the ventricle
appeared smaller at 36 hpf and was significantly smaller than in
wild-type siblings at 48 hpf, whereas atrial development appeared
to be unaffected. Interestingly, the heart appeared to loop partially,
even though the ventricle was reduced. At 72 hpf, the ventricle
remained small and the bulbus arteriosus was reduced in size
and dysmorphic (Fig. 8G-I). To assess any changes in the
differentiation of arterial pole progenitors in the mutant embryos,
we looked at Bmp signaling as a gauge of the number of
differentiating cells at the arterial pole. We counted the cells that
were positive for pSmad1/5/8 at the arterial pole in serial sections
DEVELOPMENT
Fig. 5. Isl1 is expressed by cells that are being incorporated into
the arterial pole up to 48 hpf. Sagittal sections of Tg(cmlc2::GFP)
zebrafish, cranial to the top, dorsal to the left. (A-C⬘) Isl1 (green) is
expressed in cells adjacent to and within the arterial pole (arrows) at 24,
36 and 48 hpf during addition of the second heart field myocardial cells
to the heart tube (GFP, red). (D,D⬘) By 72 hpf, Isl1 is absent from the
myocardium and is expressed in the cranial-most portion of the smooth
muscle of the bulbus arteriosus. A, atrium; V, ventricle; Y, yolk. Scale
bar: 5m.
Fig. 6. Phosphorylated Smad1/5/8 is present in cells being
incorporated into the arterial pole up to 72 hpf. Sagittal sections
of wild-type zebrafish, cranial to the top, dorsal to the left.
(A-C⬘) pSmad1/5/8 is detected in cells adjacent to and within the
arterial pole (arrows) at 24, 36 and 48 hpf during addition of the
second heart field myocardial cells to the heart tube (MF20).
(D)pSmad1/5/8 (red) is expressed in a subset of Isl1-positive cells
(green) closest to the myocardium (CT3) at 30 hpf. (E,E⬘) By 72 hpf,
pSmad1/5/8 (green) is expressed in the myocardium and the smooth
muscle (red) of the bulbus arteriosus. A, atrium; V, ventricle; Y, yolk.
Scale bar: 5m.
of mutant and wild-type embryos at 30 hpf (Fig. 8J-L). There was
a significant reduction in pSmad1/5/8 in cells adjacent to the
arterial pole in both mutants, indicating a significantly reduced
population of differentiating cells (Fig. 8M).
To further understand the arterial pole phenotypes in these
mutants, we labeled cells adjacent to the arterial pole with DiI to
see whether they would move into the heart. DiI labeling was
performed at 24 hpf in wild-type embryos (Fig. 9A-C) and smo
mutants (Fig. 9D-F) and at 30 hpf in wild-type controls (Fig. 9GI) and tbx1 mutants (Fig. 9J-L) owing to the difference in the
severity of the cardiac phenotype of these mutants. Incorporated
cells were again observed at 48 hpf and confirmed at 72 hpf (as in
Fig. 4). Only 17% (2/12) of Tg(cmlc2::GFP); smob577 mutants and
14% (2/14) of Tg(cmlc2::GFP); tbx1tu285 mutants incorporated any
DiI-labeled cells into the myocardium (Table 2). In the absence of
functional Tbx1 or Hedgehog signaling, the addition of labeled
cells from the pericardial wall to the arterial pole was significantly
reduced after heart tube formation.
RESEARCH ARTICLE 2395
DISCUSSION
In the epiblast of chick and mouse, the craniocaudal location of
cardiac progenitors is collinear with their organization in the heart
tube (Hatada and Stern, 1994; Tam et al., 1997). Because
gastrulation occurs craniocaudally, the most cranial epiblast cells,
which represent the arterial pole progenitors, are the first to
gastrulate. Although a dorsal midline primitive streak does not exist
in zebrafish, the location of cardiac progenitors in the blastoderm
is comparable because the first tier of cells gastrulate first with the
subsequent tiers following (Warga and Kimmel, 1990). Thus, our
data support the work of Keegan et al. (Keegan et al., 2004) and
show that the location of myocardial progenitors within the
blastoderm and their order of gastrulation are consistent among all
vertebrate models studied. Our data show that arterial pole
progenitors represent a subset of the ventricular myocardial
progenitors in the blastoderm, which would be among the first
cardiac progenitor cells to undergo gastrulation. Although we did
not observe arterial pole smooth muscle progenitors in the blastula,
none of our tracing experiments found labeled smooth muscle cells
in the absence of labeled myocardium and this supports the idea
that some smooth muscle precursors are at least located in the
vicinity of myocardial precursors.
