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First published online 8 October 2008
doi: 10.1242/dev.024083
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1 Developmental Genetics Program and Department of Cell Biology, Kimmel Center
for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New
York University School of Medicine, New York, NY 10016, USA.
2 Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan
8, 3584CT, Utrecht, The Netherlands.
3 University of Sheffield, Department of Biomedical Science, Western Bank,
Sheffield S10 2TN, UK.
Author for correspondence (e-mail:
yelon{at}saturn.med.nyu.edu)
Accepted 12 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Hedgehog, Cardiac progenitor specification, Cyclopamine, Heart development, Smoothened, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Nerem, 2007). It is therefore
desirable to elucidate the entire network of signals that govern cardiac
progenitor specification in the early embryo. Which signals cooperate to
establish cardiac fate, when do these signals act and where do key signaling
events take place?
Previous studies have established important roles for several signaling
pathways in the specification of the cardiac progenitor pool. The Fgf, Bmp and
Nodal pathways play inductive roles and are thought to promote cardiac fate
assignment (Brand, 2003;
Solloway and Harvey, 2003;
Yelon, 2001). By contrast, the
retinoic acid pathway restricts the acquisition of cardiac identity
(Keegan et al., 2005), and Wnt
signaling promotes or represses cardiogenesis depending on the developmental
stage (Klaus et al., 2007;
Ueno et al., 2007). These
inductive and repressive signaling pathways presumably collaborate to ensure
formation of the appropriate number of cardiomyocytes. However, as the roster
of relevant signals remains incomplete, it is difficult to decipher the
precise recipe for creating cardiac identity.
The Hedgehog (Hh) signaling pathway regulates specification, patterning and
growth of multiple embryonic organs
(Ingham and McMahon, 2001),
and has been implicated in cardiac development. In mouse, loss of Shh
causes several cardiac abnormalities, including ventricular hypoplasia,
septation defects and outflow tract (OFT) shortening
(Chiang et al., 1996;
Tsukui et al., 1999;
Washington Smoak et al.,
2005). Tissue-specific removal of Hh pathway components has
demonstrated that Hh signaling is required within the cardiac neural crest and
the second heart field (the origin of OFT myocardium) for OFT morphogenesis
(Goddeeris et al., 2007;
Lin et al., 2006;
Washington Smoak et al.,
2005), and that Hh signaling within the dorsal mesocardium is
required for atrioventricular septation
(Goddeeris et al., 2008).
Less is known about earlier roles that Hh may play during cardiac
progenitor specification. In Drosophila, ectodermal Hh influences the
specification and positioning of cardiac progenitor cells within the
cardiogenic mesoderm (Jagla et al.,
1997; Liu et al.,
2006; Ponzielli et al.,
2002). Mice that lack smoothened (Smo), the obligate Hh
co-receptor, and mice lacking both Shh and Indian hedgehog
(Ihh) exhibit more severe cardiac defects than mice lacking only
Shh (Zhang et al.,
2001). These defects include aberrant cardiac morphogenesis,
reduced heart size and delayed initiation of expression of the pre-cardiac
marker Nkx2-5 (Zhang et al.,
2001). Expanded Nkx2-5 expression is observed in mice
lacking the inhibitory patched 1 (Ptch1) receptor. Although these
studies implicate Hh signaling in early steps of heart formation, it remains
unclear if, when, where or how Hh signaling impacts cardiac fate
assignment.
In this study, we demonstrate that Hh signaling plays an important role in driving cardiac specification in the zebrafish embryo. Using both loss-of-function and gain-of-function approaches, we demonstrate that Hh signaling promotes cardiomyocyte formation. This influence of Hh takes place during and shortly after gastrulation, when Hh signaling ensures specification of the proper number of myocardial progenitors. We use genetic inducible fate mapping in the mouse embryo to show a direct response to Hh signaling within the myocardial progenitors. Finally, transplantation experiments in zebrafish identify a cell-autonomous role for Hh signaling in promoting the contribution of cells to the myocardial lineage. Together, these results reveal that Hh signaling has an early and direct impact on cardiac progenitors, and identify Hh as an essential component of the optimal signaling milieu for cardiac specification.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), smohi1640
(Chen et al., 2001),
ptchhu1602 (Koudijs et
al., 2008), ptc2/leptj222
(Koudijs et al., 2005),
Tg(cmlc2:DsRed2-nuc)f2
(Mably et al., 2003) and
Tg(cmlc2:egfp)twu277
(Huang et al., 2003). The
cardiac phenotype of embryos mutant for the null allele
smohi1640 was found to be comparable with that of
smob577 embryos at the 18-somite stage, at 24 hours
post-fertilization (hpf) and at 48 hpf. All zebrafish work followed protocols
approved by the NYU School of Medicine IACUC.
