Identifying the Evolutionary Building Blocks of the Cardiac Conduction System
et al. (2012) Identifying the Evolutionary Building Blocks of the Cardiac Conduction
System. PLoS ONE 7(9): e44231. doi:10.1371/journal.pone.0044231
Identifying the Evolutionary Building Blocks of the Cardiac Conduction System
Bjarke Jensen 0
Bastiaan J. D. Boukens 0
Alex V. Postma 0
Quinn D. Gunst 0
Maurice J. B. van den Hoff 0
Antoon F. M. Moorman 0
Tobias Wang 0
Vincent M. Christoffels 0
Marie Jose Goumans, Leiden University Medical Center, The Netherlands
0 1 Department of Anatomy, Embryology & Physiology, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands , 2 Department of Biological Sciences , Zoophysiology , Aarhus University , Aarhus , Denmark
The endothermic state of mammals and birds requires high heart rates to accommodate the high rates of oxygen consumption. These high heart rates are driven by very similar conduction systems consisting of an atrioventricular node that slows the electrical impulse and a His-Purkinje system that efficiently activates the ventricular chambers. While ectothermic vertebrates have similar contraction patterns, they do not possess anatomical evidence for a conduction system. This lack amongst extant ectotherms is surprising because mammals and birds evolved independently from reptilelike ancestors. Using conserved genetic markers, we found that the conduction system design of lizard (Anolis carolinensis and A. sagrei), frog (Xenopus laevis) and zebrafish (Danio rerio) adults is strikingly similar to that of embryos of mammals (mouse Mus musculus, and man) and chicken (Gallus gallus). Thus, in ectothermic adults, the slow conducting atrioventricular canal muscle is present, no fibrous insulating plane is formed, and the spongy ventricle serves the dual purpose of conduction and contraction. Optical mapping showed base-to-apex activation of the ventricles of the ectothermic animals, similar to the activation pattern of mammalian and avian embryonic ventricles and to the His-Purkinje systems of the formed hearts. Mammalian and avian ventricles uniquely develop thick compact walls and septum and, hence, form a discrete ventricular conduction system from the embryonic spongy ventricle. Our study uncovers the evolutionary building plan of heart and indicates that the building blocks of the conduction system of adult ectothermic vertebrates and embryos of endotherms are similar.
Funding: This work was supported by grants from the European Communitys Seventh Framework Programme contract (CardioGeNet 223463 to VMC) and the
Netherlands Heart Foundation (NHS 2008B062 to VMC). Tobias Wang and Bjarke Jensen were supported by The Danish Council for Independent Research |
Natural Sciences. 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.
The hearts of mammals and birds maintain high rates of
contraction  that in concert with high systemic blood pressures
accommodate their high rates of oxygen consumption due to their
endothermic state . The high heart rates, the timing of
sequential atrial and ventricular contractions and the rapid spread
of the activating impulse over the avian and mammalian ventricles
are possible because of a specialized cardiac conduction system
. However, while the sequential activation of the cardiac
chambers and appropriate matching of the atrial and ventricular
contractions are similar across all vertebrate groups, there is no
anatomical evidence for a specialized conduction system in hearts
of reptiles or other ectothermic vertebrates [4,5]. Because
mammals and birds evolved independently from reptilian
ancestors, the evolutionary origin of their specialized conduction
systems has remained unclear; either their conduction systems
evolved independently or primordial components of the system
were already present in the ancestral reptiles (Fig. 1).
Like the hearts of ectothermic vertebrates, embryonic
mammalian and avian hearts also exhibit regulated sequential activation
patterns in the absence of a morphological conduction system
[2,6,7]. This suggests that the functional components for
conduction system are established early in development and in
evolution, but are not represented by anatomically distinguishable
components as in the mature hearts of endothermic vertebrates.
Instead, the components may reflect an intrinsic part of the
building plan of the heart.
