Development of the Hearts of Lizards and Snakes and Perspectives to Cardiac Evolution

Abstract

Birds and mammals both developed high performance hearts from a heart that must have been reptile-like and the hearts of extant reptiles have an unmatched variability in design. Yet, studies on cardiac development in reptiles are largely old and further studies are much needed as reptiles are starting to become used in molecular studies. We studied the growth of cardiac compartments and changes in morphology principally in the model organism corn snake (Pantherophis guttatus), but also in the genotyped anole (Anolis carolinenis and A. sagrei) and the Philippine sailfin lizard (Hydrosaurus pustulatus). Structures and chambers of the formed heart were traced back in development and annotated in interactive 3D pdfs. In the corn snake, we found that the ventricle and atria grow exponentially, whereas the myocardial volumes of the atrioventricular canal and the muscular outflow tract are stable. Ventricular development occurs, as in other amniotes, by an early growth at the outer curvature and later, and in parallel, by incorporation of the muscular outflow tract. With the exception of the late completion of the atrial septum, the adult design of the squamate heart is essentially reached halfway through development. This design strongly resembles the developing hearts of human, mouse and chicken around the time of initial ventricular septation. Subsequent to this stage, and in contrast to the squamates, hearts of endothermic vertebrates completely septate their ventricles, develop an insulating atrioventricular plane, shift and expand their atrioventricular canal toward the right and incorporate the systemic and pulmonary venous myocardium into the atria.

Full text

Development of the Hearts of Lizards and Snakes and
Perspectives to Cardiac Evolution
Bjarke Jensen
1,2
*, Gert van den Berg
2
, Rick van den Doel
2
, Roelof-Jan Oostra
2
, Tobias Wang
1
,
Antoon F. M. Moorman
2
1Department of Bioscience – Zoophysiology, Aarhus University, Aarhus, Denmark, 2Department of Anatomy, Embryology & Physiology, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Abstract
Birds and mammals both developed high performance hearts from a heart that must have been reptile-like and the hearts
of extant reptiles have an unmatched variability in design. Yet, studies on cardiac development in reptiles are largely old
and further studies are much needed as reptiles are starting to become used in molecular studies. We studied the growth of
cardiac compartments and changes in morphology principally in the model organism corn snake (Pantherophis guttatus),
but also in the genotyped anole (Anolis carolinenis and A. sagrei) and the Philippine sailfin lizard (Hydrosaurus pustulatus).
Structures and chambers of the formed heart were traced back in development and annotated in interactive 3D pdfs. In the
corn snake, we found that the ventricle and atria grow exponentially, whereas the myocardial volumes of the
atrioventricular canal and the muscular outflow tract are stable. Ventricular development occurs, as in other amniotes, by an
early growth at the outer curvature and later, and in parallel, by incorporation of the muscular outflow tract. With the
exception of the late completion of the atrial septum, the adult design of the squamate heart is essentially reached halfway
through development. This design strongly resembles the developing hearts of human, mouse and chicken around the time
of initial ventricular septation. Subsequent to this stage, and in contrast to the squamates, hearts of endothermic
vertebrates completely septate their ventricles, develop an insulating atrioventricular plane, shift and expand their
atrioventricular canal toward the right and incorporate the systemic and pulmonary venous myocardium into the atria.
Citation: Jensen B, van den Berg G, van den Doel R, Oostra R-J, Wang T, et al. (2013) Development of the Hearts of Lizards and Snakes and Perspectives to Cardiac
Evolution. PLoS ONE 8(6): e63651. doi:10.1371/journal.pone.0063651
Editor: Leonard Eisenberg, New York Medical College, United States of America
Received January 25, 2013; Accepted April 4, 2013; Published June 5, 2013
Copyright: ß2013 Jensen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Bjarke Jensen and Tobias Wang 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.
* E-mail: bjarke.jensen@biology.au.dk
Introduction
Mammals and the common lineage of birds and crocodilians
(the archosaurs) are the only vertebrates where the cardiac
ventricle has complete anatomical division of the systemic and
pulmonary sides. Both mammals and birds stem from a lizard-like
ancestor (Fig. 1) and while all seem to agree that the divided hearts
of mammals and birds evolved independently from undivided
reptile-like hearts [1,2] several competing hypotheses seek to
identify the primordial structures of the reptile ventricle that gave
rise to the advanced state of mammals and birds. Some studies
emphasize a right-sided partial septum called the muscular ridge
[1,3], while others emphasize a left-sided trabeculation (the
vertical septum) because of its proximity to the atrioventricular
valve [4]. The validity of these competing hypotheses remains
difficult to assess because little is known about the embryological
development of the reptilian heart.
