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First published online 15 February 2006
doi: 10.1242/dev.02279
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1 Cardiovascular Research Center and Cardiology Division, Massachusetts General
Hospital and Harvard Medical School, Boston, MA, USA.
2 Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue,
Cambridge, MA, USA.
* Author for correspondence (e-mail: dmilan{at}partners.org)
Accepted 10 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Notch, Neuregulin, Cardiac conduction system, Development, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Failure of any single component of this complex network has
immediate adverse effects, including heart failure and sudden cardiac death
(Jongbloed et al., 2004;
Kleber and Rudy, 2004).
Cardiac electrical impulses arise in the sinoatrial node and pass rapidly
through the atria to the atrioventricular (AV) node. Within the AV node, the
impulse is significantly delayed prior to entering the His-Purkinje network or
the distal conduction system. The AV node is one of the most complex
structures in the cardiac conduction system and slows impulse propagation
between atrium and ventricle, as well as setting an upper limit on the
frequency of conducted impulses. The anatomy and physiology of the AV
conduction tissue have been well described
(Anderson and Ho, 1998;
Kleber and Rudy, 2004), but
the factors regulating its development are largely unknown
(Gourdie et al., 1999;
Moorman and Christoffels,
2003; Pennisi et al.,
2002). The AV node is thought to arise from tissue that emerges at
the boundary between atrium and ventricle. Evidence of such tissue has been
observed in chick, manifest as an impulse delay between the atrium and
ventricle in the tubular heart (de Jong et
al., 1992; Sedmera et al.,
2004), but study of the formation of the central conduction system
has proven difficult, as it arises so early in cardiac development.
Lineage analyses have demonstrated that the entire cardiac conduction
system arises from cardiomyocyte progenitors
(Gourdie et al., 1995;
Mikawa and Fischman, 1996).
Conduction tissues can be classified, on the basis of function, into two broad
divisions: the central conduction system, characterized by slow impulse
propagation and prolonged refractory periods; and the peripheral conduction
system, with rapid impulse propagation. Some of the molecular signals involved
in the induction of the peripheral conduction system have been identified.
Peripheral conduction fiber markers can be induced in cultured embryonic
myocytes by treatment with exogenous endothelin 1
(Gourdie et al., 1998;
Patel and Kos, 2005).
Likewise, addition of exogenous neuregulin in whole mouse embryo culture
causes expansion of conduction system marker expression and changes in the
ventricular electrical activation pattern consistent with a recruitment of
cells to the conduction system (Rentschler
et al., 2002). The study of the effects of inactivation of these
same pathways on the conduction system has proven less straightforward:
endothelin 1-null mice display ventriculo-septal defects in 50% of
pups, but there are no data on conduction system function
(Kurihara et al., 1995).
neuregulin knockout mice die around day 8.5 of generalized myocardial
failure, but prior to a time when electrophysiological analysis is feasible
(Kramer et al., 1996;
Liu et al., 1998;
Meyer and Birchmeier,
1995).
By contrast, little is known of the signals required for the
differentiation of the proximal conduction system
(Gourdie et al., 2003;
Moorman and Christoffels,
2003; Pennisi et al.,
2002). An ongoing requirement for nkx2.5 expression has
been established for the persistence of the central conduction system through
adulthood (Pashmforoush et al.,
2004), and haploinsufficiency of tbx5 causes defects in
the postnatal maturation of this tissue
(Moskowitz et al., 2004).
However, the earliest steps in AV conduction system formation remain unclear,
largely because these stages of development are inaccessible in most
models.
Traditional analyses of development have focused largely on molecular
markers to categorize cell identity. However, the relationship between such
markers and the ultimate cellular phenotype is not always straightforward
(Cleaver and Melton, 2003;
Morshead, 2004;
Parmacek and Epstein, 2005).
As methods are developed for the study of embryonic physiology, the
relationship between molecular markers and cell function will become more
clearly defined. There are few known markers of the early conduction system,
and their relationship to cellular function is undefined. In order to better
understand central conduction tissue formation, we developed techniques to
study the electrophysiological function of differentiating cardiomyocytes in
vivo throughout development.