Tbx2 and Tbx3 belong to an ancient family of transcription
factors  expressed in the embryonic atrioventricular canal from
human to primitive fish [3,9]. In the embryo, the atrioventricular
canal delays the impulse from atrium to ventricle. Tbx2/3
suppresses chamber genes including Nppa and Gja5, encodes for
connexin40 that is required for fast conduction , and hence
inhibits differentiation of the atrioventricular canal to
fastconducting chamber myocardium [3,11]. Tbx3 remains expressed
in the mature conduction system components of mammals,
including the atrioventricular node that derives from the
atrioventricular canal . Bmp2/4 are expressed in the
atrioventricular canal of early embryonic mammals, birds and fish, and are
crucial for activation of Tbx2/3 (Fig. S1) . Hence, Tbx2/3,
Bmp2/4 and Gja5/Nppa represent evolutionary conserved positive
and negative markers, respectively, that discriminate the
embryonic slow-conducting atrioventricular myocardium and
fastconducting chamber myocardium. The ventricular His-Purkinje
system of mammals and birds is specifically marked by expression
of Gja5 (and Nppa in mammals) [3,10].
Here, we carried out in situ hybridization analysis using
evolutionary conserved genetic markers and provide a
threedimensional reconstruction of the key components of the
conduction system. The ventricular conduction pattern was
visualized using optical imaging of activation. The cardiac
expression and conduction patterns of a reptile were then
compared to those of mammals, chicken, and other ectothermic
vertebrates, frog and fish. We find an anatomic, genetic and
physiologic conserved building plan where hearts of adult
ectothermic vertebrates are similar to embryos of the endothermic
mammals and birds. This indicates that primordial components of
the cardiac conduction system were present in the ancestral
Figure 3. The phenotype of the slow propagating atrioventricular canal is evolutionary conserved. Numbers in the phylogenetic tree
indicate time in millions of years since major splits in tetrapod evolution. (AE) The hearts of mature ectotherms (blue) and embryonic endotherms
(red) maintain complete muscular connection in the atrioventricular canal (avc). (FJ) Markers of fast propagating chamber myocardium (Nppa and
Gja5) are absent from the atrioventricular canal (arrowheads). Note that the specimen in H is contracted, obscuring the spongy design otherwise
visible. Scale bars in (AE), 300 mm; (FJ), 100 mm.
Materials and Methods
All experimental procedures on adult material complied with
national and institutional guidelines and were approved by
Institutional Animal Care and Use Committee of the University
of Amsterdam. The approval is registered as DAE101617 for
optical mapping of the ectotherms and DAE101532 for optical
mapping of developing mice. In The Netherlands experiments
Figure 4. The developmental gene programme of amniotes is maintained in the mature heart of Anolis. (AD) Stage 17/19 Anolis hearts
show complementary expressions of Bmp2 and Tbx3 to Gja5 in the developing myocardial atrioventricular canal (arrowheads). (EH) The
developmental expression of Bmp2, Tbx3 and Gja5 is maintained in the mature myocardial atrioventricular canal (left side shown). la, left atrium; ra,
right atrium; ven, ventricle. Scale bars are 100 mm.
with non-mammalian embryos (that are not autonomously viable)
do not require approval from the Institutional Animal Care and
Adult zebrafish were provided by the Hubrecht Laboratory,
Utrecht, the Netherlands, and adult Xenopus laevis from Leiden
University, the Netherlands. Mice and Xenopus laevis embryos were
raised in the AMC. Green and brown anole (Anolis carolinensis and
A. sagrei) eggs and adults and fertilized chicken eggs, were bought
commercially in the Netherlands. Xenopus embryos were staged
according to Nieuwkoop , Anolis embryos according to Sanger
et al. , chicken according to Hamburger and Hamilton 
and mice from days post coitus.
Optical mapping was performed at 25uC in the ectothermic
vertebrates and we used specific ringer solutions for zebrafish and
Xenopus (in mmol/l: NaCl 115, Tris 5, NaH2PO4 1, KCl 2.5,
MgSO4 1, CaCl2 1.5, Glucose 5, pH adjusted to 7.2 with HCl) and
Anolis (adopted from ; in mmol/l: NaCl 95, Tris 5, NaH2PO4
1, KCl 2.5, MgSO4 1, CaCl2 1.5, Glucose 5, pH adjusted to 7.5
with HCl). For embryonic mouse hearts we used Tyrodes solution
at 37uC (in mmol/l: NaCl 128, KCl 4.7, CaCl2 1.45, MgCl2 0.6,
NaHCO3 27, NaH2PO4 0.4, Glucose 11[pH maintained at 7.4 by
equilibration with a mixture of 95% O2 and 5% CO2]). Excised
hearts from sedated animals were incubated in the specific solution
containing 15 mmol/l di-4-ANEPPS (voltage sensitive). 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). Activation patterns were measured during sinus
rhythm. Optical action potentials were analyzed with custom
In situ Hybridization
All embryos and hearts were fixed in 4% paraformaldehyde
for one day and then kept in 70% ethanol until imbedding in
paraffin and then sectioned at 712 mm for in situ hybridization.
