Download Notch1b and neuregulin are required for

RESEARCH ARTICLE 1125
Development 133, 1125-1132 (2006) doi:10.1242/dev.02279
Notch1b and neuregulin are required for specification of
central cardiac conduction tissue
David J. Milan1,*, Andrea C. Giokas1, Fabrizio C. Serluca2, Randall T. Peterson1 and Calum A. MacRae1
Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells:
the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the
conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue
develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial
signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and
valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the
development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for
patterning central ‘slow’ conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions
within the developing heart tube.
INTRODUCTION
There is an increasing realization of the importance of the cardiac
conduction system in human disease (Moorman and Christoffels,
2003). 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
1
Cardiovascular Research Center and Cardiology Division, Massachusetts General
Hospital and Harvard Medical School, Boston, MA, USA. 2Novartis Institutes for
Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 10 January 2006
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;
DEVELOPMENT
Key words: Notch, Neuregulin, Cardiac conduction system, Development, Zebrafish
1126 RESEARCH ARTICLE
MATERIALS AND METHODS
Fish
All experiments were performed in wild-type zebrafish (Tübingen AB) or the
mutant lines silent heart (tc3006) and cloche (m39), raised and maintained
using standard methods. Embryos were staged according to morphological
criteria (somite number) and by timing in hours post-fertilization.
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 ␮g/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 ␮g/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 ␮M with 1⫻Danieau’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:
EDN-1, 5⬘-GTAGTATGCAAGTCCCGTATTCCAG-3⬘;
Troponin T, 5⬘-CATGTTTGCTCTGATCTGACACGCA-3⬘;
Neuregulin exon 6 acceptor, 5⬘-TGCTGGTGGCTGCTGCACAGAGGAA-3⬘;
Neuregulin exon 6 donor, 5⬘-ATGCGGATTTGCGGCAATACCTGCA3⬘;
Notch 1b exon 27 donor, 5⬘-AATCTCAAACTGACCTCAAACCGAC3⬘; and
Notch 1b exon 27 acceptor, 5⬘-TCGATCTCACCTGCCACAGGAACAA-3⬘.
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 digoxigeninlabeled 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).
RESULTS
Slow conduction tissue develops in the
atrioventricular ring at 40 hpf
The two defining characteristics of central conduction tissue are
slow conduction and prolonged refractory periods. To assess these
physiological parameters in the developing zebrafish heart we
employed calcium-sensitive fluorophores. At present, signal-tonoise considerations prevent the use of voltage-sensitive dyes in
the study of cardiac activation patterns in zebrafish embryos.
Although they can become dissociated at high frequencies or
during arrhythmias, calcium activation and membrane
depolarization are tightly coupled under most physiological
situations (Laurita and Singal, 2001). 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
DEVELOPMENT
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.
Development 133 (6)
RESEARCH ARTICLE 1127
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 ␮g/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.
In addition to the physiological evidence for specialized
conduction tissue at this point in development, the AV ring exhibits
a unique pattern of gene expression. We examined the in situ
expression of the genes notch1b and bmp4 in wild-type embryos
(Walsh and Stainier, 2001; 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.
Fig. 1. Normal development of AV conduction tissue.
(A,B) Calcium activation maps from a single cardiac cycle in wild-type
zebrafish at 36 hpf (A) and 48 hpf (B). Isochronal lines (50 mseconds)
obtained by fluorescence microscopy are superimposed on maximum
intensity projection images. These data demonstrate smooth
conduction throughout the heart with ventricular acceleration at 36 hpf
(A). Marked slowing at the AV junction can be seen at 48 hpf (B). Scale
bars: 25 ␮m in A; 50 ␮m in B. (C) Time course of onset of 2:1 or higher
grade AV block in response to atrial pacing (bars) or terfenadine (line)
expressed as a percentage of the embryos studied. (D,E) In situ
expression patterns of notch1b (D) and bmp4 (E) in wild-type embryos
at 48 hpf. Arrowheads indicate expression in the AV ring a dotted line
outlines the cardiac silhouette in D.
Fig. 2. Characteristics of retrograde conduction in the zebrafish
heart at 48 hpf. (A,B) Calcium activation maps for normal
anterograde cardiac cycle (A) and spontaneous premature ventricular
beat (B). Both anterograde and retrograde AV delay are evident as
compressed spacing of isochronal lines (100 msecond intervals). Scale
bars: 50 ␮m. (C) Contemporaneous recordings of atrial and ventricular
contraction using intensity-time plots from regions over the respective
chambers, during ventricular pacing at 48 hpf. The ladder diagram
depicts the resulting Wenckebach-type retrograde ventriculo-atrial
block.