Generation of maternal-zygotic smo embryos
Germline replacement chimeras were generated as previously described
(Ciruna et al., 2002). Donor
embryos were generated from an intercross of fish heterozygous for
smob577, and wild-type embryos were used as hosts. At 24
hpf, host embryos were screened for the presence of donor-derived
Tg(nanos:gfp)-expressing cells, and the identity of the corresponding
donor embryo for each host was determined phenotypically at this stage. Three
fertile female chimeras were successfully raised out of 67
Tg(nanos:gfp)-positive hosts. MZsmo embryos presented the
same characteristic morphology seen in zygotic smo, including
U-shaped somites, mild cyclopia and ventral tail curvature.
Cyclopamine treatment
Embryos in their chorions were treated with 75 µM CyA (Toronto Research
Chemicals) in egg water, diluted from a 10 mM stock in ethanol. Unless stated
otherwise, CyA was added at 40-50% epiboly. Wash-out experiments were
conducted by replacing the embryo media three times with fresh egg water,
which has been shown to restore Hh signaling after CyA exposure
(Chen et al., 2001). For
fate-mapping experiments, dechorionated embryos were treated with 50 µM
CyA. Control embryos for the CyA treatments were exposed to 0.0075% ethanol in
egg water.
RNA injections
Embryos were injected with 100 pg of zebrafish shha mRNA
(Ekker et al., 1995) at the
one-cell stage.
Immunofluorescence and cardiomyocyte counting
MF20 and S46 whole-mount immunofluorescence of embryos was conducted as
previously described (Alexander et al.,
1998; Yelon et al.,
1999). Cardiomyocyte counting using the transgene
Tg(cmlc2:DsRed2-nuc) was conducted as previously described
(Schoenebeck et al., 2007). To
generate embryos for counting, Tg(cmlc2:DsRed2-nuc)+/-;
smob577/+ fish were intercrossed to generate zygotic
smo mutant embryos and were crossed to MZsmo germ line
chimeras to generate MZsmo embryos.
In situ hybridization
In situ hybridization was conducted as previously described
(Berdougo et al., 2003;
Concordet et al., 1996;
Thompson et al., 1998;
Yelon et al., 1999). Mutant
embryos were identified after imaging via PCR genotyping; protocols are
available upon request. To count cells at 18-somite or 22-somite stages, we
scored cells positive for the NBT/BCIP precipitate in each heart field (see
Fig. S1 in the supplementary material). Individual cells are easily identified
as the precipitate is excluded from the nucleus and the cells are arranged in
epithelial sheets, typically one cell thick
(Trinh and Stainier,
2004).
Fate mapping with caged fluorescein
Fate-mapping experiments in tier 1 of the 40% epiboly embryo were conducted
using previously described protocols
(Keegan et al., 2004). In each
experiment, we labeled neighboring blastomeres along the embryo margin. After
recording the positions of labeled blastomeres, individual embryos were placed
in half-dram glass vials with egg water. For CyA-treated embryos, 50 µM
cyclopamine was added at this stage. To enhance identification of labeled
cardiomyocytes, we performed in situ hybridization for cmlc2 prior to
detection of the fluorescein lineage tracer
(Keegan et al., 2004).
Genetic inducible fate mapping
Fate mapping in mouse embryos was conducted as described previously
(Ahn and Joyner, 2004).
Gli1CreERT2/+; R26R/R26R males were mated with
Swiss Webster females to generate Gli1CreERT2/+;
R26R/+ embryos. Noon on the day of a plug was designated embryonic
day (E) 0.5. To initiate labeling at 
E7.0, TM was administered by gavage
at 5 pm on E6.5. For fate mapping of Gli1-derived cells between E8.0 and
E9.0, TM was given at 5 pm on E7.5. Hearts (E18.5) or upper bodies (E15.5)
were dissected in ice-cold PBS, fixed in 4% paraformaldehyde in PBS for 20
minutes, cryoprotected and embedded in OCT
(Blaess et al., 2006). Sections
(12 µm) of the dissected tissues were cut on a cryostat, and
β-galactosidase staining and nuclear fast red counterstaining were
performed (Ahn and Joyner,
2004). All mouse work followed protocols approved by the NYU
School of Medicine IACUC.
Transplantation
Blastula stage transplantations were conducted as previously described
(Ho and Kane, 1990;
Parker and Stainier, 1999).