The external fertilization and development of the zebrafish enables the
observation and manipulation of cardiac physiology at the earliest stages
(Stainier et al., 1996;
Warren and Fishman, 1998;
Yelon, 2001). By using three
independent techniques, we have established that myocytes within the AV ring
differentiate into slowly conducting cells with prolonged refractory periods
at 40 hours post-fertilization (hpf) in zebrafish. These functional assays
were employed as physiological reporters of cellular differentiation to
explore endothelial-myocardial signaling in conduction tissue development.
Using a combination of known mutants and antisense morpholino knockdown
(Nasevicius and Ekker, 2000),
we demonstrate that the formation of central `slow' conduction tissue is not
dependent on blood flow, but does require endocardial signals, including
neuregulin and notch.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pacing
Tungsten 0.5 M
bipolar fork electrodes (WPI, Sarasota, FL) mounted
on a Narishige micromanipulator were introduced into the pericardial space of
zebrafish embryos anesthetized with propofol at 10 µg/ml in E3 medium at
room temperature. Pacing was performed with a Medtronic 5328 stimulator
(Medtronic, Minneapolis, MN) at the cycle lengths indicated and at outputs of
twice diastolic threshold.
Drug treatment
Embryos were treated with a dose of terfenadine (12 µg/ml; Sigma
Chemicals) empirically defined to cause 2:1 AV block in 100% of wild-type
embryos at 48 hpf. Fish were exposed to drug at 36 hpf and observed at 48 hpf
(or the indicated time) by video microscopy using a Nikon TE200 inverted
microscope and an ORCA-ER CCD camera (Hamamatsu, Hamamatsu City, Japan).
Images were captured and analyzed using commercially available image
processing software (Metamorph, Universal Imaging Corporation, Downingtown,
PA) (Milan et al., 2003).
Morpholino injections
Morpholinos (Gene Tools, Philomath, OR) were resuspended in sterile water
to a concentration of 1 mM and diluted to 10-100 µM with 1xDanieau's
[58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM
Ca(NO3)2, 0.5 mM Hepes, pH 7.6]. The morpholinos were
injected at the single-cell stage in a volume of 
5 nl.
Morpholino sequences were as follows:
For each experiment, appropriate four-base mismatch morpholino controls were employed. The effects of morpholinos on mRNA were assayed independently using quantitative real-time RT-PCR from pooled, injected embryos.
Calcium imaging
Embryos at the single-cell stage were injected (into the body of the cell)
with dextran-coupled calcium-green (Molecular Probes, Eugene, OR), of an
average Mr of 3000, at a concentration of 3 mM. Embryos
then were allowed to develop in a dark environment before imaging at the
indicated time on an inverted fluorescence microscope with video capture using
a CCD camera. In all cases, except for the silent heart mutant
embryos, motion artifact was suppressed with an antisense morpholino targeted
against the cardiac troponin T transcript
(Sehnert et al., 2002). Images
were analyzed off-line and isochronal maps were generated with Metamorph
software (Universal Imaging Corporation, Downington, PA). Calcium wavefront
velocities in the atrium were calculated by measuring the distance along the
greater curvature of the atrium and then dividing by the elapsed time. To
minimize the inherent inaccuracies imposed by foreshortening, we have reported
the maximum velocity averaged over several beats. At the video frame rates
used, we were unable to reliably measure the ventricular velocities in
wild-type fish at 48 hpf; therefore we have reported the lower limit for these
datapoints.
In situ analyses of expression
Whole-mount in situ hybridization and immunohistochemistry were carried out
according to standard procedures (Jowett
and Lettice, 1994). Images were acquired using a color digital CCD
camera and processed with Adobe Photoshop (Adobe Systems, San Jose, CA). To
synthesize digoxigenin-labeled antisense RNA probes, a plasmid containing
neuregulin cDNA (pCR-II:Neuregulin-full length) was amplified with
M13 forward and reverse primers to yield a linear template, prior to
transcription with T7 RNA polymerase (Promega). The plasmid for
notch1b was a kind gift from Michael Lardelli (University of
Adelaide, Australia). The plasmid for bmp4 was a kind gift from Dr
Didier Stainier (Walsh and Stainier,
2001). The S46 antibody was obtained from the Developmental
Studies Hybridoma Bank.