After gastrulation, we show that a common progenitor pool
within the mesodermal cardiogenic fields gives rise to the smooth
muscle and myocardial cells that comprise the definitive arterial
pole of the zebrafish, as has been shown in chick (Hutson et al.,
2010). In fate-mapping studies, the secondary heart field has been
localized to the mediocaudal edge of the cardiogenic field in chick
(Abu-Issa and Kirby, 2008). By contrast, our data show a
mediocranial location of the secondary heart field progenitors in
the cardiogenic field in zebrafish. This difference reflects the
manner in which the heart tube is formed in these species. In chick,
heart fields fold 120°-130° along with the coelom during migration
towards the midline, bending ventrally to reorient towards the
midline (Abu-Issa and Kirby, 2008). As the zebrafish is an
anamniote, folding of the coelom and the heart fields does not
occur. Instead, the heart fields merge to form a cardiac cone that
becomes a tube by a process of asymmetric involution of the right
side of the cone (Rohr et al., 2008; Stainier et al., 1993).
For the heart to continue to elongate and loop after early heart
tube formation, second heart field progenitors must remain
undifferentiated until the appropriate time for addition to the
myocardium of the heart tube. The arterial pole progenitors that we
have identified differentiate after heart tube formation and can be
observed in the pericardial wall at 30 hpf, similar to what is
observed in chick and mouse (Kelly et al., 2001; Mjaatvedt et al.,
2001; Waldo et al., 2001). Previously, de Pater et al. demonstrated
Fig. 7. The absence of Bmp signaling induces an
expansion of the bulbus arteriosus at the expense
of the ventricle. (A-C)Ventral view of 72 hpf zebrafish
treated with dorsomorphin (B,C) or DMSO carrier (A)
from 24 to 72 hpf. (D)The bulbus arteriosus can be
detected at 52 hpf as shown by anti-Eln2 labeling. A,
atrium (S46); V, ventricle (MF20). (E,F)Ventral view of 48
hpf embryos. Dorsomorphin treatment (F) from 24 to 48
hpf leads to smaller ventricles (red) at 48 hpf.
(G,H)Sagittal sections of embryos with dorsal to the top.
Dorsomorphin treatment from 24 to 30 hpf (H) results in
a significant loss of phosphorylation of Smad1/5/8 (green
nuclei) at the arterial pole (arrow). Scale bars: 5m.
DEVELOPMENT
Zebrafish arterial pole development
2396 RESEARCH ARTICLE
Development 138 (11)
that cells are added to the poles of the heart tube between 24 and
48 hpf in zebrafish (de Pater et al., 2009). We have observed cells
labeled at the pericardial wall being incorporated into the arterial
pole in the same time frame. This is consistent with the idea of a
true secondary heart field in zebrafish. Additionally, after
acceptance of this article for publication, Lazic and Scott (Lazic
and Scott, 2011) showed that cells adjacent to and within the
Fig. 9. Labeled cells in the pericardial wall are not incorporated
into the developing heart in smo and tbx1 mutants.
(A-C,G-I) Cells labeled with DiI (pseudocolored green) in the branchial
region are incorporated into the myocardium (pseudocolored red) in
wild-type zebrafish hearts after labeling at 24 hpf (A-C) and 30 hpf
(G-I). DiI colocalized with the myocardium in wild-type fish at 48 and
72 hpf (arrows). (D-F,J-L) This incorporation of cells does not occur in
most smo (D-F) or tbx1 (J-L) mutants. The green cells that appear to
overlap with red cardiac cells (K) do not overlap with the myocardium
24 hours later (L). All embryos expressed cmlc2-GFP. Scale bar: 25m.
developing cardiac arterial pole express mef2cb. In the absence of
this zebrafish homolog of Mef2c, ventricular cell number is reduced
significantly at 48 hpf.
The arterial pole of the zebrafish includes the most cranial
myocardium of the ventricle and the large smooth muscle bulbus
arteriosus that connects to the ventral aorta (Grimes and Kirby,
2009). The bulbus arteriosus is thought to protect the gills from
high blood pressure arising at the ventricle and to prevent backflow
(Albrecht et al., 2003; Santer, 1985). Although the function of this
vessel differs from that of the base of the great arteries of
mammalian and avian hearts, its developmental origin seems to be
at least partially derived from the heart fields. The interface of the
zebrafish bulbus arteriosus with the most cranial myocardium at
the outflow is strikingly similar to that in other vertebrates (Grimes
et al., 2006). The homology of the secondary heart field and the
resulting arterial pole structure suggests that the molecular
mechanisms involved are conserved.
To explore these mechanisms, we first looked at Isl1 as an early
marker of the second heart field and showed that it is expressed in
zebrafish in the mesenchyme adjacent to the arterial pole. In mice,
Isl1 is restricted to the second heart field after the first heart field
begins to differentiate, and Isl1 expression ceases as cells are added
to the growing heart tube (Cai et al., 2003; Ma et al., 2008). In the
zebrafish, Isl1 is expressed by differentiating myocardial cells that
are in the process of forming the cardiac cone, as well as in
mesoderm where undifferentiated cardiac cells are located at the
14-somite stage. This is similar to the enduring expression of Isl1
observed in the fusing heart tissue of the chick early heart tube
(Yuan and Schoenwolf, 2000). Whereas knockdown of isl1
produces no phenotype at the arterial pole in zebrafish (de Pater et
al., 2009), the Isl1-null mouse has a dramatic arterial and venous
pole phenotype (Cai et al., 2003). This suggests non-conservation
of Isl1 function in zebrafish heart development or, alternatively,
that another gene fulfills the same function but is yet to be
recognized as a result of the ancient polyploidy event that took
place in teleosts (Semon and Wolfe, 2007; Van de Peer et al.,
2003).
