Identification and Functional Characterization of Cardiac Pacemaker Cells in Zebrafish
et al. (2012) Identification and Functional Characterization of Cardiac Pacemaker Cells in
Zebrafish. PLoS ONE 7(10): e47644. doi:10.1371/journal.pone.0047644
Identification and Functional Characterization of Cardiac Pacemaker Cells in Zebrafish
Federico Tessadori 0
Jan Hendrik van Weerd 0
Silja B. Burkhard 0
Arie O. Verkerk 0
Emma de Pater 0
Bastiaan J. Boukens 0
Aryan Vink 0
Vincent M. Christoffels 0
Jeroen Bakkers 0
Andrea Barbuti, University of Milan, Italy
0 1 Hubrecht Institute-KNAW, University Medical Centre Utrecht , Utrecht , The Netherlands , 2 Department of Anatomy, Embryology and Physiology, Heart Failure Research Center, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands , 3 Department of Pathology, University Medical Center Utrecht , Utrecht , The Netherlands , 4 Interuniversity Cardiology Institute of the Netherlands , Utrecht , The Netherlands
In the mammalian heart a conduction system of nodes and conducting cells generates and transduces the electrical signals evoking myocardial contractions. Specialized pacemaker cells initiating and controlling cardiac contraction rhythmicity are localized in an anatomically identifiable structure of myocardial origin, the sinus node. We previously showed that in mammalian embryos sinus node cells originate from cardiac progenitors expressing the transcription factors T-box transcription factor 3 (Tbx3) and Islet-1 (Isl1). Although cardiac development and function are strikingly conserved amongst animal classes, in lower vertebrates neither structural nor molecular distinguishable components of a conduction system have been identified, questioning its evolutionary origin. Here we show that zebrafish embryos lacking the LIM/ homeodomain-containing transcription factor Isl1 display heart rate defects related to pacemaker dysfunction. Moreover, 3D reconstructions of gene expression patterns in the embryonic and adult zebrafish heart led us to uncover a previously unidentified, Isl1-positive and Tbx2b-positive region in the myocardium at the junction of the sinus venosus and atrium. Through their long interconnecting cellular protrusions the identified Isl1-positive cells form a ring-shaped structure. In vivo labeling of the Isl1-positive cells by transgenic technology allowed their isolation and electrophysiological characterization, revealing their unique pacemaker activity. In conclusion we demonstrate that Isl1-expressing cells, organized as a ringshaped structure around the venous pole, hold the pacemaker function in the adult zebrafish heart. We have thereby identified an evolutionary conserved, structural and molecular distinguishable component of the cardiac conduction system in a lower vertebrate.
Funding: Work in J.Bakkers laboratory was supported by the Royal Dutch Academy of Arts and Sciences (KNAW) and the Netherlands Organization for Scientific
Research (NWO/ALW) grant 864.08.009. Work in V. Christoffels laboratory was supported by the European Commission under the FP7 Integrated Project
CardioGeNet (HEALTH-2007-B-223463) and by the Rembrandt Institute. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The cardiac conduction system comprises several components,
amongst which the sinus node, the site of electrical impulse
generation, and the Purkinje fibers transducing the impulse rapidly
through the myocardium . The sinus node harbors specialized
pacemaker cells which, due to regular and spontaneous membrane
depolarization, generate the electrical signal necessary to induce
cardiomyocyte contractions . Although the sinus node was
described histologically and functionally more than a century ago
, the molecular regulators required for pacemaker cell
differentiation and function are not fully understood. Nonetheless,
several recent developments have provided new insights. These
include identification of the embryonic origins of the sinus- and
atrioventricular nodes [4,5] and of several transcriptional
regulators involved in their development (reviewed in ). A major
advance for the field was the identification of T-box transcription
factor 3 (Tbx3) in pacemaker cells, and the subsequent
demonstration that it is required for sinus- and atrioventricular node
development and postnatal homeostasis [6,7]. Other
transcriptional regulators that have been identified for their role in sinus
node development are Nkx2.5, Tbx5, Pitx2 and Shox2 .
