1 Developmental Genetics Program and Department of Cell Biology, Skirball
Institute of Biomolecular Medicine, New York University School of Medicine,
New York, NY 10016, USA
2 Department of Biochemistry and Biophysics and Programs in Developmental
Biology, Genetics, and Human Genetics, University of California, San
Francisco, San Francisco, CA 94143, USA
* Author for correspondence (e-mail: yelon{at}saturn.med.nyu.edu)
Accepted 28 August 2003
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SUMMARY |
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Key words: Zebrafish, Ventricle, Atrium, Cardiac myosin heavy chain, Chamber formation, Atrial natriuretic factor
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Introduction |
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Intrinsic chamber-specific differentiation pathways clearly play a major
role in the acquisition of chamber morphology. For example, in mice, the bHLH
transcription factors Hand1 and Hand2 are required for normal ventricular
growth and morphology (Firulli et al.,
1998; Riley et al.,
1998
; Riley et al.,
2000
; Srivastava et al.,
1997
), and the T-box transcription factor Tbx5 is essential for
normal atrial morphogenesis (Bruneau et
al., 2001
).
In addition to its genetic regulation, chamber morphology may also be
susceptible to epigenetic influences. The morphology of the adult heart is
known to be responsive to increased functional demand; for example, pressure
overload can stimulate ventricular hypertrophy
(Seidman and Seidman, 2001).
The embryonic heart can also respond to hemodynamic changes; one striking
example comes from a recent study in which a physical blockade of blood flow
was shown to cause defects in valve formation, bulbus arteriosus formation and
cardiac looping in the zebrafish embryo
(Hove et al., 2003
). Can
embryonic hemodynamics also influence the morphology of the ventricle and the
atrium the number of cells in each chamber, the thickness of the
chamber wall and the dimensions of the chamber lumen?
To understand the regulation of chamber morphogenesis, we have identified a
number of zebrafish mutations that cause cardiac chamber defects
(Alexander et al., 1998;
Stainier et al., 1996
). One of
these mutations, weak atrium (wea), exhibits defects in both
chambers: contractility defects in the atrium and morphological defects in the
ventricle. Through candidate gene analysis, we demonstrate that wea
mutations disrupt the zebrafish atrial myosin heavy chain
(amhc) gene. Loss of amhc function can explain the atrial
contractile defects in wea mutants. However, because expression of
amhc is restricted to the atrium, the wea mutant ventricular
phenotype, including defects in chamber circumference, wall thickness, lumen
size and gene expression, represents a ventricular response to atrial
dysfunction. Thus, our studies of wea mutants clearly indicate that
function of one chamber can influence morphogenesis of the other, which
implicates epigenetic factors in the regulation of chamber morphology.
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Materials and methods |
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Immunofluorescence, in situ hybridization and photography
Whole-mount immunofluorescence experiments were performed as previously
described (Yelon et al.,
1999), using the monoclonal antibodies MF20
(Bader et al., 1982
), S46
(generous gift from F. Stockdale) and CH1
(Lin et al., 1985
). MF20 and
CH1 were obtained from the Developmental Studies Hybridoma Bank, maintained by
the Department of Biological Sciences, University of Iowa, under contract
NO1-HD-2-3144 from the NICHD.
In situ hybridization experiments with vmhc and cmlc2
antisense probes were performed as previously described
(Yelon et al., 1999). An
antisense amhc probe was synthesized from a 645 bp fragment of
amhc cDNA (beginning at nucleotide 2802). An antisense anf
probe was synthesized from a 422 bp fragment of anf cDNA (see
below).
Stained embryos were examined with Zeiss Axioplan and M2Bio microscopes, and photographed with a Zeiss Axiocam digital camera. Images were processed using Zeiss Axiovision and Adobe Photoshop software. Live embryo videos were recorded and processed using an Optronics DEI750 video camera and an Axioplan microscope, and Pixelink, QuickTimePro and iMovie software.
Cloning of zebrafish amhc, vmhc and anf cDNAs
To identify a zebrafish amhc gene, we evaluated the expression
patterns of available zebrafish ESTs (RZPD, Berlin) resembling myosin heavy
chain genes, found one that was atrium-specific (fc52a03)
(Clark et al., 2001), and
cloned a corresponding full-length cDNA using previously described reverse
transcription and RACE techniques (Keegan
et al., 2002
). To detect amhc mutations, we amplified
fragments of amhc cDNA from mutant embryos using previously described
RT-PCR strategies (Keegan et al.,
2002
). For these experiments, mutant embryos were generated by
mating wea homozygotes that had survived to adulthood. All sequences
were confirmed in at least two independent amplifications of each region of
cDNA. Oligonucleotides used for amhc amplification were:
We also cloned a full-length vmhc cDNA, using RACE initiated from
our previously reported partial cDNA clone (AF114427)
(Yelon et al., 1999).