We have also shown that Bmp signaling is highly active in
arterial pole progenitors and in the myocardium, particularly in a
subset of Isl1-positive progenitors, and also in smooth muscle
closest to the ventricle at 72 hpf. Exogenous Noggin, an inhibitor
of Bmp signaling, has been shown to delay differentiation in
cultured secondary heart field progenitors (Waldo et al., 2001). Our
DEVELOPMENT
Fig. 8. Zebrafish tbx1 and smo mutants
display arterial pole defects.
(A-C)Ventral view of whole-mount wildtype zebrafish, with cranial to the top. The
heart is looped by 36 hpf and the bulbus
arteriosus (Eln2) is formed by 72 hpf.
Ventricle (MF20, red); atrium (S46, yellow).
(D-I)In smo (D-F) and tbx1 (G-I) mutants,
cardiac development is impaired. (J)Sagittal
sections from the left show pSmad1/5/8
(green and arrows) near the arterial pole of
the wild-type heart (CT3) at 30 hpf. (K,L)In
the smo (K) and tbx1 (L) mutants,
pSmad1/5/8 is significantly reduced.
(M)Quantification of pSmad1/5/8-positive
cells in wild-type versus smo and tbx1
mutants. Numbers in bars indicate the
number of fish sampled. *, P1.23⫻10–3.
Error bars indicate s.d. Scale bars: 5m.
RESEARCH ARTICLE 2397
Table 2. Summary of Di-labeled cells at the pericardial wall
that moved into the ventricle
Labeled at:
Genotype
24 hpf
30 hpf
Wild type
smo
tbx1
82%
17%
–
62%
–
14%
The percentage of wild type, smo and tbx1 mutants that incorporated any DiIlabeled cells into the myocardium following DiI labeling at 24 or 30 hpf.
data are also consistent with the observation of restricted Bmp2/4
expression in the mesoderm adjacent to the arterial pole after
myocardial addition has ceased in chick (Somi et al., 2004).
Previous work has shown that Bmp signaling drives myocardial
differentiation in vivo in chick (Tirosh-Finkel et al., 2010). Bmp
signaling also induces myocardial differentiation in chick
secondary heart field explants, which can separately be induced to
differentiate as smooth muscle with exogenous Fgf8 (Hutson et al.,
2010). We confirm that Bmp signaling drives myocardial
differentiation in zebrafish and further show that loss of Bmp
signaling increases the size of the smooth muscle bulbus arteriosus,
suggesting that the progenitor pool is temporally restricted in that
a reduced contribution to the arterial pole myocardium results in an
enhanced contribution to the bulbus smooth muscle.
In addition to these markers, we have also explored the roles of
Hedgehog signaling and Tbx1 transcriptional regulation. Tbx1 is
expressed in the mesoderm and endoderm of the pharyngeal region
and is known to be necessary for myocardial and endocardial
addition to the arterial pole (Chapman et al., 1996; Huynh et al.,
2007). The Shh pathway is also known to regulate Tbx1 activity in
the pharyngeal region through Forkhead transcription factors
(Yamagishi et al., 2003). Smoothened is a transmembrane protein
that is activated in the presence of Hedgehog ligands and is
required for all Hedgehog signaling. We used the smo mutant, in
which all Hedgehog signaling is eliminated (Varga et al., 2001).
Recent work has shown that the Shh pathway promotes
proliferation in the chick secondary heart field progenitors in vivo
(Dyer and Kirby, 2009a). Additionally, the loss of Hedgehog
signaling has been shown to reduce the size of the heart fields in
the zebrafish blastula (Thomas et al., 2008). Given the lack of
secondary heart field structures in the zebrafish mutants that we
examined, our data support the preliminary investigations of
Grimes and Kirby (Grimes and Kirby, 2009) and indicate that the
role of these genes in arterial pole development is conserved in
teleosts.
In conclusion, our data demonstrate conservation of all the steps
in the development of arterial pole progenitors prior to septation.
Abnormal development of secondary heart field progenitors has
been shown to underlie many conotruncal defects that are seen in
neonates (Hutson et al., 2006; Ward et al., 2005). The conservation
of the pathways needed for arterial pole development in an
organism that lacks outflow septation establishes the importance
of these pathways in tissue patterning and strongly suggests that
the process of elongation of the arterial pole preceded, and plays
a key role in, the process of outflow septation in evolutionary
history.
Acknowledgements
We thank Mary R. Hutson, John Klingensmith, Elwood Linney and Kenneth D.
Poss for discussions and comments on the manuscript; and Harriett A. Stadt,
Kyle N. Erwin and Martha R. Alonzo for technical help. This work was
supported by NIH grant HL084413 (M.L.K.), by an AHA predoctoral fellowship
0815074E (D.H.) and by the Jean and George Brumley, Jr Neonatal Perinatal
Research Institute at Duke University. Deposited in PMC for release after 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.061473/-/DC1
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