DEVELOPMENT
Central conduction system development
Similarly, it is possible that the conduction block seen with atrial
pacing is a result of a prolonged refractory period, not in the AV ring,
but in the ventricular myocardium. To discriminate between these
two possibilities, we performed incremental ventricular pacing in
wild-type embryos at 48 hpf. We observed retrograde ventriculoatrial conduction block of a Wenckebach type (characterized by a
prolongation of ventriculo-atrial conduction times prior to the failure
of impulse propagation; Fig. 2C). The combination of anterograde
and retrograde block can only be explained by the presence of tissue
in the AV ring with a refractory period longer than that of the
chambers on either side. These results confirm the existence of
specialized conduction tissue in the AV junction.
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
Fig. 3. Endocardial signaling is required for the development of
AV conduction tissue. (A) Calcium activation map of a single
representative cardiac cycle in cloche mutant embryos at 48 hpf.
Isochronal lines (black) obtained by fluorescence microscopy are
superimposed on a maximum intensity projection of the same heart
and demonstrate the failure of AV conduction tissue development in
the absence of endocardium. (B) Lack of AV conduction block in cloche
embryos compared with wild-type siblings is demonstrated by atrial
pacing (white bars) and terfenadine exposure (gray bars). Inset of
contemporaneous recordings of atrial and ventricular contraction using
intensity-time plots from regions over the respective chambers in a
terfenadine treated cloche embryo demonstrating 1:1 AV conduction.
(C,D). In situ expression patterns of notch1b (C) and bmp4 (D) in cloche
embryos at 48 hpf. Despite overstaining in the brain there is no
evidence of notch1b signal in the heart (C). In D, the black arrowhead
indicates the location of the AV ring; the white arrowhead denotes
intense expression of bmp4 at the sinus venosus.
Development 133 (6)
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).
Blood flow and endothelin 1 are not required for
AV conduction development tissue
Given the requirements for blood flow in the development of the
peripheral conduction system, we determined the role of flow in the
development of central AV conduction tissue (Gourdie et al., 1998;
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 2monoxime to prevent cardiac contraction for the first 48 hours postfertilization (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
Fig. 4. Flow is not required for the development of AV
conduction tissue. (A) Time line of the experiments with silent heart
embryos. (B) Calcium imaging of silent heart embryo at 48 hpf.
Compressed spacing of isochronal lines (50 mseconds) in the AV ring
demonstrates physiological conduction delay. Scale bar: 50 ␮m.
(C) silent heart mutant embryos develop 2:1 block at 48 hpf in response
to terfenadine treatment, as demonstrated in this plot of atrial and
ventricular fluorescence intensity.
DEVELOPMENT
1128 RESEARCH ARTICLE
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.
Neuregulin signaling is necessary for
development of AV conduction tissue
We cloned the zebrafish neuregulin cDNA and characterized its
expression pattern in 48 hpf embryos. Zebrafish neuregulin expression
is strongest in neural tissue, but can also be seen in the heart with
accentuated expression in the AV ring (Fig. 6A) that is restricted to the
endocardium (Fig. 6A, inset). We designed a splice-acceptor
morpholino to target exon 6 of the neuregulin gene (Falls, 2003). 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
Fig. 5. Endothelin 1 is not required for AV conduction tissue
development. (A) Representative images of wild-type and endothelin
1 morphant fish. endothelin 1 morphants fail to develop structures
derived from the pharyngeal arches, including the mandible.
(B) endothelin 1 morphant fish develop normal AV conduction block on
treatment with terfenadine at 48 hpf. Inset of contemporaneous
recordings of atrial and ventricular contraction using intensity-time plots
from regions over the respective chambers in an endothelin 1 morphant
embryo demonstrating 1:1 AV conduction. (C) endothelin 1 morphant
fish develop a normal AV conduction delay as seen in this calcium
activation map of a single cardiac cycle at 48 hpf. Isochronal lines (50
mseconds) obtained by fluorescence microscopy are superimposed on a
maximum intensity projection image.
RESEARCH ARTICLE 1129
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 ␮M morpholino injection, and 100%
failing to develop 2:1 AV block at 100 ␮M. 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).