neuregulin cDNA cloning
Using the human, mouse and chick neuregulin cDNAs, degenerate
primers were synthesized and a zebrafish 
Zap cDNA library was
screened. A 2840 bp clone was identified and sequenced completely in both
directions, revealing a 1797 bp open-reading frame encoding a 599 amino acid
protein with high homology to known neuregulins (GenBank Accession Number
DQ366108).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
the current studies we have employed calcium imaging to study the excitation
pattern during normal baseline rhythm and therefore anticipate calcium
excitation to closely parallel voltage activation. Injection of calcium green
at the single-cell stage allows the visualization of cardiac excitation in
vivo, as well as the analysis of evolving patterns of excitation during
development. In Fig. 1A, the
calcium activation sequence of the embryonic heart at 36 hpf is illustrated
using isochronal lines superimposed on the maximum intensity projection from
the complete stack of fluorescent images. These lines are spaced at 50
milliseconds intervals. The uniform spacing of isochronal lines in the atrium
demonstrates smooth progression of the activation wave front, which proceeds
to the ventricle without pause. Acceleration of the calcium transient in the
ventricle is visible as increased spacing between the isochronal lines. The
atrial average maximal wavefront velocity was 403±67 microns/second,
whereas the ventricular average velocity was 912±109 microns/second.
The activation pattern of a 48 hpf embryo is shown in
Fig. 1B. In contrast to the 36
hpf heart, there is a significant slowing of the excitation wave front in the
AV ring, indicated by the compressed spacing of the isochronal lines. At 48
hpf, the atrial average maximal wavefront velocity was 2853±580
microns/second, whereas the lower limit for the ventricular average velocity
was 2051±203 microns/second. These data demonstrate that, between 36
and 48 hours of development, myocytes in the AV ring differentiate to a more
slowly conducting phenotype. Refractory periods in any excitable tissue are revealed only when the input stimulus interval encroaches on the recovery time of the tissue. In the central conduction system this manifests as an AV block, when atrial impulses arrive at the AV conduction tissue while it is still refractory. Thus, a simple method to define the functional refractory period of AV tissue is to pace the atrium at incrementally shorter stimulus intervals until some of the impulses no longer conduct to the ventricle. We performed atrial pacing, using a bipolar needle electrode, in embryos at multiple time points over the first 48 hours of development. At 36 hpf, none of the embryos exhibited a pacing-induced 2:1 AV block (Fig. 1C), but, by 40 hpf, 80% of the embryos displayed 2:1 AV conduction. At 48 hpf, all of the embryos studied developed 2:1 heart block with incremental atrial pacing. Ten embryos were studied at each time point. The mean cycle length at which 2:1 AV block developed in the 40 hpf embryos was 754±46 milliseconds, and at 48 hpf was 777±72 milliseconds. The shortest cycle length required for 2:1 block in the 48 hpf embryos was 690 milliseconds. All other embryos were paced at incrementally faster rates until a loss of atrial capture was demonstrated. In all cases, this occurred at cycle lengths at or below 690 milliseconds.
Previously, we observed that treatment with drugs that inhibit the
repolarizing potassium current IKr results in 2:1 AV block in zebrafish
embryos at 48 hpf but not at 24 hpf (Milan
et al., 2003). In order to better understand the relationship
between this drug response and the onset of physiological AV delay, we defined
the timing of the onset of the drug-mediated 2:1 block using video microscopy
(Milan et al., 2003). We
monitored embryos exposed to the IKr blocker terfenadine (12 µg/ml) every
hour after the initiation of a heartbeat. The embryos developed heart block
over a narrow time window, so that, by 40 hpf, 90% of the embryos exhibit 2:1
AV block (n=40; Fig.
1C). Taken together, these results demonstrate that, between 37
and 41 hpf, myocytes within the AV ring develop the key electrophysiological
properties of central conduction tissue: slowed conduction and prolonged
refractory periods.
|
;
Westin and Lardelli, 1997).
The expression of notch1b is highest in the central nervous system,
but is also evident in the heart, where it is accentuated in the endocardium
of the AV ring (Fig. 1D)
(Walsh and Stainier, 2001).