Methodology of the non-radioactive in situ hybridization analysis
has been described previously [22,23] and so has probes for
Zebrafish , Xenopus [24,25], chicken  and mouse
. Probes for Anolis were made in house based on the
following coordinates using UCSC Genome Browser on Lizard
May 2010 (Broad AnoCar2.0/anoCar2) Assembly; Tnnt2
(chr4:131,362,830131,375,015), Bmp2 (chr1:136,434,179
136,445,741), Gja5 (chr3:162,064,640162,065,729), Tbx3
(chrUn_GL343338:1,255,3541,255,812), Tbx2 (regarded as
Tbx3, chrLGb:3,261,7913,267,757), Tbx5
(chrUn_GL343338:1,127,7241,145,671). Briefly, we generated
Figure 5. Three-dimensional reconstructions of Tbx3 (yellow) expression in amniotes reveal a shared design. Reconstructions are
based on in-situ hybridizations of serial sections, except in human (based on immunohistochemistry, modified from [57,58]). The Tbx3 domains are
strikingly similar in the early phases of chamber formation (upper panel). The Tbx3 expression of the Anolis ventricle is very similar to that associated
with ventricular septation (black arrows) in the other amniotes (lower panel).
a cDNA library with standard TRIzol RNA extractions 
from freeze-fixed specimens of developmental stages 59 and
GOI cDNA were obtained by PCR amplification and cloned
into pBluescript SK_ (Stratagene, La Jolla, CA).
Digoxigeninelabeled antisense mRNA were then produced by in vitro
transcription according to the manufacturers instructions
(Roche, Mannheim, Germany).
7, 10 and 12 mm serial sections were stained by in situ
hybridization and 3D reconstructions were performed as described
previously using AmiraH version 5.2 software . The interactive
3D pdfs were created using Adobe Acrobat Pro ExtendedH version
9.3. The 3D pdf can be viewed with the freeware version: Adobe
Results and Discussion
In adult lizards, the sequential chamber contractions and an
atrioventricular delay are well-established , but we found no
insulating plane or insulated atrioventricular node in Anolis (Fig. 2).
Instead, the atrioventricular canal was entirely myocardial (Fig. 3).
This differs from the adult hearts of mammals and birds, where the
atrioventricular myocardium has largely disappeared and an
insulating plane of fibrous-fatty tissue has ingressed between the
atria and ventricles except at the atrioventricular node and His
bundle, which provide the sole electrical communication between
the atria and the ventricles [12,34,35]. To explain the
atrioventricular delay in reptiles, we hypothesized, therefore, that the
atrioventricular canal of Anolis has a molecular phenotype that
differs from that of the chambers.
In embryos of mammals, birds, frog and fish, Nppa and/or Gja5
mark the rapid propagating atrial and ventricular chamber
myocardium, whereas the atrioventricular canal is negative for
these markers [3,15] (Nppa is lost in birds and all reptiles except
turtles [36,37]). Focusing on the atrioventricular gene programme
during Anolis development, we found Tbx3 and Bmp2 to be
expressed in the developing atrioventricular canal myocardium,
exactly complementary to Gja5 in the adjacent chambers (Fig. 4A
D). Tbx5, known to promote differentiation into Nppa- and
Gja5expressing chamber myocardium , was also present in the
atrioventricular canal and the chambers (Fig. S2) .
We then examined the adult atrioventricular region in Anolis,
and observed that Bmp2, which in mammals and birds is expressed
in the atrioventricular canal only at embryonic stages, remained
expressed throughout ontogeny (Fig. 4B,F). Tbx3, which marks the
cardiac conduction system in mature mammals , was found
within the same Bmp2-positive atrioventricular domain (Fig. 4C,G).