Between 3 and 4 hpf, 10-20 donor cells were transplanted from the margin of a
donor embryo and placed into the margins of two wild-type host embryos. Donor
embryos carried the Tg(cmlc2:egfp) transgene to allow evaluation of
cardiac contribution at 2 days post-fertilization. To ensure that both donor
populations (wild-type and MZsmo) were exposed to the same
experimental conditions, including developmental stage at time of transplant,
we generated clutches that were made up of 50% wild-type
(smo+/-) and 50% MZsmo donor embryos by crossing
a germ line chimera MZsmo female to a smo+/- male
carrying two copies of Tg(cmlc2:egfp). The genotype of each donor
embryo was determined post-transplant at 24 hpf. For experiments with a
lineage tracer, 1 nl of fluorescein-dextran (MW 10,000, Invitrogen), from a 5
mg/ml stock in Phenol Red with 0.2 M KCl, was injected into donor embryos.
Imaging
Images were captured with Zeiss Axiocam digital cameras on Zeiss Axioplan
and M2Bio microscopes and were processed with AxioVision (v4.6.3 and v3.0.6),
Adobe Photoshop 7 and Adobe CS3 software, except for live fate mapping images,
which were captured with MetaMorph Imaging software (v6.1r3, Universal
Imaging).
Statistical analyses
Statistical tests were run with GraphPad Prism v.4, Microsoft Excel, and as
by Zar (Zar, 1999). To compare
the means of cell counting data sets, we used two-tailed, unpaired
t-tests. To compare transplant contribution frequencies, we used the
normal approximation of the 
2 test to evaluate the difference
between the two proportions. Because of the smaller number of experiments
conducted, we used Fisher's exact test to compare the frequencies of labeling
myocardial progenitors in our fate-mapping studies.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Varga et al., 2001). To
investigate the involvement of Hh signaling in cardiac development, we
inhibited Smoothened function, both genetically and pharmacologically, and
analyzed the resulting cardiac phenotypes.
First, we examined the phenotypes caused by two different
smoothened mutant alleles, smob577 and
smohi1640 (Chen et
al., 2001; Varga et al.,
2001): in both cases, mutant embryos exhibit small, dysmorphic,
improperly looped hearts at 48 hpf (Fig.
1A,B; N.A.T. and D.Y., unpublished). The mutant ventricle appears
compact and tubular, and the mutant atrium is misshapen. However, these
phenotypes may not reflect all functions of Hh during heart formation, owing
to residual signaling activity provided by maternally supplied
smoothened (Chen et al.,
2001; Varga et al.,
2001). To deplete Hh signaling activity in the early embryo, we
generated maternal-zygotic smob577 mutants
(MZsmo) via germline replacement
(Ciruna et al., 2002). As a
complementary approach, we treated embryos with cyclopamine (CyA), a
pharmacological inhibitor of Smoothened
(Chen et al., 2002), from the
30% epiboly stage, prior to the initiation of gastrulation. Both approaches
consistently yield cardiac chambers that are reduced in size and misshapen.
The hearts of MZsmo mutant embryos resemble slightly smaller versions
of zygotic smo mutant hearts (Fig.
1B,C), whereas CyA treatment causes a greater and more variable
disruption of cardiac morphology and size
(Fig. 1D). The exacerbation of
the cardiac phenotype in CyA-treated embryos might be attributable to as yet
uncharacterized off-target effects of CyA, as the smob577
allele is thought to be a functional null that disrupts protein folding and
localization (Barresi et al.,
2000; Eberhart et al.,
2006; Feng et al.,
2006; Varga et al.,
2001).
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; Lawson et al.,
2002), and alterations in circulation can impact cardiac chamber
morphogenesis (Auman et al.,
2007). Thus, to determine whether the cardiac chamber defects
simply reflected defects in morphogenesis, we quantified the number of
cardiomyocytes at 52 hpf. Examining all three loss-of-function scenarios, we
found significant reductions in the number of cardiac cells
(Fig. 1E; see Table S1 in the
supplementary material). In all cases, the number of ventricular
cardiomyocytes is more affected than the number of atrial cardiomyocytes; for
example, CyA-treated embryos display 65% fewer ventricular cardiomyocytes and
56% fewer atrial cardiomyocytes than are found in wild-type embryos.
Altogether, the consistent trend of reduced cardiac cell number in embryos
with disrupted Hh signaling demonstrates a role for Hh in promoting
cardiomyocyte formation.