The molecular signature of the mouse sinus node primordium has
been confirmed in human embryonic hearts, indicating
evolutionary conservation of the developmental mechanism .
The LIM domain transcription factor Isl1 is expressed in the
mammalian cardiac progenitor cells [15,16]. Isl1 expression
gradually decreases during differentiation and is eventually lost
in mature cardiomyocytes [15,17,18], except for myocytes
pertaining to the sinus node [19,20]. Due to structural heart
defects and early lethality of mouse embryos deficient for Isl1, its
putative role in the developing and mature sinus node has
Although the presence of specialized conduction system
components in the heart of lower vertebrates has been suggested
by functional analysis , their identification has remained
elusive due to the lack of morphologically distinctive structures and
Our research presented in this manuscript resolved this issue by
describing the first molecular and structural identification of
specialized cardiac pacemaker cells in the embryonic and adult
zebrafish heart, utilizing a combination of in vivo microscopic
examination, 3D gene expression pattern reconstruction, reporter
transgenics and ex vivo electrophysiology. Our findings establish
that Islet-1 is required for pacemaker activity in the embryonic
heart and that Islet-1 marks the pacemaker cells in the adult heart,
which represents a previously unappreciated role for Isl1 in the
cardiac conduction system.
Results and Discussion
Cardiac pacemaker activity is affected in Islet-1 mutant
Zebrafish, embryos lacking functional Isl1 protein are immobile
and display reduced heart rate (bradycardia) at 2 days post
fertilization (dpf) . Unlike the mouse Isl1 mutant hearts that
fail to loop and lack recognizable chambers , zebrafish isl1
mutant hearts loop normally and are morphologically
indistinguishable from their wild-type siblings at 2 days post fertilization
(dpf), allowing their functional characterization. To identify the
primary defect responsible for the previously reported bradycardia
phenotype we further investigated the heart rhythm in isl1 mutant
embryos by high-speed video imaging combined with functional
image analysis. Both the isl1 mutant and wild-type sibling embryos
showed initiation of contraction in the inflow region (venous pole)
continuing in the atrium, followed by rapid contraction of the
ventricle (Movies S1 and S2). To quantify heart rhythm, we drew
kymographs for atrium and ventricle of sibling and mutant hearts
using a 1 pixel-thick collection of lines positioned perpendicularly
to the blood flow (Fig. 1A, white dotted lines). The resulting
kymographs (Fig.1B) readily confirmed the bradycardia of the isl1
mutant, as the distance between two dotted lines encompassing a
full cardiac cycle (Fig.1B, white double arrows) is much longer in
the mutant than in the sibling. Moreover, the kymograph-based
quantification of the cardiac cycle (interval necessary for one
complete contraction) illustrates the slow and irregular heart
rhythm in isl1 mutant embryos (Fig. 1C). Cardiac cycles of the
imaged isl1 mutants oscillated between about 450 ms and 800 ms,
showing not only high variability within a single embryo (Fig. 1C,
box-whisker plots for mutants; maximal and minimal values vary
of about 200 ms for all mutants analyzed), but also between
different individuals (Fig.1C, mutants 1,2 and 3 display average
cardiac cycle period times of 586 ms, 661 ms and 494 ms
respectively over the recording time). In comparison, siblings
displayed a maximal variation in cardiac cycle measured at the
atrium of about 20 ms and average cardiac cycle periods of
325 ms (Fig. 1C, Movie S1). We never observed uncoupling of the
atrial and ventricular contraction, fibrillation or atrioventricular
block, as every atrial contraction was followed by a ventricular
contraction (Fig. 1B; Movie S2).
As development proceeded, the severity of the heart beat
phenotype increased. We frequently observed a sinus block in isl1
mutant hearts at 34 dpf resulting in the absence of atrial and
ventricular contraction for 1020 seconds (Fig. 1D). Altogether,
the combination of phenotypes displayed by the isl1 mutant is
compatible with defective initiation of contraction, suggesting
faulty pacemaker activity.