Likewise bmp4 is expressed in the myocardium of the AV ring
(Fig. 1E). These restricted
patterns of expression further support uniquely specified populations of
endocardial and myocardial cells at the boundary between atrium and
ventricle.
It has been proposed that early AV conduction delay is a result of a
source-sink mismatch due to differing tissue architecture between the
thin-walled atrium and thicker-walled ventricle
(Markhasin et al., 2003;
Rohr et al., 1999). If this
were the case, it would be expected that impulses arising in the ventricle and
conducting retrogradely would not exhibit conduction slowing at the AV
interface. During calcium imaging, we observed spontaneous ventricular
premature beats that conducted to the atrium. These retrograde impulses
displayed conduction slowing in the AV ring
(Fig. 2A,B; see also Movie 1 in
the supplementary material). This demonstration of both anterograde and
retrograde conduction slowing effectively excludes source-sink mismatch as the
operative mechanism of AV delay.
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Endocardial signaling is required for the development of AV conduction tissue
The zebrafish mutant cloche fails to develop endothelium and,
therefore, provided a unique opportunity to study the development of
specialized AV conduction tissue in the absence of any endocardium
(Stainier et al., 1995).
Calcium activation patterns in 52 hpf mutant embryos showed no evidence of AV
delay, but rather exhibited virtually constant calcium transient velocities
throughout the heart (Fig. 3A).
Similarly, incremental atrial pacing in cloche embryos failed to
elicit 2:1 AV block at cycle lengths consistently lower than those resulting
in a conduction block in all wild-type embryos
(Fig. 3B). In addition,
treatment with terfenadine over a broad range of doses failed to cause AV
block (Fig. 3B). As expected,
notch1b, one of the potential endocardial signals for myocyte
differentiation in the AV ring, is absent from the hearts of cloche
mutants (Fig. 3C).
Cloche embryos also display abnormal localization of the marker
bmp4, which is absent from the AV ring, and which instead shows more
prominent expression in myocytes in the inflow tract of the heart
(Fig. 3D).
|
; Hall et al.,
2004; Hyer et al.,
1999; Kanzawa et al.,
2002). Owing to a missense substitution in the cardiac troponin T
gene, the zebrafish mutant silent heart never exhibits cardiac
contraction (Sehnert et al.,
2002). Calcium imaging of silent heart mutant embryos at
48 hpf revealed normal conduction slowing in the AV ring
(Fig. 4B). Furthermore,
treatment of these embryos with terfenadine resulted in 2:1 AV block that
could be visualized by calcium imaging (see Movie 2 in the supplementary
material). The atrial and ventricular rates from a representative fish are
seen in Fig. 4C, confirming 2:1
AV block. Similar results were obtained using 2,3-butanedione 2-monoxime to
prevent cardiac contraction for the first 48 hours post-fertilization (data
not shown). These findings confirm the formation of normal AV conduction
tissue in hearts that have never contracted nor been exposed to physiological
blood flow.
Endothelin 1 is a known endocardial signaling molecule that has been
implicated in the flow-mediated induction of the peripheral conduction system
phenotype (Gourdie et al.,
1998; Hyer et al.,
1999; Patel and Kos,
2005). We therefore sought to determine the role of endothelin 1
in the development of AV conduction tissue. Using a previously described
antisense morpholino, we knocked down endothelin 1 mRNA translation
(Miller and Kimmel, 2001). The
resulting embryos exhibited the characteristic pharyngeal arch defects
(Fig. 5A) seen in the genetic
mutant sucker, which is null at the endothelin 1 locus
(Miller et al., 2000). Despite
phenocopy of the sucker mutant, the morphants exhibited normal AV
conduction physiology (Fig.
5B,C), suggesting that, in contrast to observations in the
peripheral conduction system, endothelin 1 is not required for the
specification of AV conduction tissue.
|
). This exon
contains the EGF-like domain that is crucial for the function of all
neuregulin splice forms (Falls,
2003). In neuregulin morphant fish, calcium activation
mapping revealed a generally slowed conduction throughout the heart, and a
loss of physiological AV delay, as demonstrated in the typical isochronal map
seen in Fig. 6B. In these
morphants, the average maximal atrial wavefront velocity was 434±14
microns/second, whereas the average ventricular wavefront velocity was
386±10 microns/second. The morphants also failed to develop 2:1 AV
block with terfenadine (Fig.