Notch signaling is required for the normal
formation of AV conduction tissue
In light of the importance of notch signaling in cell fate
determination, and of the localization of notch1b to AV
endocardium, we investigated the role of this isoform in the
development of AV conduction tissue. We targeted the splice donor
Fig. 6. Neuregulin is necessary for the induction of AV
conduction tissue. (A) Neuregulin is expressed widely in the
developing brain (seen here at 48 hpf) and in the AV ring endocardium
(arrowhead) from 36 hpf onwards. Inset shows a Hematoxylin and
Eosin stained section of the AV canal in cross section with neuregulin
staining (purple) in the AV ring endocardium. (B) neuregulin
knockdown embryos fail to develop an AV conduction delay, as shown
in this calcium activation map of a single cardiac cycle in a neuregulin
morphant at 48 hpf. Isochronal lines (50 mseconds) obtained by
fluorescence microscopy are superimposed on maximum intensity
projection image. (C) neuregulin morphant embryos fail to develop an
AV conduction block in the presence of terfenadine, as seen in
contemporaneous recordings of atrial and ventricular contraction.
(D) The morpholino effect is dose related, as seen in a histogram of the
proportion of embryos that develop 2:1 AV block in the presence of
terfenadine at 48 hpf. This proportion correlates with the amount of
neuregulin mRNA detectable by RT-PCR from the respective embryos.
DEVELOPMENT
Central conduction system development
of exon 27, which contains the intracellular signaling domain.
Calcium imaging in notch1b morphant embryos revealed a loss of
AV conduction slowing, as shown in Fig. 7A. At doses that knocked
down 69% of processed mRNA, 28% of embryos (30/108) failed to
develop 2:1 AV block on treatment with terfenadine (Fig. 7B).
Higher doses of morpholino were uniformly lethal, causing
developmental arrest in the first 24 hours post-fertilization. In order
to confirm the specificity of these results, a second non-overlapping
morpholino targeting the splice acceptor of exon 27 was designed.
This morpholino resulted in the failure of AV delay development in
injected embryos, both by calcium imaging (6 out of 10 injected
embryos) and by terfenadine treatment (25% of injected embryos
failed to develop 2:1 AV block, n=28; Fig. 7B). Failure of AV delay
development was never seen in any of the control embryos (n=50).
These results confirm a requirement for notch1b in the formation of
central conduction tissue. Because notch signaling is an important
determinant of cell fate, we considered whether its inhibition might
have more global effects on cardiac chamber specification. We
therefore examined known atrial and ventricular specific markers,
which demonstrated normal patterns of expression and excluded any
major effects on chamber fate (see Fig. S1 in the supplementary
material).
In order to determine the relationship between neuregulin and
notch in AV myocyte differentiation, we characterized notch1b
expression in neuregulin morphants. Cardiac notch1b expression is
normal in these fish (Fig. 7C). Interestingly, neuregulin expression
was also normal in notch 1b morphant embryos that failed to develop
Fig. 7. Notch is required for the induction of AV conduction
tissue. (A) notch1b knockdown embryos fail to develop an AV
conduction delay, as shown in this calcium activation map of a single
cardiac cycle in a notch1b morphant at 48 hpf. Isochronal lines (50
mseconds) obtained by fluorescence microscopy are superimposed on a
maximum intensity projection image. (B) notch1b morphants fail to
develop an AV conduction block on treatment with terfenadine, as
seen in this histogram of the proportion of embryos developing 2:1 AV
block at 48 hpf. Error bars indicate s.e.m. (C) neuregulin morphants
exhibit normal expression of notch1b, as seen in this in situ
hybridization. Arrowhead indicates cardiac expression in the AV ring.
(D) notch morphants exhibit normal expression of neuregulin, as seen
in this in situ hybridization. Arrowhead indicates cardiac expression in
the AV ring.
Development 133 (6)
2:1 AV block (Fig. 7D). The expression pattern of bmp4 was
unperturbed in affected neuregulin and notch1b morphants (data not
shown). These data suggest that there is no interaction between
notch1b and neuregulin at the mRNA level, but do not exclude posttranscriptional cross-talk between these pathways.
DISCUSSION
Several elegant genetic screens designed to identify the crucial
pathways in cardiogenesis have taken advantage of the zebrafish
(Fishman and Olson, 1997; 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.
DEVELOPMENT
1130 RESEARCH ARTICLE
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
RESEARCH ARTICLE 1131
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.
The authors would like to thank Drs Patrick Ellinor, Charles Hong, Jordan Shin,
Ashok Srinivasan and Jeremy Ruskin for their thoughtful comments and
suggestions. This work was funded by NIH grants HL076361 (D.J.M.),
GM075946 (C.A.M. and D.J.M.), HL065962 (C.A.M.) and HL079267 (R.T.P.),
and by a grant from the Cardiovascular Research Foundation (D.J.M.).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/6/1125/DC1
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