Oligonucleotides used for vmhc RACE were:
5'-TTGCACTGCCTCTTCCACTTCTGTCTG-3' and
5'-GTTCTTCTTCATCCTCTCCAGGTGAGC-3'.
The coding sequences of both amhc and vmhc appear polymorphic in wild-type zebrafish strains from our fish facility (E.B. and D.Y., unpublished); reported sequences represent the most common wild-type allele.
To identify a zebrafish anf gene, we assembled a consensus cDNA sequence from available ESTs (zeh1304, zeh1366, zah4805, zah5977, zeh11098 and bb02c03) and then amplified a 422 bp cDNA fragment using the oligonucleotides 5'-ACACGTTGAGCAGACACAGC-3' and 5'-TGTTAACAAATTAAGCCGTATTGT-3'.
GenBank Accession Numbers are AY138982 (amhc), AY138983 (vmhc) and AY319419 (anf).
Radiation hybrid mapping and linkage analysis
Physical mapping of amhc with a radiation hybrid panel and meiotic
mapping of wea with SSLP markers were performed using previously
described protocols (Keegan et al.,
2002; Yelon et al.,
2000
). Linkage of amhc and wea was also
confirmed by demonstrating that the weam58 mutant
phenotype and the single-base deletion detected in weam58
mutants are tightly linked (0 recombinants in 188 meioses). Additional
radiation hybrid mapping placed vmhc on LG2 near Z8517.
Morpholino microinjection
Wild-type embryos were injected at the one-cell or two-cell stage with 1-3
ng of anti-amhc morpholino (GeneTools). The anti-amhc
morpholino (5'-ACTCTGCCATTAAAGCATCACCCAT-3') is predicted to block
translation of Amhc.
Transmission electron microscopy
Embryos were fixed at 48 hours postfertilization (hpf) with 2%
paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer,
postfixed with 1% osmium tetraoxide followed by 1% uranyl acetate, dehydrated
through a graded series of ethanol washes, and embedded in LX112 resin (LADD
Research Industries, Burlington, VT). Ultrathin (80 nm) sections were cut on a
Reichert Ultracut UCT, stained with uranyl acetate followed by lead citrate,
and viewed on a JEOL 1200EX transmission electron microscope at 80 kV.
Histology
Prior to sectioning, embryos were fixed in 4% paraformaldehyde, dehydrated
through an ethanol series, cleared in xylene and embedded in paraffin wax. 4
µm longitudinal sections were cut, dewaxed, dried, and stained with
Hematoxylin and Eosin.
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Results |
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wea is required for assembly of the atrial contractile
apparatus
The functional defects in wea mutants suggest that the atrial
contractile apparatus is abnormal. We compared sarcomere assembly in wild-type
and wea mutant embryos at 48 hpf. At this stage, the wild-type atrium
exhibits nascent sarcomeres containing both thick and thin filaments
(Fig. 2A) (Wanga et al., 2001). By
contrast, the wea mutant atrium lacks organized myofibrillar arrays:
some mutant atrial cells contain no obvious myofilaments (data not shown),
whereas others contain a small number of poorly organized myofilaments
(Fig. 2B, arrow). Ventricular
sarcomere assembly is intact in both wild-type and wea mutant embryos
(Fig. 2C,D). These data suggest
that wea encodes an atrium-specific component of the sarcomere.
|
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In wild-type embryos, amhc expression begins around the 19-somite
stage, slightly later than the initiation of vmhc expression, which
begins around the 13-somite stage (Yelon
et al., 1999). From its onset, amhc expression is
complementary to vmhc expression: the expression patterns of
vmhc and amhc subdivide the myocardial precursors into two
separate populations that are likely to represent the ventricular and atrial
precursors (Fig. 4)
(Yelon et al., 1999
). For
example, at the 21-somite stage, the inner portion of the cardiac myosin light
chain 2 (cmlc2; mylc2a Zebrafish Information
Network)-expressing myocardial cone expresses vmhc, and the outer
portion expresses amhc (Fig.
4A-C). The cardiac cone then elongates to form a heart tube with
vmhc-expressing cells at one end and amhc-expressing cells
at the other (Fig. 4E-G). By 48
hpf, the vmhc-expressing ventricle and amhc-expressing
atrium are morphologically distinct within the looped heart
(Fig. 4I-K). We have never
observed amhc expression in the ventricle, nor have we observed
vmhc expression in the atrium.
|
The wea locus encodes Amhc
Hypothesizing that wea could encode Amhc or a regulator of
amhc expression, we chose to map both amhc and wea.
Using radiation hybrid panels (Geisler et
al., 1999; Hukriede et al.,
2001
), we mapped amhc to zebrafish LG20 near the SSLP
marker Z4329 (Shimoda et al.,
1999
). Through meiotic mapping, we mapped wea to the same
region of LG20 (near Z7568). The concordance of these map positions made
amhc a strong candidate gene for the wea locus.