Hedgehog signaling is required for the initial establishment of cardiomyocytes
The observed reduction in cardiomyocyte number could be the result of an
early shortage of myocardial progenitors, inefficiency of myocardial
differentiation and/or later defects in the proliferation or survival of
differentiated cardiomyocytes. To determine when the cardiac deficit
originates, we evaluated the expression of nkx2.5 in myocardial
progenitors (Schoenebeck et al.,
2007). Compared with wild-type embryos, MZsmo mutants and
CyA-treated embryos have smaller fields of nkx2.5-expressing cells in
the anterior lateral plate mesoderm (ALPM), as well as decreased intensity of
nkx2.5 expression (Fig.
2A-C), suggesting an early defect in myocardial progenitor
specification. Evidence of a myocardial deficiency persists after terminal
differentiation is under way. At the 18-somite stage, the number of
cmlc2-expressing differentiated cardiomyocytes and the intensity of
cmlc2 expression are decreased in MZsmo mutants and
CyA-treated embryos (Fig.
2D-F). Similar defects were apparent when examining
chamber-specific gene expression (Fig.
2G-L).
For a more quantitative understanding of the extent of the early
cardiomyocyte deficit, we counted the number of cells present prior to heart
tube assembly. In these studies, we used CyA treatment to generate large
numbers of embryos deficient in Hh signaling. Quantification of the reduction
in cmlc2, vmhc or amhc demonstrated similar effects on both
ventricular and atrial lineages. CyA-treated embryos displayed 50% of the
number of cmlc2-expressing cells, 50% of the number of
vmhc-expressing cells and slightly more than half the number of
amhc-expressing cells seen in wild-type embryos (see Table S2 in the
supplementary material). Thus, reduction of Hh signaling causes a significant
early reduction in the number of ventricular and atrial cardiomyocytes,
comparable in scale with the deficits observed in the cardiac chambers at 52
hpf.
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;
Concordet et al., 1996;
Ekker et al., 1995;
Krauss et al., 1993;
Lewis et al., 1999). In
ptc1;ptc2 double mutants, the loss of both Ptc gene products results
in overactive Hh signaling throughout the embryo, including in the ALPM, where
ptc2 is normally expressed at low levels
(Koudijs et al., 2008). In
both gain-of-function scenarios, we found a subtle expansion of
nkx2.5 expression (Fig.
3A-C) and a more evident expansion of cmlc2 expression
(Fig. 3D-F). Examination of
vmhc and amhc expression revealed similar expansions of both
genes, indicating that both chamber lineages are sensitive to increased Hh
signaling (Fig. 4A-D). The early expansion of the cardiomyocyte population in embryos with increased Hh signaling could lead to an increase in the number of cells in the cardiac chambers; alternatively, the cardiomyocyte surplus might not be maintained. Quantification of cells in the cardiac chambers of shh-injected embryos revealed an increased number of atrial cardiomyocytes (Fig. 4G; see Table S1 in the supplementary material). However, although the ventricles of shh-injected embryos often appeared enlarged, the increase in ventricular cell number was not statistically significant (Fig. 4E-G). As shh-injected embryos display an excess of vmhc-expressing cells at earlier stages (Fig. 4A,B), there may be additional consequences of ectopic Hh signaling that affect ventricular cardiomyocyte maintenance. Combining these results with our loss-of-function data, we conclude that the level of Hh signaling activity is an important determinant of the number of cardiomyocytes that arise from the heart fields.
Hedgehog signaling is required prior to myocardial differentiation
To determine when Hh signaling is required to promote cardiomyocyte
formation, we administered CyA during discrete periods of development. First,
we added CyA at different stages and allowed embryos to develop until the
18-somite stage, when we evaluated the number of vmhc-expressing
ventricular cardiomyocytes. We analyzed vmhc instead of
cmlc2 in order to simplify quantification, as our data indicated that
atrial and ventricular populations respond similarly to reduced Hh signaling
(Figs 1,
2). Addition of CyA at the dome
or germ ring stage caused a reduction of more than half of the number of
ventricular cardiomyocytes at the 18-somite stage
(Fig. 5A,B; see Table S3 in the
supplementary material). Treatment with CyA at successively later stages
significantly reduced the number of ventricular cardiomyocytes; however, the
extent of cardiomyocyte loss decreased with later application of CyA
(Fig. 5A,B; see Table S3 in the
supplementary material). Finally, adding CyA at the 10-somite stage did not
change the number of ventricular cardiomyocytes
(Fig. 5A,B; see Table S3 in the
supplementary material). These data suggest that the requirement for Hh
signaling during cardiomyocyte formation begins during gastrulation and ends
by the 10-somite stage.
|
). To
inhibit and then restore signaling, we exposed embryos to CyA for defined time
intervals; subsequent media changes allowed significant restoration of Hh
signaling within 2 hours, as assessed by recovery of ptc1 expression
(see Fig. S2 in the supplementary material). Consistent with our prior results
(Fig. 5A,B), inhibition of Hh
signaling from the dome stage until the six-somite stage has the same effect
as continuous inhibition from the dome stage until the 18-somite stage
(Fig. 5C,D; see Table S4 in the
supplementary material). Together, our data suggest that formation of a normal
number of cardiomyocytes requires continual Hh signaling during gastrulation
and early somitogenesis stages. Interestingly, this time period leads up to
the initiation of robust expression of nkx2.5 around the six-somite
stage (Schoenebeck et al.,
2007) and far precedes the onset of myocardial differentiation
(Yelon et al., 1999). Thus,
this temporal requirement suggests that Hh signaling may influence progenitor
specification.