The fully formed heart of non-crocodilian reptiles (i.e.
squamates and chelonians) receives inflow from three systemic
veins to the right atrium as well as one or two pulmonary veins
that feeds into a single orifice to the left atrium, while the outflow
occurs through three arteries, the left and right aortae and a single
pulmonary artery [5]. The two atrial chambers are fully separated,
and the sinus venosus is situated upstream of the right atrium. In
diastole the ventricular receives blood from the left atrium to its
left-most compartment, the cavum arteriosum, whereas blood of
the right atrium is received in central cavum venosum and the
right-most compartment, the cavum pulmonale. The right atrial
blood is then predominantly directed towards the pulmonary
artery and the left atrial blood towards the aortae. In most reptiles,
however, the ventricle is not divided into a low pressure right
ventricle and a high pressure left ventricle, but functions as a single
pressure pump [6]. It is therefore pulmonary to systemic outflow
resistances that determine where the ventricular blood is ejected
to. Resistance is typically highest in the pulmonary circulation, at
least in resting animals, and cardiac output is thus disproportion-
ally directed to the systemic circulation, a so-called right-to-left
shunt. Blood flows are nonetheless well separated within the
ventricle, probably due to the septa [5–9], and admixture of
oxygen-poor and oxygen-rich blood is minimized. Being ectother-
mic, the cardiac output, heart rate and blood pressures of reptiles
are generally much lower than in the endothermic mammals and
birds, but similar to those of amphibians [9–11].
The ventricle is anatomically the most complex chamber and
the nomenclature of the structures and compartments are
introduced in Figure 1. Much of our results will be discussed in
the context of cardiac evolution, so we extend the nomenclature to
avian and mammalian hearts (Fig. 1G–H). Also, we provide a
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glossary of the described structures, which includes definitions and
synonyms. There is no standardized nomenclature on embryonic
cardiac structures for amniotes. Because we focus on reptiles, we
adopt the nomenclature of previous works on reptile cardiac
development, which, unfortunately, is not standardized as well.
Figure 1 is based on anole specimens halfway through develop-
ment where many early structures are still distinct and the fully
formed heart is outlined. The formed squamate heart is partially
divided into three compartments, called cava, by three structures,
usually referred to as septa. From left to right, the cava are the
cavum arteriosum, the cavum venosum and the cavum pulmonale.
The cavum arteriosum is partially separated from the cavum
venosum by a sheet-like aggregation of trabeculations called the
vertical septum. The cavum venosum is partially separated from
the cavum pulmonale by a spiraling septum called the muscular
ridge (also known as the horizontal septum [3,12]), Muskelleiste
Figure 1. Terminology of the ventricle based on
Anolis sagrei
St9/19 embryos. A. Phylogenetic tree of amniote evolution, with ectotherms
in blue and endotherms in red. Reptilia includes birds but we use ‘reptile’to mean ectothermic members of Reptilia. B.A10mm thick section, close to
the transverse plane, stained for Anolis cardiac troponin 2. Smooth-walled myocardium (white stipulated lines) extends from the otherwise much
trabeculated ventricle (ven) to the myocardial outflow tract (mot) and is reminiscent of the earlier heart tube myocardium. Mesenchyme is associated
with the smooth-walled myocardium and can be categorized as dorsal (1) and ventral (2) atrioventricular cushion and parietal (3) and septal (4)
cushion (nomenclature of the mammalian heart [53]. C. Sister section to A stained for Anolis Tbx5, which is absent from the mot in all vertebrates. The
mot is incorporated to the ventricle during development, which creates the bulbuslamelle (red stipulated line) and a fold in the ventricular wall also
known as the muscular ridge (orange dot). D. A 3D reconstruction, of a different specimen, cut in a plane corresponding to the sections of B–C
(mesenchyme in yellow). It shows the complete part of the muscular ridge (asterisk) and its free-standing part (orange dot). Oppositely, the
bulbuslamelle is found and notice the associated cushion. The position of the atrioventricular canal is indicated by the black stipulated line, the
probable position of the future vertical septum is indicated with the white arrow and the positions of the future cavum arteriosum (‘ca’), cavum
venosum (‘cv’) and cavum pulmonale (‘cp’) can accordingly be designated. E. Same sectioning plane as in C, looking towards the atria, with the atrial
septum indicated (blue arrow) F. Slightly more tilted sectioning plane than in C looking towards the deeper right parts of the muscular ridge (asterisk)
from a skewed angle (the mesenchyme is removed, but shown in the miniature). Also shown, is where the atrioventricular canal meets the ventricle
(arrow). G. Chicken hearts of Hamburger/Hamilton stage 28–29 (embryonic day 6) also have a reptilian-like design (modified from [23]). H. Human
heart, Carnegie stage 14 (embryonic days 31–35) has essentially the same design as the reptilian heart, with the ventricular septum (vs) being an
important variation. Notice that the bulbuslamelle (red stipulated line) is defined by the presence of mesenchyme (modified from [51]). l(r)a, left(right)
atrium; sv, sinus venosus.