Isl1 is expressed at the putative sinoatrial boundary of
the embryonic and adult heart
In mammals, including humans, the primary pacemaker
function is held by the sinus node, which resides at the junction
of the superior caval vein and right atrium . For example,
in mouse, the right sinus venosus was shown to form the sinus
node, which includes the venous lining of the right venous valve
[4,13]. Isl1-expressing cells are found in the developing and
mature mammalian sinus node [14,19,20].
We hypothesized that Isl1 expression marks the pacemaker
tissue in the zebrafish heart, as no molecular markers for the
zebrafish sinus node or the pacemaker cells within it have been
identified to date. Using an antibody recognizing both zebrafish
Isl1 and Isl2  we observed few Isl-positive cells in both the
dorsal and ventral regions of the proposed sinoatrial junction at 2
dpf (Fig. 2A, B, C, D), consistent with the proposed pacemaker
function at the sinoatrial junction in the zebrafish heart . The
Isl-expressing cells expressed Tg(myl7:eGFP), which marks
cardiomyoctes. Isl expression at the sinoatrial junction was detected
continuously during development and was maintained in the same
region in the adult zebrafish heart (Fig. 2E, F, G, H). This
indicates that Isl expression in the zebrafish proposed sinoatrial
junction is constant between embryonic stages and adulthood. We
continued by molecular characterization of the proposed sinoatrial
junction region in the adult heart using in situ hybridization (ISH).
Contrary to what has been observed in mammals, the zebrafish
sinus venosus did not express the myocardial marker myl7.
Consequently, the upstream border of myocardium muscle was
at the venous valves and atrium (Fig. 3A, B). We observed that isl1
expression was confined to the myocardium located at the base of
the venous valves (Fig. 3C). The zebrafish ortologue of hcn4
encodes a member of the family of ion channels responsible for the
hyperpolarization-activated current, If, in pacemaker cells. Its
expression is enriched in pacemaker tissue of mammalian hearts
[28,3033]. In the zebrafish adult heart hcn4 had a broader
expression pattern than isl1 (Fig. 3D), similar to observations made
in the mammalian embryonic heart [13,14]. Expression of the two
genes overlapped at the sinoatrial junction (Fig. 3D). A 3D
reconstruction based on a series of ISH on sagittal adult heart
sections revealed that isl1 expression is confined to a ring-like
structure within the myocardial tissue at the base of the venous
valves, around the proposed sinoatrial junction (Fig. 3F). In the
mammalian heart expression of Nppa is specific for the fast
conducting working myocardium of the atrium and ventricle but is
not expressed in the slow conducting primitive myocardium of the
pacemaker tissue at the sinoatrial junction . We observed a
very similar mutually exclusive expression pattern of nppa and isl1
at the proposed sinoatrial junction of the zebrafish heart (Fig. 3E).
Furthermore, since bmp4 and tbx2b are expressed at the venous
pole of the embryonic heart ( and Figure S1) and since the
bmp4-tbx2b regulatory axis suppresses chamber differentiation
and nppa expression, we analyzed their expression in the adult
heart. Interestingly, expression of bmp4 and tbx2b was maintained
in the myocardium of the proposed sinoatrial junction of the adult
heart and restricted to the base of the venous valves or the entire
venous valve tissue, respectively (Fig. 3G, H). In summary, we
identified an isl1+cell population in the hcn4+tbx2b+nppa-negative
myocardium that is organized in a ring-shape at the proposed
sinoatrial junction. Together with the above-described observation
that isl1 mutants display irregular heart rhythms, this suggests that
isl1 expression identifies cardiac pacemaker cells in the zebrafish
Using an Isl1-LacZ knock-in model, Isl1/LacZ activity was
observed in the sinus node of the adult mouse heart .
Corroborating these findings we detected endogenous Isl1
expressing cells in the adult sinus node of the mouse heart using
an anti-Isl antibody (Figure S2). Using a similar approach we also
detected Isl1 expressing cells in the sinus node of the adult human
Figure 1. Characterization of the embryonic Isl12/2 cardiac phenotype in vivo. (A) Zebrafish embryonic heart at 2 dpf. The embryonic heart
is highlighted in black dotted contour; white dotted lines through the atrium (A) and the ventricle (V) are placed at kymograph positions. (B) Atrial (A)
and ventricular (V) kymographs from 2 dpf embryonic hearts spanning approximately 2.8 s. Note the much longer period of the Isl2/2 heart when
compared to the sibling and the irregularity of the period (double arrow and white dotted vertical lines). Movies are available as Movies S1 and S2,
respectively. (C) Box-whisker plots representation of 20 successive heartbeats of 2 dpf Isl12/2 and sibling embryos. (D) Kymograph recorded at 3 dpf
covering a period of about 16 s of absent heart contractions. For all panels a: atrium; v: ventricle.