Three-dimensional reconstructions of the expression patterns of
Tbx3 in early developing hearts of Anolis, chicken and mammals
revealed a striking resemblance (Fig. 5, Fig. S3). In all species, a
similarly-shaped atrioventricular ring was observed. The Tbx3
expression domain extended into the sinus venosus, marking the
sinus node primordium. This expression pattern did not
substantially change in lizards just prior to hatching, whereas in mammals
and birds, it became more complex with further development as
the morphology of the heart, and particularly the sinu-atrial
region, changed (Fig. 5).
We then pursued this building plan of the atrioventricular canal
to older vertebrate classes, represented by Xenopus, an amphibian
and thus a non-amniotic member of tetrapods, as well as zebrafish.
In both species, the atrioventricular canal is composed of
myocardium in continuity with the atrium and ventricle (Fig. 3).
An insulating plane and an insulated atrioventricular node were
not found (Fig. 2). In adult zebrafish and stage 48 Xenopus, we
found Tbx2 in the atrioventricular canal and Nppa (whose
spatiotemporal pattern strongly resembles that of Gja5 in mammals) in
the atria and ventricles, the patterns resembling those of Tbx3 and
Gja5, respectively, in the amniotic vertebrates (Figs. 6, 7). No
cardiac Tbx3 expression was found (Fig. S4).
Next, we examined the His-Purkinje system in Anolis. Gja5 was
used as marker for the mature His-Purkinje system conserved in
mammals and chicken . The developing His bundle does not
express Gja5 until late stages in mammals  and birds , but
can nonetheless be identified very early by the expression of Tbx3
. In mouse  and chicken, the Tbx3-positive and
Gja5negative myocardium of the developing His bundle extends from
the atrioventricular canal ventrally and dorsally into the
ventricular myocardium and unto the crest of the ventricular septum
(Fig. 5). The region formed by the dorsal and ventral extension
Figure 7. Tbx2 expression (in-situ hybridization) in developing Xenopus (st 48). Tbx2 is expressed in the atrioventricular canal and base of
the myocardial outflow tract and complementary to Nppa, marker of chamber myocardium. In ectotherms primitive myocardium (p), remnants of the
embryonic heart tube, can be recognized by its smooth surface as opposed to the trabeculated myocardium (t) formed during chamber formation.
The primitive myocardium of the ventricular base of st 48 Xenopus hearts already has the adult configuration. The trabecular component is far from
fully developed. a, atrium; avc, atrioventricular canal; c, conus arteriosus (myocardial outflow tract); p, primitive (atrabecular) myocardium; t,
ventricular trabeculated myocardium; ven, ventricle.
and the crest is referred to as the primary ring . The Anolis
ventricle is not septated, but shows Tbx3 expression into the
ventricle ventrally and dorsally, indicating the presence of a
primordial, but incomplete, primary ring that lacks the septal crest
component (Fig. 5).
The ventricular wall in mammalian and avian embryos is
composed of a trabecular inner layer and a thin compact outer
layer. Initially, both of these layers express natriuretic peptides
(Nppa and Nppb) and Gja5 [27,42], but halfway through
development, Nppa and Gja5 expression ceases in the strongly expanding
compact layer. After birth (or hatching in birds) the expression of
Nppa and Gja5 is limited to the His-Purkinje network that
eventually constitutes only a small fraction of the ventricular mass
(Fig. 8) [10,43]. The ventricular wall of fish, amphibian and
reptilian hearts does not display such an overt distinction in
expression pattern between an inner trabecular wall and a
compact outer layer. Their ventricular wall typically is composed
of a spongy or trabecular type of myocardium (Figs. 2, 3). In adult
Anolis hearts, Gja5 was homogenously expressed throughout the
trabecular ventricular wall. This suggests an absence of tracts of
preferential conduction leading to the ventricular apex and such
condition resembles that of early mammalian and avian embryos
(Fig. 9). Further back in evolution, as represented by Xenopus and
zebrafish, we observed homogenous Nppa expression in their
trabecular ventricular wall (Fig. 9A,D), while Tbx3 did not identify
a primordial atrioventricular bundle (Fig. S4).