Hedgehog signaling regulates the specification of myocardial progenitors
Although our timing experiments suggested that Hh signaling might impact
the specification of myocardial progenitors, it was also possible that the
cardiomyocyte reduction in embryos with depleted Hh signaling represented a
deficit in the proliferation of myocardial progenitors and their progeny, as
Hh promotes cell proliferation in many developmental contexts
(Ingham, 1995;
Roy and Ingham, 2002). In
order to distinguish between these possibilities, we monitored myocardial
progenitors in CyA-treated embryos by labeling cells in the lateral marginal
zone (LMZ) at 40% epiboly. In prior fate-mapping studies, we have shown that
the LMZ contains a mix of multipotent mesendodermal progenitor cells,
including myocardial progenitors (Keegan
et al., 2004). Examining the fates of cells from the LMZ allows
the determination of myocardial progenitor distribution and productivity
(Keegan et al., 2004). By
comparing experiments in wild-type and CyA-treated embryos, we sought to
determine if Hh signaling influences myocardial progenitors via an effect on
progenitor specification and/or proliferation.
In each experiment, we labeled neighboring blastomeres in tier 1 (the first
row of cells above the blastoderm margin at 40% epiboly) via photoactivation
of caged fluorescein-dextran (Fig.
6A). After recording the location of the labeled cells, we raised
embryos until 48 hpf. CyA was administered to individual embryos subsequent to
blastomere labeling, allowing us to monitor any potential alterations to
progenitor distribution or proliferation in response to a loss of Hh
signaling. We scored whether labeled cells contributed to the myocardium and
counted the number of labeled cardiomyocytes
(Fig. 6B,C). As we sampled
blastomeres in tier 1, we found ventricular myocardial progenitors but not
atrial myocardial progenitors (Fig.
6D,E; Tables S5-S7 in the supplementary material), consistent with
our prior demonstration of separate spatial origins for ventricular and atrial
lineages (Keegan et al.,
2004). We compared the frequency of finding ventricular
progenitors in wild-type and CyA-treated embryos, focusing on experiments
labeling cells between 50°-125° from dorsal, where ventricular
progenitors are known to reside (Keegan et
al., 2004). We found wild-type myocardial progenitors at a
frequency of 48%: out of 33 labeling experiments in wild-type embryos, we
labeled a myocardial progenitor on 16 occasions
(Fig. 6D,E). By comparison, we
labeled a myocardial progenitor in eight out of 35 such experiments in
CyA-treated embryos, a frequency of 23%
(Fig. 6D,E). Thus, CyA
treatment significantly reduced the frequency of labeling a ventricular
progenitor to half that of wild type, indicating that inhibition of Hh
signaling results in a reduced density of myocardial progenitors.
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). We
found no significant difference in progenitor productivity following CyA
treatment: we detected an average of 4.6 cardiomyocytes per wild-type
progenitor and an average of 4.5 cardiomyocytes per CyA-treated progenitor
(Fig. 6E; see Table S5 in the
supplementary material). Thus, extrapolating from our tier 1 fate map data, we
conclude that reduced Hh signaling causes a reduction in myocardial progenitor
specification, but not in progenitor proliferation.
Complementary changes in endothelial patterning in response to altered Hedgehog signaling
Given the reduction in myocardial progenitors observed in CyA-treated
embryos, we next asked whether this loss correlates with an expansion of
another embryonic lineage. We focused on lineages that originate near the
myocardial progenitors (Keegan et al.,
2004), including the endocardial, anterior endothelial, myeloid,
pectoral fin mesenchyme and pharyngeal mesendodermal lineages. No obvious
gains were evident from gross morphological assessment or from our
fate-mapping experiments. However, careful examination of molecular markers
revealed slight alterations in endothelial patterning. Specifically, we
noticed a broadened common cardinal vein (CCV) in smo mutants and
CyA-treated embryos (Fig. 7A,B;
N.A.T. and D.Y., unpublished). Conversely, shh-injected embryos seem
to lack the CCV (Fig. 7C).