6C). The morpholino effect was dose dependent, with 25% of embryos
(n=20) lacking functional AV conduction tissue following a 50 µM
morpholino injection, and 100% failing to develop 2:1 AV block at 100 µM.
Progressive knockdown of neuregulin mRNA in these embryos was
confirmed by quantitative RT-PCR (Fig.
6D). Finally, we designed a second non-overlapping antisense
morpholino targeting the donor splice site of exon 6, which reproduced the
failure of AV delay phenotype in calcium imaging experiments (5 out of 10
fish). A four-basepair mismatch morpholino had no effect on the development of
AV delay (data not shown). All control-injected fish developed 2:1 AV block at
40 hpf. Taken together, these data define an early requirement for neuregulin
in the differentiation of the AV conduction tissue, while also suggesting a
more general requirement for neuregulin in the electrical maturation of the
chambers (Liu et al., 1998;
Meyer and Birchmeier, 1995;
Rentschler et al., 2002;
Zhao et al., 1998).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sehnert and Stainier, 2002;
Stainier et al., 1996). Recent
efforts have extended into developmental physiology, where early work has
suggested parallels between the zebrafish and higher vertebrates
(Milan et al., 2003). In the
work presented here, we have developed methods to study the formation of
specialized AV conduction tissue in zebrafish, using physiological endpoints
rather than expression analyses. We were able to use these techniques in
combination with well-characterized zebrafish mutants and morpholino gene
knockdown to explore the role of endocardial signals regulating the induction
of AV conduction tissue.
Functional imaging of myocyte differentiation
By using three independent techniques, we have demonstrated that myocytes
in the AV ring undergo differentiation into cells with slow conduction
properties and prolonged refractory periods. These are the defining features
of central `slow' conduction tissue. The propagation of electrical activity in
the embryonic heart is much slower than in the adult. It has been proposed
that the entire embryonic heart is a `nodal' structure, and that, as the
chambers differentiate, they develop more rapid conduction, while AV ring
conduction remains slow. In this model, the properties of the AV conduction
tissue are the default pathway (de Jong et
al., 1992). However, our results demonstrate that the development
of conduction delay in the AV ring is an active process, rather than the
persistence of a primitive myocardial phenotype.
Flow-independent endocardial signals induce AV conduction tissue
The zebrafish mutant cloche is a valuable tool with which to
explore the role of endothelial tissue interactions during development, as it
lacks all endocardial tissue and almost all endothelial cells
(Stainier et al., 1995). We
found that the cloche mutants failed to develop AV conduction tissue,
highlighting an inductive role for the endocardium in this process. There is a
growing recognition that endothelial cells participate in many developmental
events (Cleaver and Melton,
2003). Endothelial signals and physiological blood flow are
required for the formation of specialized vascular structures, including renal
glomeruli (Majumdar and Drummond,
1999; Patel and Kos,
2005; Serluca et al.,
2002; Stainier et al.,
1995). Co-culture experiments, and in some cases in vivo studies,
have demonstrated that endothelial cells induce the differentiation of many
cell types, particularly neural and neuroendocrine fates
(Lammert et al., 2001;
Lammert et al., 2003). In many
of these settings, there is evidence for bidirectional signaling between
endothelial cells and surrounding tissues, and there are several examples
where these signals are dependent on mechanical forces including physiological
blood flow (Bartman et al.,
2004; Hove et al.,
2003), e.g. the development of the peripheral conduction system
(Hyer et al., 1999). Here, it
is thought that flow triggers endothelin 1 release, which in conjunction with
other cues, causes surrounding myocytes to differentiate into Purkinje fibers
(Gourdie et al., 1998;
Hall et al., 2004;
Kanzawa et al., 2002). In vivo
data directly implicating blood flow as a signal for conduction system
development have not been previously available.