We proceeded to look for amhc mutations in cDNA isolated from
weam58 and weask7 mutants. cDNA from
weam58 mutants has a deletion of a single T at position
4024 of the amhc orf (Fig.
5A), creating a frame-shift that would produce 66 missense codons
followed by a stop codon. cDNA from weask7 mutants
contains a T to A substitution at position 4577 of the amhc orf,
creating a stop codon (Fig.
5B). The premature stop codons found in both
weam58 and weask7 suggest that the
decreased stability of the mutant amhc RNA
(Fig. 4H,L) could be the result
of nonsense-mediated decay (Culbertson,
1999; Hentze and Kulozik,
1999
). Based on the lack of MF20 and S46 immunoreactivity
(Fig. 3B), and the low levels
of amhc RNA (Fig.
4H,L), in the wea mutant atrium, it is unlikely that
wea mutants contain much Amhc protein. Even so, both mutant
amhc cDNAs would be predicted to encode truncated Amhc proteins that,
if stable, could be deficient in dimerization and/or aggregation
(Fig. 5C). Together, our data
suggest that weam58 and weask7 are
strong hypomorphic, if not null, alleles of amhc.
|
Ventricular morphology responds to atrial dysfunction
As myosin heavy chains are essential for myofibrillogenesis, the lack of
functional Amhc in wea mutants can account for the observed defects
in atrial myofibrillar organization and contractility. In addition to their
atrial defects, wea mutants exhibit significant ventricular defects.
As amhc expression is restricted to the atrium
(Fig. 4), any ventricular
phenotypes in wea mutants are likely to represent secondary
consequences of atrial dysfunction.
During the first 36 hours of development, ventricular form and function appear normal in wea mutants (Fig. 6A-C, and data not shown). By 48 hpf, although the rhythm of ventricular contractions remains normal, the wea mutant ventricle acquires an unusual and variable morphology (Fig. 6D-F; also see Movie 1 at http://dev.biologists.org/supplemental/, Fig. 1C,D, Fig. 3 and Fig. 5D-F). Specifically, the wea mutant ventricle becomes more compact, with a smaller circumference than the wild-type ventricle. Sections through the wea mutant heart reveal significant thickening of the ventricular wall and narrowing of the ventricular lumen (Fig. 7). The thickness of the wea ventricular wall varies between, and within, individual embryos, which is in contrast to the consistent and uniform structure of the wild-type ventricular wall (Fig. 7C,D). Increased ventricular thickness in wea mutants does not seem to be caused by excess proliferation. The number of ventricular myocardial cells varies between wea mutant embryos, but is not greater than the number found in wild-type embryos [at 72 hpf, the total number of ventricular myocardial nuclei in serial sections from wild-type embryos was 350±15 (n=2) compared with 274±70 (n=3) in sections from wea mutant embryos]. All characterized features of the wea ventricular phenotype are found in both weam58 and weask7 mutants, and in embryos injected with the anti-amhc morpholino. Overall, the wea ventricular phenotype indicates that a loss of atrial function indirectly stimulates reorganization of the ventricular myocardium, producing significant changes in ventricular form.
|
|
As in other species (Zeller et al.,
1987; Small and Krieg,
2000
; Houweling et al.,
2002
), the zebrafish anf gene is expressed in both the
ventricle and atrium before becoming restricted to the atrium
(Fig. 8A, and data not shown).
In comparison with wild-type embryos, wea mutants exhibit striking
upregulation of anf in both chambers
(Fig. 8A,B). Similarly,
cmlc2 is expressed in both chambers in wild-type embryos and is
significantly upregulated throughout the wea mutant heart
(Fig. 8C,D). Thus, the response
to cardiac dysfunction in wea mutants shares molecular
characteristics with mammalian cardiomyopathies.
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Discussion |
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Although the wea mutant atrium lacks contractile force, it retains passive function throughout the life of wea mutants, serving as a passageway for blood headed into the ventricle. Thus, wea mutants provide an opportunity to evaluate the secondary consequences of a loss of atrial function.
Atrial function influences ventricular development
Although intrinsic gene expression programs dictate aspects of cardiac
chamber formation, our data indicate that cardiac chambers can also respond to
epigenetic influences during their development. Specifically, the changes in
shape, size, organization and gene expression of the wea mutant
ventricle indicate that the ventricular myocardium can respond to atrial
failure. In order to conclude that ventricular aspects of the wea
mutant phenotype are secondary to the loss of amhc function, it is
essential to establish that amhc is not expressed in the ventricle.
Although it is difficult to address precisely whether there is overlap of
amhc and vmhc expression at the atrioventricular boundary,
it is clear that there are two separate zones of amhc and
vmhc expression that are compatible with the expected locations of
atrial and ventricular myocardiocytes throughout development
(Yelon et al., 1999).