Figure 2. Isl1 expression in the embryonic and adult zebrafish heart. Single confocal scans of a fluorescent antibody labeling of Isl1 and
eGFP in embryonic (2 dpf) (AD) and adult (EH) zebrafish expressing Tg(myl7:eGFP) in all cardiomyocytes. GFP+ cardiomyocytes are displayed in
grey (B, F) and in green in (D, H). Isl1 is shown in grey (C, G) and in red (D, H). Arrowheads indicate Isl+/GFP+ cells. Illustrations of a lateral view of a 2
dpf (A) and adult (E) zebrafish heart indicate the location of Isl1+ cells (red). The box in panel E represents the area shown in (FH). (BD) Fluorescent
immunolabeling of Isl1 and eGFP in a 2 dpf embryo (sagittal section 100 mm). At this time point Isl1+/GFP+ cells were only found in the IFT of the
heart. (FH) Fluorescent immunolabeling of Isl1 and eGFP in an adult zebrafish heart (sagittal section 100 mm). Isl1+/GFP+ cells are located at the
junction of the sinus venosus and atrium in the inflow region of the heart (arrowheads). Isl1+ cells showed low expression of myl7. v, ventricle; a,
atrium; oft, outflow tract; ift, inflow tract; ba, bulbus arteriosus; sv, sinus venosus; a, anterior; p, posterior; d, dorsal; v, ventral. Scale bars represent
heart (Figure S3). Interestingly, we observed that only a
subpopulation of sinus node cells expresses Isl1, suggesting that the
Isl1 expressing cells have different properties compared to the
isl1negative sinus node cells.
Isl1-expressing cells display electrical pacemaker activity
To functionally characterize the Isl1+ cells in the zebrafish
heart, we generated an Isl1-GFP reporter line using the binary
Gal4/UAS expression system (Figure S4). To validate the reporter
line we confirmed that all GFP+ cells were co-labeled by Isl1
immunostaining (Fig. 3I, J, K arrowheads). In vibratome sections
GFP+ cells visualized by fluorescent antibody labeling were
located in bilateral cell populations at the proposed sinoatrial
junction (Fig. 3I, J, K, arrows), conform the ISH staining.
Neighboring GFP+/Isl1+ cells displayed cytoplasmic protrusions,
which may connect them between each other (Fig. 3K,
arrowheads). 3D reconstruction of confocal image stacks revealed that
Isl1+ pacemaker cells are interspersed with Isl-negative cells.
However, they form a coherent structure (Fig. 3L). It is known that
isolated groups of cells with residual pacemaker activity, such as
remnant embryonic nodal atrioventricular canal myocytes, may
cause cardiac arrhythmias (reviewed in [34,35]). The cell-to-cell
interconnection could therefore be an essential feature for proper
pacemaker function, likely coordinating a synchronous activation
of the myocardium. Moreover, the expression of myosin is low in
Isl+ cells, which is supportive for a primitive myocardial identity of
these cells, typical for pacemaker cells (Figure S5) [27,36,37].
To elucidate whether the GFP+ cells localize to the region in
which the electrical activation is initiated, we performed optical
mapping of epicardial activation patterns on adult zebrafish
hearts. First moment of atrial activation corresponded with the
localization of the GFP+ cells (Fig. 4A, B, C), which is compatible
with pacemaker function for isl1 expressing cells.