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Figure 2. Generation of 3D reconstructions. A. Example of a 10 mm section from P. guttatus 10 days stained for myocardium with antibodies
against rabbit cardiac troponin I. B. Same section as in A with annotations made in AmiraH. C. 3D reconstruction based on 160 sections projected out
of the section of A with mesenchyme and transparent lumen (myocardium not shown). D. Same 3D reconstruction as in C visualizing the annotated
compartments. E. Five reconstructions exemplifying the transition from the youngest to the oldest stage.
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[13] or other names [5]). Exactly opposite the muscular ridge is the
bulbuslamelle e.g. [14] (‘Bulbuslamelle’was coined by Greil [15]
instead of Bru¨ckes name of ‘Fleischpolster’ [16]). Very little is
known about when and how these structures appear in the
ontogenetic development of any reptile.
The few embryological studies on reptile hearts are in stark
contrast to the vast number of studies of cardiac development in
fish, amphibians, birds and mammals [4,11,17,18]. Nonetheless,
the embryology of reptile hearts remains pivotal for our
understanding of vertebrate cardiac evolution [4,12,15,19–23].
For instance, Greil [15] argued that the myocardial outflow tract
contributes to the right ventricle in reptiles, mammals and birds
and this has been verified experimentally multiple times by gene
expression, cell fate and lineage tracings [24–26]. Here, we
describe the growth and morphological changes after the early
heart tube formation to the formed heart in the corn snake
(Pantherophis guttatus guttatus) and in the green and brown anoles
(Anolis carolinensis and A. sagrei). Genome sequencing was recently
completed in the green anole [27] and is underway in the corn
snake [28], which can be expected to greatly facilitate gene
expression studies.
This study provides a developmental series from the onset of
chamber formation of some of the most commonly used squamate
models, the corn snake and the anole. The reptile heart and the
ventricle in particular have a very complex anatomy and we have
therefore provided comprehensively annotated 3D models in pdf
format of all investigated stages of the corn snake from 2 to
42 days after egg laying. Secondly, we discuss separately the
findings in the context of cardiac evolution. It is the ventricular
anatomy that varies the most between reptiles, mammals and
birds. This study emphasizes the ventricle.
Materials and Methods
Specimens
Fertilized eggs of the corn snake (P. guttatus guttatus) and two
fertilized eggs of the Philippine sailfin lizard (Hydrosaurus pustulatus)
were incubated on gravel to prevent contact with water in an
incubator at 29uC and 85% humidity. Fertilized eggs of the green
and brown anoles (Anolis carolinensis and A. sagrei) were bought
commercially and fixed immediately in a 4% paraformaldehyde
phosphate-buffered saline solution overnight and then 70%
ethanol followed by embedding in paraplast. Corn snake embryos
were fixed similarly at 2, 10, 12, 14, 16, 20, 26, 35 and 42 days
post egg laying (referred to as days) out of ca. 60 days, sailfin lizard
embryos at 4 and 7 days out of ca. 60 days and anoles at stages 5,
7, 9, 12 and 17 out of 19 (anole development, from egg laying, lasts
ca. 25 days and includes stages 4–19, [29]). The hearts of a
3 months-old corn snake and an adult green anole, ca. 1 year,
were included as fully formed hearts. A heart of an adult Burmese
python and an adult ostrich were included in the comparative
analyses. All adult hearts were fixed as above.
Ethics statement
In The Netherlands experiments with non-mammalian embryos
(that are not autonomously viable) do not require approval from
the Institutional Animal Care and Use Committee. The fertilized
corn snake and sailfin lizard eggs were donated to us (R-JO) from
the Diergaarde Blijdorp (Rotterdam, the Netherlands) whereas
fertilized anole lizard eggs were obtained commercially. All
embryos were sacrificed by immersion in 4% paraformaldehyde
except in the late developmental stages, corn snake 42 days and
anole lizard stages 17 and 19, where the embryo was first
decapitated and the head split saggittally to stop all brain activity.
Adult reptiles were commercially obtained and sacrificed in
Figure 3. Absolute (mm
3
) and relative (%) myocardial volumes of cardiac compartments rendered from 3D reconstructions.