Although atrial contractility is aberrant in wea mutants from the initiation of the embryonic heartbeat (around 24 hpf), the wea mutant ventricular defects first become apparent around 48 hpf. This sequence of events fits a model in which the ventricular myocardium responds to a physiological stimulus that is the result of atrial dysfunction. There are several possibilities for the nature of the signal received by the wea mutant ventricle. For example, ventricular mechanosensation of hemodynamic changes produced by atrial failure could trigger changes in ventricular morphology and gene expression. Alternatively, oxygen sensors could provide feedback to the ventricle regarding inefficient circulation, or the nonfunctional atrial myocardium could emit stress signals that are perceived by the ventricle.
A comparison of wea mutants with other zebrafish mutants with
sarcomere defects yields insights regarding potential triggers of the
wea ventricular phenotype. The zebrafish silent heart/tnnt2
(sih) locus encodes cardiac troponin T
(Sehnert et al., 2002), and
the pickwick/ttn (pik) locus encodes titin
(Xu et al., 2002
). In
sih and pik mutants, neither the atrium nor the ventricle
contract or assemble sarcomeres normally
(Sehnert et al., 2002
;
Xu et al., 2002
).
Additionally, neither the sih ventricle nor the pik
ventricle acquire the compact and thick morphology typical of the wea
ventricle (Sehnert et al.,
2002
; Xu et al.,
2002
). Indeed, pik mutants feature a contrasting
phenotype, a dilated cardiomyopathy in which the embryonic ventricle becomes
unusually thin (Xu et al.,
2002
). We have also examined the roles of two other zebrafish
loci, both of which are required for ventricular sarcomere formation and are
dispensable for atrial sarcomere formation (H.C., C. Fabricant and D.Y.,
unpublished). Like sih and pik, neither of these ventricular
mutants exhibit ventricular thickening. These comparisons indicate that
reduced blood flow is not necessarily sufficient to provoke the ventricular
phenotypes observed in wea mutants. Perhaps a specific type of
hemodynamic alteration elicits the wea ventricular response, and this
physiological circumstance is not replicated when the ventricle, or the entire
heart, fails to contract. Alternatively, ventricular contractility and/or
sarcomere assembly might provide a degree of cellular integrity that is a
prerequisite for the type of chamber morphogenesis observed in wea
mutants.
Conservation of chamber responsiveness and relevance to congenital
heart disease
Zebrafish wea mutants demonstrate that epigenetic parameters can
play key roles in regulating ventricular morphogenesis. A recent study
suggests that the relationship between atrial function and ventricular
development may be conserved among vertebrate species. Specifically, analysis
of mice lacking MLC2a, an atrial regulatory myosin light chain gene
essential for atrial myofibrillogenesis, indicates that loss of atrial
function affects ventricular morphology in the mouse embryo
(Huang et al., 2003). The
conserved influence of function on form is likely to be relevant to the causes
of congenital abnormalities in cardiac chamber formation.
When considering the relationship of zebrafish wea mutations with
human disease, it is important to note that autosomal dominant mutations in
human cardiac myosin heavy chain genes can cause either hypertrophic or
dilated cardiomyopathy, depending on the specific gene, mutation and
individual (Seidman and Seidman,
2001). In affected individuals, missense mutations are thought to
be responsible for the production of malfunctional myosin heavy chain proteins
that act cell-autonomously to evoke cardiomyopathy. By contrast,
loss-of-function wea mutations appear entirely recessive, as
heterozygotes are phenotypically wild-type. Thus, wea mutants
demonstrate an alternate method by which a sarcomere defect can trigger a
morphogenetic response not only by directly affecting the cells
expressing the mutant gene, but also by indirectly affecting another
chamber.
Alterations in circulation, as observed in wea mutants, can
trigger significant changes in chamber size, shape, cellular organization and
gene expression. Our data suggest that similar scenarios could be responsible
for congenital heart defects. For example, our studies lend credence to the
proposal that hypoplastic left heart syndrome, which includes defects in left
ventricular morphogenesis, can be caused by reduced blood flow through the
left ventricle (Harh et al.,
1973; Grossfeld,
1999
; Sedmera et al.,
1999
; Sedmera et al.,
2002
). Just as reduced ventricular loading, caused by mitral
atresia, is suggested to trigger hypoplastic left heart syndrome
(Grossfeld, 1999
), reduced
ventricular loading in wea mutants, caused by atrial failure, is
associated with changes in ventricular morphology. Altogether, because of
their striking ventricular phenotypes and relevance to congenital heart
defects, zebrafish wea mutants provide a valuable genetic model for
the analysis of the epigenetic mechanisms that influence cardiac chamber
formation.
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ACKNOWLEDGMENTS |
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Footnotes |
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