To unequivocally discriminate whether indeed isl1 expression
marks cells with pacemaker activity, dissociated single GFP+ and
GFP- cells from micro-dissected sinoatrial tissue were
patchclamped (Fig.4D). All measured GFP+ cells (n = 6) were
spontaneously active, while all measured GFP- cells (n = 8) were typically
quiescent. In GFP- cells, action potentials could be elicited by
current pulses through the patch pipette. Figure 4D shows typical
spontaneous action potentials of a GFP+ cell as well as typical
action potentials recorded from a GFP- cell that was stimulated at
3 Hz, i.e. with a cycle length similar close to that of the GFP+ cell
(Table 1). All parameters, except for action potential duration at
90% repolarization (APD90), differed significantly between both
cell types (Table 1). GFP- cells had a stable resting membrane
potential of -79.061.6 mV, while GFP+ cells showed a
spontaneous diastolic depolarization rate (64617 mV/s) with a
maximum diastolic potential of 265.063.0 mV. In GFP+ cells, this
diastolic depolarization resulted in pacemaker activity with an
intrinsic cycle length of 378658 ms. In GFP+ cells, the maximum
upstroke velocity was typically low (7.462.6 V/s) as opposed to
GFP- cells (112614 V/s). In both cell types, action potentials
overshot the zero potential value, but the action potential
amplitude was higher in GFP- cells. Action potentials of
GFPcells repolarized earlier and faster, resulting in shorter APD20 and
APD50. Thus, the results obtained on the optical mapping and
patch-clamp experiments revealed that while GFP- cells display
characteristics specific to cardiac chamber myocytes, pacemaker
activity resides in Isl+/GFP+ cells.
We present here the molecular and functional identification of
cardiac pacemaker cells in the embryonic and adult zebrafish
heart. The embryonic cardiac expression pattern (Fig.2) and
knockout phenotype of Isl1 in zebrafish (Fig.1) hinted at a role for
this gene in pacemaker function. Indeed, analysis of 3D
reconstructions of expression pattern and reporter transgenics in
adult fish showed a previously unidentified ring-shaped region of
Isl1 expression within the myocardium at the proposed sinoatrial
junction of the mature zebrafish heart (Fig.3). Optical mapping of
the activation sequence and electrophysiological characterization
of single Isl1+ myocytes demonstrated the presence of pacemaker
activity in the Isl1-expressing cells (Fig. 4). Altogether, our data
allow us to establish that (1) Isl1 is the first identified molecular
marker for pacemaker cells in the zebrafish heart, (2) the
functional pacemaker of the adult zebrafish heart is organized as
a ring around the venous pole and (3) expression of Isl1 in the
pacemaker cells of the adult heart is conserved from fish to human.
Cells pertaining to the primary pacemaker structure in zebrafish
and mammals share a number of molecular markers, relative
Figure 4. Isl1 cells have pacemaker activity. (AC) Optical mapping on an explanted, contracting adult zebrafish tg(isl1BAC:GalFF; UAS:GFP)
heart. Arrow indicates the sinus venosus in all panels. (A) Explanted adult zebrafish heart. (B) GFP-fluorescent cells reporting Isl1 expression are
situated at the sinus venosus. (C) The activation pattern measured by di-4-ANEPPS fluorescence shows that the GFP+ myocytes are situated in the
area of earliest activation. (D) Typical action potentials of freshly isolated GFP+ and GFP2 myocytes. The GFP2 cell was stimulated at 3 Hz. The inset
displays a representative example of a GFP+ myocyte.
GFP+ cells (n = 6)
GFP2 cells (n = 8)
Data are mean6SEM; n = number of cells, MDP = maximal diastolic potential,
DDR50 = diastolic depolarization rate over the 50-ms time interval starting at
MDP+1 mV, dV/dtmax = maximal upstroke velocity, APA = action potential
amplitude, APD20, APD50, and APD90 = action potential duration at 20, 50, and
90% repolarization. *p,0.05.
position in the heart, and functional identity. However, while in
zebrafish the Isl1-expressing pacemaker cells are few and
organized in a ring-shaped structure of interconnected cells in
the venous valves, the mammalian sinus node is a more compact,
spindle-shaped (also referred to as comma-shaped) clearly
demarcated structure. Although at this stage we do not know whether the
ring-shaped pacemaker can be extended to other lower
vertebrates, the diverging and unpronounced structure of the zebrafish
sinus node and the absence of molecular markers until now may
explain why a pacemaker structure in lower vertebrates has not
previously been identified.