Mammalian and avian hearts have an elaborate
Gja5/Nppaexpressing ventricular conduction system that activates the
ventricles from apex to base [42,44,45]. The homogenous Gja5/
Nppa expression patterns in Anolis, Xenopus and zebrafish trabecular
ventricles suggest that the electrical activation front may spread
from the vicinity of the atrioventricular canal, i.e. the ventricular
base, and reach the apex later. Such an activation pattern would
be reminiscent of early embryonic mammalian and avian
ventricles . We used optical mapping to measure epicardial
activation patterns in Anolis. The first point of activation always
occurred in the cranial third of the ventricle, i.e. the base, and later
at the apex (Fig. 9H). This activation pattern is consistent with
most previous ECG and electrode investigations on reptiles and
very similar to the activation patterns of chamber-forming hearts
of mouse (E810) and chicken (E25) (Table 1) . At these
stages in mammals and birds, a morphologically distinct
conduction system has yet to form and ventricular septation is only
starting to take place [48,49]. Assuming that the dorsal and ventral
activation patterns share the same time point of activation at the
apex, we could synchronize the activation patterns and infer that
the dorsal base is activated prior to the ventral base in the
ectothermic vertebrates (Fig. 9B,E). Dorsal activation of the
ventricular base has been reported in chicken hearts prior to
septation and seemingly occurs in embryonic mouse as well
[42,4549]. In Xenopus and zebrafish, the activation front travels
from the dorsal base to the apex. In Anolis, Xenopus and zebrafish
the location of the first point of activation varied within the
dorsoFigure 8. Development of compact walls. The development of the compact walls (Nppa and Gja5 negative) of mammals and birds leaves the
trabeculated myocardium (Nppa and Gja5 positive) as a thin inner lining of the ventricular lumina in the fully formed hearts. Nppa is not expressed
birds. Scalebars, 100 mm. ivs, interventricular septum; lv, left ventricle.
basal region (Fig. S5). The activation maps and expression data
indicate that the trabeculated ventricular wall of the ectothermic
vertebrates function essentially as an isotropic conduction network.
Consistently, conduction on the luminal surface of the adult
mammalian and avian ventricles through the Gja5-positive
HisPurkinje system also proceeds from base-to-apex [49,50]. On the
epicardial side, however, the activation front reaches the apex first
and then the base (Fig. 9JL) . In mammals and birds, the
developmental change in activation pattern to apex-to-base
observed at the epicardial side coincides with the development
of the Gja5-negative compact ventricular wall, which therefore
may contribute to this developmental change in activation pattern.
Hearts of ectothermic species and of embryos of endothermic
species do not have anatomically marked conduction system
components. In this study we used expression patterns of
conserved genetic markers and identified molecular conduction
system components in developing and adult lizards. We found
them to be similar to the components in embryonic mammals and
birds, indicating they constitute an integral part of the building
plan of the heart. Therefore, the conduction systems found in
mature mammals and birds most likely evolved from the
components of this shared building plan, and did not evolved
In mature birds and mammals, left-over traces of the
atrioventricular canal muscle in addition to the atrioventricular
node can be found. Birds have a well-developed right-sided
atrioventricular ring bundle that communicates with the ventricle
anteriorly through the so-called recurrent branch [4,52]. The
mammalian heart also maintains a molecularly distinct
atrioventricular ring bundle above the insulating plane . Interestingly,
in congenital corrected transposition of the human heart, the
insulating plane disrupts the normal posterior atrioventricular
communication, whereas the anterior communication is
abnormally maintained [53,54]. The anterior part, then, resembles the
recurrent branch of the bird heart.
The adult lizards and ectothermic vertebrates in general
maintain important aspects of the embryonic vertebrate building
plan. The Bmp2/4-Tbx2/3-positive, Gja5/Nppa-negative
atrioventricular canal myocardium is maintained in adult ectothermic
vertebrates. This provides an electrical insulation between atrium
and ventricle in these hearts that lack an insulating plane of
connective tissue. The developing hearts of mammals and birds
have great tolerance to ischemia and regenerative potential, which
Figure 9. Trabeculated ventricles are activated from base to apex. (A, D, G, J) Markers of fast propagating myocardium (Nppa and Gja5) are
homogenously expressed in the ventricular trabeculated myocardium from base to apex (ap). (B, E, H) Ventricular activation occurs from base to apex.