Prior to vessel formation, subtle changes in endothelial progenitor marker
expression reveal similar phenotypes (see Fig. S3 in the supplementary
material).
These data suggest the possibility that Hh signaling influences the choices
made by multipotent cardiovascular progenitor cells that are able to become
either myocardium or particular subsets of endothelium
(Kattman et al., 2007;
Martin-Puig et al., 2008;
Wu et al., 2008).
Alternatively, these endothelial patterning defects could reflect aberrant
morphogenesis of the anterior vessels, as defects in trunk vessel
morphogenesis are seen in shha mutants
(Brown et al., 2000;
Gering and Patient, 2005). In
favor of the former hypothesis, we note that addition of CyA from dome stage
until the 6-somite stage is sufficient to broaden the CCV (N.A.T. and D.Y.,
unpublished). Even so, it remains possible that the complementary changes in
myocardial and endothelial lineages in response to altered Hh signaling
reflect independent functions of Hh, rather than transformations between
myocardial and endothelial fates. Precise identification of the fates
exchanged when Hh signaling is altered awaits a more detailed understanding of
the lineage relationships, gene expression patterns and morphogenetic
behaviors of specific subsets of anterior endothelial cells.
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;
Stainier et al., 1993),
whereas shha and shhb are expressed in dorsal mesoderm
(Ekker et al., 1995;
Krauss et al., 1993). During
early somitogenesis stages, cardiac progenitors are located in bilateral
fields of the ALPM (Fishman and Chien,
1997; Schoenebeck et al.,
2007), and Hh ligands are found in midline structures
(Concordet et al., 1996;
Currie and Ingham, 1996;
Krauss et al., 1993;
Lewis et al., 1999). Although
it is known that Hh can act as a long-range signal in some contexts
(Goetz et al., 2002), we have
been unable to detect ptc1 or gli1 expression in the regions
of the zebrafish embryo that contain myocardial progenitors (N.A.T. and D.Y.,
unpublished), although ptc2 expression has been reported at low
levels in the ALPM (Koudijs et al.,
2008). Difficulties detecting ptc1 or gli1 may
be due to limitations in detecting low levels of gene expression via in situ
hybridization.
As another approach to examine whether myocardial progenitor cells receive
Hh signals, we pursued genetic inducible fate mapping (GIFM) in the mouse
embryo (Joyner and Zervas,
2006). GIFM enables labeling of cells directly responding to Hh
signaling during discrete periods of development owing to an inducible Cre
recombinase expressed from the Gli1 locus
(Gli1-CreERT2) (Ahn and
Joyner, 2004). As Gli1 is a direct transcriptional target
of Hh signaling and is not expressed in the absence of Hh signaling
(Bai et al., 2002;
Dai et al., 1999;
Lee et al., 1997;
Marigo et al., 1996),
Gli1 expression indicates a positive response to Hh signaling. Upon
tamoxifen (TM) administration, Cre recombination activity is induced within
8-12 hours in Gli1-expressing cells, and remains active for 24
hours (Hayashi and McMahon,
2002; Zervas et al.,
2004). Recombination of the R26R reporter transgene
permanently marks these Gli1-derived cells and their progeny with
lacZ. However, owing to mosaic expression of Cre, particularly at the
low doses of TM used, the GIFM technique does not mark all of the
Hh-responding cells in a given tissue (Ahn
and Joyner, 2004; Joyner and
Zervas, 2006; Zervas et al.,
2004).
Use of a Ptch1LacZ allele in mouse has demonstrated
Ptch1 expression in the OFT at E9.5 and in the dorsal mesenchymal
protrusion within the atria at E10.5
(Goddeeris et al., 2008;
Washington Smoak et al.,
2005). However, it is unknown whether mouse myocardial progenitors
directly respond to Hh signaling prior to myocardial differentiation
(Zhang et al., 2001). We
therefore labeled Hh-responding cells from E7.0-E8.0 by administering TM
at 5 pm on E6.5; this period encompasses the formation of the cardiac crescent
and the transition to a linear heart tube
(Fig. 8A) and precedes by one
full day the reported Ptch1lacZ expression in the
lengthening OFT (Washington Smoak et al.,
2005). When labeled cells were analyzed at E18.5, we found
Gli1-derived cardiomyocytes in all embryos examined
(Fig. 8B-D). Furthermore, we
observed Gli1-derived cardiomyocytes in all four chambers (see Table S8 in the
supplementary material). These GIFM experiments indicate that myocardial
progenitors directly receive and respond to Hh signaling during early stages
of heart formation in the mouse embryo, potentially beginning as early as
E7.0.