Finally, the primitive myocardial identity of Isl1+ cells in adult
zebrafish is intriguingly accompanied by a capacity to
spontaneously depolarize, which is absent in working cardiomyocytes.
Future work focusing on Isl1 and its gene targets will help to
elucidate whether Isl1 expression in the pacemaker cells is
necessary to maintain the primitive myocardial fate of these cells,
similarly to its suggested role in cardiac progenitor cells
[15,17,18,38]. Alternatively, Isl1 could be required to drive a
pacemaker gene program, in a similar fashion to what was shown
for Tbx3 .
Materials and Methods
All animal and human work conformed to ethical guidelines
and was approved by the relevant
local animal ethics committees.
Human Tissue Samples
The study met the criteria of the code of proper use human
tissue that is used in the Netherlands for the use of human tissue.
The study was approved by the scientific advisory board of the
biobank of the University Medical Center Utrecht (RP2012-03).
Fish used in this study were kept in standard conditions as
previously described . The Tg(myl7:gfp) and isl1 mutant line
isl1K88X (isl1SA0029) were described previously [24,40].
Generation of the Tg(Isl1BAC:GalFF; UAS:GFP) is described in more detail
below. All animal and human work conformed to ethical
guidelines and was approved by the relevant local animal ethics
Generation of the tg(isl1BAC:Gal4ff) transgenic line
Recombineering of BAC clone CH211-219F7 was performed
following the manufacturers protocol with minor modifications, as
described in . Primers used were: isl1_Gal4FF_F
BAC DNA was injected at 300 ng/ml in the presence of 0.75U
PI-SceI meganuclease (New England Biolabs) into Tg(UAS:GFP)
embryos . Healthy embryos displaying robust isl1-specific
fluorescence were grown to adulthood. Tg(Isl1BAC:GalFF;
UAS:RFP) were obtained by outcross to a tg(UAS:RFP) line .
High-speed imaging and analysis
2 Dpf and 4 dpf isl1K88X mutant and sibling embryos were
mounted in 0.25% agarose (Life Technologies BV) prepared in E3
medium embryonic medium with 16 mg/ml 3-amino benzoic acid
ethylester. Embryonic hearts were imaged with a Hamamatsu
C9300-221 high speed CCD camera (Hamamatsu Photonics,
Hamamatsu City, Japan) at 150 fps mounted on a Leica AF7000
microscope (Leica Microsystems GmbH, Wetzlar, Germany) in a
controlled temperature chamber (28.5uC) using Hokawo 2.1
imaging software (Hamamatsu Photonics GmbH, Herrsching am
Ammersee, Germany). Image analysis was carried out with ImageJ
(http://rsbweb.nih.gov/ij/). Statistical analysis and drawing of the
box-whisker plot were carried out in Excel 2007 (Microsoft,
Redmond, WA, USA).
In situ hybridisation and immunohistochemistry
ISH on embryos was carried out as previously described .
ISH on adult heart tissue was carried out as previously described
 with minor modifications. 3D reconstructions of serial
ISHlabeled sections were performed as described in .
Immunohistochemistry was carried out as previously described .
Embryos and adult hearts for immunocytochemistry were fixed in
2% paraformaldehyde, embedded in 3% agarose/1% gelatine and
sectioned at 100 mm thickness. The primary antibodies used were
mouse anti-Isl1 (Developmental Studies Hybridoma Bank, Iowa
City, IA, USA, clone 39.4D5, 1:100), mouse-anti-tropomyosin
(Sigma-Aldrich, Zwijndrecht, the Netherlands, Cat. No. T9283,
1:100), rabbit anti-GFP (Torrey Pines Biolabs Inc., Secaucus, NJ,
USA, Cat. No. TP401, 1:200) and rabbit-anti-DsRed (Clontech
Laboratories Inc., Mountain View, CA, USA, Cat No. 632496,
Hearts from adult tg(isl1:GalFF; UAS:GFP) fish were excised and
incubated in 10 ml Ringers solution (composition in mM: NaCl
115, Tris 5, NaH2PO4 1, KCl 2.5, MgSO4 1, CaCl2 1.5, glucose
5, pH adjusted to 7.2 with HCl) containing 15 mM Di-4 ANEPPS
for 5 minutes and placed in an inverted microscope. Excitation
light was provided by a 5-Watt power LED (filtered 510620 nm).