Myocardial volume of the ventricle and the atria increases exponentially. Neither the conus nor the atrioventricular canal changes substantially in size
and come to contribute only miniscule proportions to the fully formed hearts represented by 150 days for the corn snake (3 months post hatching)
and ‘Adult’ for the anoles (the mm
3
values for the adult anole are written onto the graph).
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Denmark in accordance with Danish Federal Regulation, where
the harvest of tissue of sacrificed animals by anesthesia does not
require approval from Institutional Animal Care and Use
Committee; the animals were anaesthetized with 100 mg pento-
barbital (Sygehusapotekerne, Denmark) per kilo body mass and
then had their hearts excised. The carcass of a euthanized ostrich
was given to us (BJ and TW) by Givskud Zoo, Denmark.
Sectioning, staining, MRI scanning and 3D
reconstructions
Most specimens were cut in 10 mm sections, except corn snakes
of 26, 35 and 42 days and 3 months that were cut in 14 mm
sections and the adult green anole which was cut in 12 mm
sections. Myocardial staining was performed as previously
described [30]. In all specimens of corn snake and sailfin lizard
the myocardium was visualized by immunohistochemistry using a
rabbit antibody to cardiac troponin I (cTnI) polyclonal antibody
(HyTest ltd., dilution 1:500) binding of which was visualized by a
fluorescently labelled secondary goat-anti-rabbit antibody coupled
to Alexa 568 (Invitrogen, dilution 1:250). Two specimens of anole
(stage 5 and 12) were cut in 7 mm sections and stained for the
myocardial marker, cTnI, as described above, and were addition-
ally stained for all nuclei with Sytox Green (1:40,000 Molecular
Probes S-7020), and for incorporation of Bromodeoxyuridine
(BrdU), a synthetic analogue of thymidine used to detect DNA
replication, with a rat-monoclonal anti-BrdU (1:600, Immuno-
source). Antibody binding was then visualized using a fluorescently
labelled secondary goat-anti-rat antibody coupled to Alexa 680
(Invitrogen, dilution 1:250). 100 ml BrdU (10 mg BrdU (Sigma)
per ml physiological salt solution (0.9% NaCl)) was injected
through the shell into the egg yolk. Incubation with BrdU was at
room temperature for one hour.
We used in-situ hybridizations for Tnnt2 (anole cardiac troponin
T) for myocardial stain and Tbx5 on anole stages 7, 9 and 17 [23].
Only the adult green anole heart was stained with picro-sirius red
for collagen. All sections were photographed, stacked, and aligned
in AmiraHv4.1.1 (or newer versions) and then reconstructed as
previously described (Fig. 2A–C) [31,32]. In AmiraHwe annotated
Figure 4. Changes in cardiac dimensions throughout development in the corn snake. A. To scale reconstructions, showing the dorsal
halve of the heart.. B. Cardiac profiles during development, arbitrarily fixed at the right margin of the atrioventricular canal. C. The ventricle is initially
wider (right-left) and deeper (dorsal-ventral) than long (caudal-cranial), but growth is primarily by lengthening. avc, atrioventricular canal; l(r)a,
left(right) atrium; sv, sinus venosus; ven, ventricle.
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the developing compartments on the following morphological
criteria; the myocardial outflow tract is the smooth-walled tract of
the arterial pole; the ventricle is the trabeculated chamber
upstream of the myocardial outflow tract and downstream of the
atrioventricular canal; the atrioventricular canal is the smooth-
walled cylindrical canal in between the more voluminous atria and
ventricle; the atria are the compartment between the atrioven-
tricular canal and the sinus venosus, i.e. the systemic inflows to the
right atrium with myocardium (Fig. 2D). By using the Material-
Statistics tool in AmiraHwe obtained volume readouts for all
annotated structures. Length of the myocardial outflow tract was
measured as described previously [33].
In our discussion of the evolutionary fates of the bulbuslamelle we
compare to python and bird hearts on the basis of 3D models made
from MRI scans. The MRI scans were performed with clinically
available Philips Achieva 1.5 T system (Philips Medical Systems,
Amsterdam, the Netherlands). The hearts were imbedded in agar
and positioned in the centre of the magnet. Data were acquired with
a dedicated radiofrequency surface coil using high-resolution 3D
gradient–echo sequence with the following parameters: Python
molurus; field-of-view: 60680680 mm
3
; repetition time: 75 ms; echo
time: 5.4 ms and excitation flip angle: 45u. Images were isotropically
acquired with a spatial resolution of 0.960.960.9 mm
3
/voxel.