Receipt of Hedgehog signaling is required cell autonomously for effective contribution to myocardium
Although we found that mouse myocardial progenitors receive Hh signals,
GIFM alone could not address whether Hh signaling is required within these
cells. We conducted transplantation experiments in zebrafish to test the cell
autonomy of the requirement for Hh signaling during cardiomyocyte formation.
Blastomeres were transplanted at the sphere stage, using donor embryos
carrying the transgene Tg(cmlc2:egfp)
(Huang et al., 2003) to
facilitate assessment of myocardial contribution. To determine whether
wild-type cells and MZsmo cells have the same ability to become
cardiomyocytes, we transplanted either wild-type or MZsmo donor cells
into wild-type hosts (Fig.
9A-E). Our data indicated that MZsmo donor cells
contributed to host myocardium less frequently than did the wild-type donor
cells (Fig. 9A). In
wild-type-to-wild-type transplants, donor-derived cells contributed to the
myocardium 17% of the time (29 out of 173 transplants). By contrast,
MZsmo donor cells transplanted into wild-type hosts contributed to
the myocardium only 9% of the time (13 out of 148 transplants), a significant
difference from the effectiveness of wild-type donor cells. In six independent
transplantation sessions, we found considerable variability in the exact
frequency of myocardial contribution (see Table S9 in the supplementary
material). However, wild-type donor cells consistently had a higher frequency
of myocardial contribution than did MZsmo donor cells (see Table S9
in the supplementary material).
One explanation for the inefficient myocardial contribution of
MZsmo cells might be a general problem with transplanted cell
survival. However, in additional experiments, we labeled donor cells with a
fluorescein-dextran lineage tracer and found that MZsmo cells
contribute robustly to many lineages in wild-type hosts. For example, we found
that wild-type and MZsmo donor cells were equally capable of fast
muscle contribution (Fig.
9F-H). This result is consistent with previously reported normal
contribution to wild-type host fast muscle by zygotic smo donor cells
(Barresi et al., 2000). Thus,
we conclude that the survival of MZsmo donor-derived cells is not
generally compromised.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Our data provide a new level of resolution of the role of Hh signaling
during heart development. Prior studies characterizing the cardiac phenotypes
of Smo and Ptch1 knockout mice indicated a relationship
between Hh signaling and heart size, but the nature of this connection - when
and where this signaling occurred and the cellular basis for the observed
cardiac phenotypes - remained unclear
(Chiang et al., 1996;
Goodrich et al., 1997;
Zhang et al., 2001). In the
zebrafish embryo, we demonstrate that Hh signaling acts during early stages to
directly promote myocardial progenitor specification. The direct requirement
for Hh signaling, rather than involvement of a relay, is interesting, given
the apparent distance between the myocardial progenitors and the known sources
of shha and shhb at the embryonic midline. This may be an
example of long-range Hh signaling, akin to that observed in
Drosophila imaginal discs, vertebrate neural tubes, vertebrate limb
buds or zebrafish endoderm, where Hh ligands are know to act at significant
distances from their origins (Briscoe et
al., 2001; Chung and Stainier,
2008; Gallet et al.,
2006; Lewis et al.,
2001; Zeng et al.,
2001). In mouse, it seems likely that Ihh could be
relevant to the specification of myocardial progenitors, as it is expressed in
the anterior endoderm adjacent to the cardiac crescent
(Zhang et al., 2001);
additionally, the cardiac phenotype of
Shh-/-Ihh-/- mice resembles the
Smo-/- phenotype more closely than the
Shh-/- phenotype
(Chiang et al., 1996;
Zhang et al., 2001).
Hh signaling is not absolutely essential for cardiomyocyte formation;
normal cardiomyocytes develop, albeit in reduced numbers, when Hh signaling is
depleted. It seems unlikely that the role of Hh signaling is to prevent
cardiac apoptosis, as caspase inhibitors fail to rescue the smo
mutant cardiac phenotype (Z. Garavito-Aguilar and D.Y., unpublished). The
alternative fate of the lost cardiac progenitors in the absence of Hh
signaling is not yet clear, although the observed responsiveness of the CCV to
Hh signaling suggests one intriguing avenue for future investigation. Overall,
we were struck by our observation that loss of Hh function consistently
produces quantitative phenotypes that represent a 50% reduction from wild
type. In MZsmo and CyA-treated embryos, we found 50% of the
normal number of cardiac cells at 2 days post-fertilization and a 50%
reduction in cells expressing cmlc2, vmhc or amhc at earlier
stages (see Tables S1 and S2 in the supplementary material). We encountered
ventricular progenitors half as frequently in our CyA-treated fate-mapping
experiments (Fig. 6E) and found
MZsmo-derived cardiomyocytes at half the wild-type frequency in
transplantation experiments (Fig.