Fluorescence (filtered.610 nm) was transmitted through a
tandem lens system on CMOS sensor (1006100 elements,
MICAM Ultima, SciMedia, Costa Mesa, CA, USA). Activation
patterns were measured during the sinus rhythm. Optical action
potentials were then analyzed with custom-made software.
Single cell preparation
Single cells were isolated from the sinoatrial node and atria as
described previously . Sinoatrial node regions were excised
from 55 adult tg(Isl1BAC:GalFF; UAS:GFP) zebrafishes and stored
in Tyrodes solution at RT, containing (in mM): NaCl 140, KCl
5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, and HEPES 5.0; pH was
set to 7.4 with NaOH. They were then transferred to Ca2+-free
Tyrodes solution (30uC), i.e., Tyrodes solution with 10 mM
CaCl2, which was refreshed two times before the addition liberase
IV (0.250.29 U/ml; Roche, Indianapolis, IN, USA) and elastase
(2.40.7 U/mL; Serva, Heidelberg, Germany) for 1215 min.
The final 6 min also contained pronase E (0.92 U/mL; Serva).
During the incubation period, the tissue was triturated through a
pipette (tip diameter: 2.0 mm). The dissociation was stopped by
transferring the strips into a modified Kraft-Bru he solution (30uC)
containing (in mM KCl 85, K2HPO4 30, MgSO4 5.0, glucose 20,
pyruvic acid 5.0, creatine 5.0, taurine 30, b-hydroxybutyric acid
5.0, succinic acid 5.0, BSA 1%, Na2ATP 2.0; pH was set to 6.9
with KOH. The tissue was triturated (pipette tip diameter:
0.8 mm) in Kraft-Bru he solution (30uC) for 4 min to obtain single
cells, which were stored at RT for 30 min in modified Kraft-Bru he
solution before patch-clamping. Cells were allowed to adhere for
5 min after which superfusion with Tyrodes solution
(28.560.2uC) was started.
Patch clamp experiments
Action potentials were recorded by the amphotericin-perforated
patch-clamp technique using an Axopatch 200B amplifier
(Molecular Devices, Sunnyvale, CA, USA). Action potentials of
GFP+ cells were low-pass filtered (cut-off frequency 5 kHz) and
digitized at 10 kHz; action potentials of GFP- cells at 5 kHz and
20 kHz, respectively. Potentials were corrected for the estimated
liquid junction potential . Data acquisition and analysis were
accomplished using custom software. Patch pipettes (borosilicate
glass; resistance 34 MV) were heat polished and filled with
pipette solution containing (in mM): K-gluc 125, KCl 20, NaCl 10,
amphotericin-B 0.22, and HEPES 10; pH was set to 7.2 with
KOH. Action potentials in GFP-negative cells were elicited at
3 Hz by 3-ms, <1.2x threshold current pulses through the patch
pipette. Parameter values obtained from 10 consecutive action
potentials were averaged.
Figure S1 Expression of tbx2b at the venous pole in
embryonic heart. Expression patterns by mRNA in situ
hybridization of tbx2b and nppa in 2 dpf embryos. Expression of
tbx2b at the venous pole (blue staining indicated with arrowheads)
does not overlap with nppa expression (red staining), which is
confined to atrium and ventricle chamber myocardium. Pictures
shown are ventral views with anterior to the top.
Figure S2 Isl1 expression in the sinus node of the adult
mouse heart. (A) 4-chamber view of section through adult
wildtype mouse heart. Boxed region indicates the region shown
enlarged in (B). (B) Expression of Isl1, depicted in red, colocalizes
with the expression of the sinus node marker Hcn4, depicted in
green. Dotted line in (B) demarcates the sinus node. ao, aorta; la,
left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle;
scv, superior caval vein.