Early activation is red, late activation is blue. Note that the time-colour coding in panel E is different from that in panels B and H. (K) In species with
thick compact myocardium, surface breakthrough of the activation front is earlier in the apical region than in the base. (C, F, I, L) Graphs show the
average activation time of the apex and base and the total ventricular activation time. Note that in zebrafish, Xenopus and Anolis, the ventricular base
is activated earlier than the apex whereas in mice the ventricular base is activated later than the apex (* Significantly different (one-way ANOVA
P,0.05)). n is 3, 6, 9 and 2, respectively. Scale bars in (B, E, H, K) indicate respectively 0.2, 1, 0.5 and 0.1 millimetre. avc, atrioventricular canal; ven,
Noseda et al, 1962 E, 1963 ECG
Vaykshnorayt et al, 2011 E
Peters and Mullen 1966
Dillon and Morad 1981 OP
Vaykshnorayt et al 2008 E
Heaton-Jones and King 1994 ECG
Zhao-Xian et al 1991 ECG
Valentinuzzi et al 1969 ECG
Meek and Eyster, 1912 E
Christian and Grigg 1999 E
McDonald and Heath 1971 ECG
Noseda et la, 1963 ECG
Dillon and Morad 1981 OP
Sedmera et al 2003 OP
Christian and Grigg 1999 E
In the formed hearts of mammals and birds, epicardial ventricular activation is from apex to base.
1Left-to-right activation is only reported in broad-hearted turtles.
2Arbel et al (1977) using ventrally placed electrodes find that as the apex becomes activated the current spreads towards the base. As they could not evaluate if the
base was activated earlier, and since the zebrafish outflow region is indeed activated later than the apex their results may be in agreement with the present study.
*Species where first point of activation definitely was neither base nor apex. From [33,5981].
is lost around birth [55,56]. Interestingly, many ectothermic
vertebrates (e.g. newts and zebrafish) retain the regenerative
capacity and ischemia tolerance throughout life . It is therefore
tempting to speculate that the maintenance of important aspects of
the embryonic programme in adult ectothermic vertebrates may
be involved in the retention of these capacities.
Our study provides a plausible scenario of the evolution of the
hearts of mammals and birds. The spongy myocardium of
ectothermic adult vertebrates, as well embryonic mammals and
birds allows for high ejection fractions and also serves to conduct
the ventricular depolarization (Fig. 10). However, a transition to
compact myocardium was necessary when pressure and heart rate
increased. This rendered the early trabecules secondary on force
generation, but available to differentiate into fibres of poor
contractility and high propagation speeds. Furthermore, mammals
and birds develop a compact ventricular septum whereby the early
trabecules come to drape the septal surfaces and thus form the
characteristic bundle branches of the His bundle (Fig. 10). Our
study, therefore, suggests that the parallel evolution of virtually
identical conduction systems and cardiac designs in birds and
mammals can be traced back to the existence of a primordial
conduction system of the ancestral reptile heart.
Figure S1 Gene program of the developing
atrioventricular canal in chicken. Tbx5, known to induce Gja5, is present in
the atrioventricular canal but Gja5 is absent where Bmp2 and Tbx3
are expressed. la, left atrium; lv, left ventricle; ra, right atrium; rv,
Figure S2 Gene program of the developing
atrioventricular canal in Anolis. Despite expression of Tbx5 the
atrioventricular canal does not initiate chamber program and
expresses the transcription repressor Tbx3 along with Bmp2. avv,
atrioventricular valves; la, left atrium; ra, right atrium; t, trachea
(positive for Tbx3).
Figure S4 Tbx3 expression (in-situ hybridization) in
developing and adult Xenopus. Tbx3 was only found outside
the heart (e.g. developing trachea, t) of stage 40 embryos (top row).
No Tbx3 was only found in the heart of adults (lower row). a,
atrium; avc, atrioventricular canal; c, conus; ven, ventricle; t,
developing trachea (positive for Tbx3).
Figure S5 Summary of individual ventricular activation
maps. Red marks the earliest epicardial breakthrough of the
activation front which was consistently at the ventricular base.
Earliest and latest activation (red and white dots respectively) from
each specimen is projected onto one specimen.
We thank Corrie de Gier-de Vries and Marc Sylva for technical assistance,
Gert van den Berg for stimulating discussions, Jeroen Bakkers for providing
zebrafish, and Tony Durston for providing adult Xenopus.
Conceived and designed the experiments: BJ AVP AFMM VMC.
Performed the experiments: BJ BJDB QDG. Analyzed the data: BJ BJDB
AFMM VMC. Contributed reagents/materials/analysis tools: MJBvdH
AFMM VMC. Wrote the paper: BJ MJBvdH AFMM TW VMC.
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