9A). One possibility is that loss of a specific subpopulation of
cardiomyocytes accounts for the missing 50%. However, we have not encountered
evidence in support of that idea, as we see that both atrial and ventricular
cardiomyocytes are significantly affected. Instead, the partial reduction of
cardiac cell number presumably reflects the mechanism by which Hh signaling
promotes specification.
There are multiple ways in which Hh signaling could directly impact the
competence of potential myocardial progenitors. One possibility is that Hh
signaling could influence the movement of myocardial progenitors, such that
its absence would prevent some cells from receiving the signals necessary for
the establishment of cardiac fate. The cardiac defects in zebrafish embryos
with altered Agtrl1b/Apelin signaling have been attributed to a similar
mechanism (Scott et al., 2007;
Zeng et al., 2007). Hh
signaling has been shown to influence cell migration and motility in several
developmental contexts, including axon guidance, and germ cell, endothelial
cell and cardiac neural crest cell migration
(Charron and Tessier-Lavigne,
2005; Deshpande et al.,
2001; Goddeeris et al.,
2007; Hochman et al.,
2006; Washington Smoak et al.,
2005). However, loss of Hh signaling has not been associated with
an effect on general gastrulation movements in zebrafish
(Solnica-Krezel et al., 1996;
van Eeden et al., 1996), and
we have not observed any obvious ALPM migration defects in MZsmo or
CyA-treated embryos (N.A.T. and D.Y., unpublished).
We favor an alternate model in which Hh signaling works in combination with
other signaling pathways that confer cardiac identity, potentially including
the Fgf, BMP and Nodal pathways. One mechanism by which Hh signaling could
affect the degree of competence imparted to a naïve cell would be to act
in a serial fashion with other inductive signals. For example, Hh signaling
could impart enhanced ability to respond to any of the complement of
specification signals that combine to confer cardiac fate. This mechanism is
reminiscent of the situation during chondrogenesis, in which Hh signaling is
required to alter the competence of somitic mesoderm to differentiate in
response to BMP signaling (Murtaugh et
al., 1999; Zeng et al.,
2002). Alternatively, instead of acting serially, Hh signaling may
act in parallel with other required signals to promote cardiac fate assignment
through regulation of shared target genes. The parallel pathways could then
partially compensate for a loss of Hh signaling. To determine the precise
nature of the relationship between Hh signaling and the competence to form
myocardium, it will be necessary to identify the targets downstream of Hh/Gli
signaling that impact cardiac specification.
Intriguingly, our gain-of-function studies are consistent with a
combinatorial signaling model. We observed an increase in cardiomyocyte number
in embryos with increased Hh signaling, although only within the ALPM and not
at separate, ectopic sites. These data suggest that the myocardial
developmental potential within the heart field is labile in response to Hh
signaling levels, but that Hh signaling alone is not sufficient to induce
cardiac fate assignment. Additionally, gain-of-function studies in P19 cells
have shown that overexpression of Shh or Gli2 can induce
cardiomyocyte formation (Gianakopoulos and
Skerjanc, 2005). However, the induction of a cardiac program
required cell aggregation, suggesting that Hh signaling might cooperate with
another signal induced only after aggregation in order to trigger
cardiomyocyte formation. In future work, it will be interesting to define
whether a comparable signal functions in the embryo.
Based on the combination of our studies in zebrafish and mouse, it is
likely that Hh signaling plays a conserved, cell-autonomous role in promoting
myocardial progenitor specification. Signaling pathways that are required cell
autonomously for cardiac specification have great potential for use in
cell-based therapies for cardiac repair, as they will provide the ability to
optimize the production of cardiomyocytes from multipotent cells in vitro
(Chen et al., 2007), a mode of
producing cardiomyocytes that remains relatively inefficient
(Laflamme and Murry, 2005).
Our finding of a cell-autonomous role for Hh signaling suggests that
manipulation of this pathway could be used to more effectively tilt the
balance towards cardiac fate assignment in undifferentiated cells. It would be
particularly interesting to determine whether Hh signaling can influence the
developmental potential of recently identified populations of mammalian
multipotent cardiovascular progenitors
(Kattman et al., 2007;
Kattman et al., 2006;
Martin-Puig et al., 2008;
Moretti et al., 2006). In the
future, a fuller understanding of how Hh signaling interfaces with additional
contributing signals will facilitate our ability to realize the goals of
regenerative medicine.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/22/3789/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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