Figure S3 Immunohistochemical detection of Islet-1 in
human cardiomyocytes in the sinoatrial node. (A)
Hematoxylin and eosin staining of the sinoatrial node. SN
indicates the area of the node showing where the specialized
cardiomyocytes are located. MC indicates myocardium adjacent
to the node. NA indicates the nodal artery. The boundary between
SN and MC is highlighted by the dotted line. Scale bar represents
400 mm. (B) Elastic van Giesen stain of a consecutive section of (A)
illustrating that the cardiomyocytes are embedded within collagen
and elastic tissue. Scale bar represents 400 mm. (C) Islet-1
immunostain of sinoatrial node. SN indicates sinoatrial node.
MC indicates myocardium adjacent to the node. The boundary
between SN and MC is highlighted by the dotted line. Scale bar
represents 160 mm. (D) Magnification of the boxed region in (C).
Islet-1 immunostain with positive brown staining of the nuclei of
the cardiomyocytes. On average 5% of the cardiomyocytes in the
sinoatrial node revealed a positive signal. Scale bar represents
40 mm. (E) Staining is absent in the myocardium next to the
sinoatrial node. Scale bar represents 80 mm.
Figure S4 Generation of a reporter transgenic line for
isl1. (A) An expression cassette containing the GalFF gene 
and kanamycin resistance gene was inserted by recombineering
into BAC CH211-219F7 at the ATG site of the 1st exon of the isl1
gene. The site of recombineering is approximately 40 kb inside the
BAC sequence, minimizing any risk of loss of isl1 regulatory
sequences. The recombined BAC was then injected in a
tg(UAS:GFP) background  to obtain the fluorescent Isl1
expression reporter line Tg(Isl1BAC:GalFF; UAS:GFP). (B)
GFP expression pattern of the Tg(Isl1BAC:GalFF; UAS:GFP)
line at 3 dpf. (C) Isl1 ISH on WT embryo at 3 dpf. The expression
pattern of GFP, reporting for isl1 expression, in the
Tg(isl1BAC:GalFF; UAS:GFP) is validated by comparison with the isl1 ISH.
Especially visible are the identical expression pattern in the eyes
Figure S5 Isl1BAC reporter activity in adult heart.
Confocal images of Tg(isl1BAC:GalFF; UAS:RFP; myl7:eGFP)
after immunolabeling with anti-RFP and anti-GFP antibodies (A),
or Tg(isl1BAC:GalFF; UAS:GFP) after immunolabeling with
antiGFP and antitropomyosin antibodies (B). Isl1 expressing cells
(indicated with arrows) are located at the base of the venous valves
and contain much lower levels of myosin light chain or
tropomyosin compared to surrounding myocardial cells. Axonal
Isl1/GFP+structures are visible at the outer surface of the
myocardium. Scale bars represent 50 mm
Movie S1 Recording of the heart beating of an isl1
sibling embryo at 2 dpf. Ventral view (anterior to the left).
Note the speed and regularity of the contractions (also see Fig.1B).
Playback speed (150 fps) is set to match recording speed to render
natural speed of the embryos heart beat.
Movie S2 Recording of the heart beating of an isl12/2
embryo at 2 dpf. Ventral view (anterior to the left). Note the
reduced speed of the contractions and their irregularity (also see
Fig.1B). Playback speed (150 fps) is set to match recording speed to
render natural speed of the embryos heart beat.
We would like to thank K. Kawakami for providing the galFF plasmid and
the Tg(UAS:GFP) and Tg(UAS:RFP) fish. The Islet-1 homeobox monoclonal
antibody 39.4D5 developed by Jessell, T.M./Brenner-Morton, S. was
obtained from the Developmental Studies Hybridoma Bank developed
under the auspices of the NICHD and maintained by The University of
Iowa, Department of Biology, Iowa City, IA 52242.
Conceived and designed the experiments: FT JHvW SBB AOV EdP VMC
JB. Performed the experiments: FT JHvW SBB AOV EdP BJB AV.
Analyzed the data: FT JHvW SBB AOV EdP BJB AV VMC JB.
Contributed reagents/materials/analysis tools: AOV AV. Wrote the paper:
FT SBB JHvW VMC JB.
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