Arresting blood flow during development is challenging in most experimental
models, but the zebrafish allows the in vivo investigation of the role of
blood flow, without the disruption of other contextual developmental signals
(Stainier et al., 1996). Under
conditions of circulatory arrest, we observed that AV conduction tissue
develops normally. This is in contrast to the available data on the peripheral
conduction system (Hall et al.,
2004; Hyer et al.,
1999), and suggests that a different set of cues is used to signal
myocytes to become central `slow' conduction tissue. Consistent with this
interpretation, knockdown of endothelin 1, a signal implicated in
hemodynamic induction of the peripheral conduction system, had no effect on
the differentiation of AV conduction tissue
(Gourdie et al., 1998;
Hall et al., 2004).
Additionally, these results demonstrate that the pathways regulating central
conduction system development are distinct from those involved in
valvulogenesis, where contraction and flow-mediated signaling are essential
(Bartman et al., 2004;
Hove et al., 2003).
Neuregulin is necessary for the induction of AV conduction tissue
A role for neuregulin in promoting the expression of conduction system
markers and in the development of rapid ventricular conduction has been
defined in the mouse (Patel and Kos,
2005; Rentschler et al.,
2002). However, mice null for neuregulin fail to develop
normal trabecular myocardium, and die as a result of `myocardial failure' at a
stage too early to observe the effects on conduction tissue. We sought to
define the effect of loss of neuregulin signaling on AV conduction tissue
development.
We have cloned the zebrafish neuregulin cDNA and demonstrate that its expression is concentrated in AV ring endocardium at a time that is critical for the development of central conduction tissue. The knockdown of neuregulin, using morpholinos targeted to the exon encoding the crucial EGF-like domain, establishes an essential role for this signaling molecule in the induction of slowly conducting AV tissue. In neuregulin morphant fish we also observed that propagation velocities throughout the heart remained at basal levels similar to those seen in the 36 hpf embryo, suggesting that neuregulin may be required for the proper electrophysiological maturation of atrial and ventricular myocytes, in addition to the AV ring.
Notch regulates the formation of AV conduction tissue
We have demonstrated that zebrafish notch1b is expressed in the AV
ring endocardium at a time point when AV conduction tissue is forming.
Targeted knockdown of notch1b expression resulted in a failure of AV
conduction tissue development in 25-28% of embryos. Higher doses of the
morpholino cause substantial lethality, presumably because of requirements for
notch1b at earlier stages of development. As is the case with the
hesr genes in mice, there may be redundancy in AV ring notch signaling
(Kokubo et al., 2005). Our
data suggest that notch signaling not only affects endocardial cushion
formation, but is also necessary for conduction tissue differentiation, and
thus is likely to be active at an early step in AV ring specification.
Expression analyses of neuregulin and notch1b in the
respective morphant fish revealed no evidence of interactions between these
pathways at the mRNA level.
Several endothelial-tissue inductive interactions, including those required
for the differentiation of insulin-producing islet cells within the pancreas,
are known to involve notch-delta pathway members
(Cleaver and Melton, 2003;
Lammert et al., 2000).
Evidence for the role of notch signaling in endocardial cushion formation
comes from several sources (Kokubo et al.,
2005; Kokubo et al.,
2004; Noseda et al.,
2004; Timmerman et al.,
2004). Recent work has identified rare dominant mutations in human
Notch 1 that cause complex congenital heart disease and aortic stenosis. No
conduction system disease was reported in these families, but it is
conceivable that there may be selection in favor of milder phenotypes or the
human mutations may act through a gain of function
(Garg et al., 2005).
At a stage of development when there are 350 or fewer cells in the entire heart, there is already functional evidence of atrial, ventricular and AV conduction tissue characterized by distinct electrophysiological properties. The functional approach outlined here may enable further characterization of the notch-delta pathway members required for the patterning of the AV ring, as well as of cross-talk with other key developmental pathways, including Bmp and Wnt signaling.
Conclusion
We have employed the zebrafish to study the earliest steps in central
conduction system development that previously have been inaccessible. Using
physiological assays, we have demonstrated for the first time that a distinct
population of slowly conducting cells arises in the AV ring of the developing
heart. The formation of this specialized conduction tissue is a result of an
active developmental process driven by flow-independent endocardial signals.
Future application of these assays will enable an unbiased investigation of
other signals involved in cardiac conduction tissue development and in the
physiological patterning of the vertebrate heart.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/1125/DC1
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