Ostrich; field-of-view: 23062306140 mm
3
, voxel size:
0.4860.4860.48 mm
3
, repetition time: 15.1 ms, echo time:
6.9 ms, excitation flip angle: 30u. Reconstructed images were
exported in DICOM format and loaded to Amira and treated as
above.
Amira models were converted to interactive 3D pdfs using
Adobe Acrobat Pro ExtendedHversion 9.3 as previously described
[34]. The 3D pdf can be viewed with the freeware version: Adobe
ReaderH(version 9.3 or higher) with JavascriptHenabled.
Results
3D reconstructions in interactive pdfs
The cardiac lumen and the following structures of the corn
snake heart are annotated in the supplementary 3D pdfs based on
morphology; sinus venosus, sinuatrial valves (10 days onwards),
septum spurium, atria (including atrial septum), atrioventricular
canal, ventricle, bulboauricularlamella (10 days onwards), bulbus-
lamelle (10 days onwards), bulbo-ventricular fold (2 days)/muscu-
lar ridge (10 days onwards), vertical septum (20 days onwards) and
myocardial outflow tract. The AmiraHfiles, which the 3D pdfs
(Figs. S1–7) are based on, are available on request and include the
editable label file and the surface file.
Supplementary videos
We recorded the beating hearts of the corn snake from days 10
and 20 with emphasis on the systemic inflow to the heart and the
sequence of chamber contractions and these videos are included in
the online material (Movies S1-S6).
Total cardiac growth
In the earliest stage available, i.e. 2 days in the corn snake,
chamber formation, i.e. the ballooning of ventricular and atrial
compartments, was already initiated (Fig. 2E). At this point the
myocardial volume is 0.2 ml, which by 42 days has increased
exponentially and more than 20 fold to a volume of 4.3 ml
(Fig. S8), whereas some 100 days later, 3 months after hatching
(i.e. juvenile snake), this volume has less than doubled to 7.2 ml
(Fig. 3). In the anoles, the heart of stage 5/19 is morphologically
similar to the corn snake 2 days, but it is much smaller, 0.024 ml. It
grows ca. 5 fold before hatching (st17/19), to 0.108 ml, and then
almost 50 fold to reach the adult condition (5.25 ml) (Fig. 3).
Cardiac growth is accompanied by an expansion of the pericardial
cavity. The distance from the pericardial wall to the atria and the
conus arteriosus respectively increases and vessels form accord-
ingly.
Table 1. Chronological appearance of cardiac structures.
Days Corn snake Sailfin lizard
2 sinus venosus
single atrium
ventricular trabeculation
bulbo-ventricular fold
4 ventricular trabeculation
bulbo-ventricular fold
endocardial atrial septum
7 sinuatrial valves
10 pulmonary vein
myocardial atrial septum
septum spurium
bulboauricularlamella
14 settled position of SA-orifice
cavum pulmonale
muscular ridge
bulbuslamelle
20 vertical septum
cavum arteriosum
cavum venosum
,60 hatching
Stage Anoles
5 sinus venosus
ventricular trabeculation
bulbo-ventricular fold
endocardial atrial septum
sinuatrial valves
7 pulmonary vein
myocardial atrial septum
septum spurium
9 bulboauricularlamella
cavum pulmonale
muscular ridge
bulbuslamelle
12 vertical septum
cavum arteriosum
cavum venosum
19 hatching
The corn snake and the sailfin lizard have been aligned because both have
similar incubation times after egg laying. In comparing squamate heart
development to other vertebrates, the developing hearts of the corn snake of 2
to 14 days and the anoles of stages 5 to 9 correspond in many regards to, we
suggest, the hearts of chicken development of Hamburger-Hamilton stages 18–
28 and of human development of Carnegie stages 14 to 18. Later stages differ
substantially.
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Growth of compartments
All compartments found in the adult heart can be recognized by
2 days in the corn snake and st5/19 in the anoles. We annotated
and measured the volumes of the myocardial outflow tract, the
ventricle, the atrioventricular canal, the atrium (later atria), the
sinus venosus and mesenchymal tissue throughout development in
both the corn snake and the anoles. Only the sinus venosus
myocardium was often damaged and thus incompletely recon-
structed. As described for mammals and birds, there are clear
compartmental differences in growth [35,36]. The myocardial
volumes of the atrioventricular canal and myocardial outflow tract
were essentially constant throughout development, whereas both
ventricular and atrial compartments increased exponentially
(Fig. 3, Fig. S8). In the two anole specimens exposed to BrdU,
an indicator of proliferation, BrdU was incorporated at the highest
rates in the regions of exponential growth, i.e. the atria and
ventricle (Fig. S9). In the corn snake, the atrioventricular and
ventricular mesenchyme, which in later stages remodel into the
atrioventricular (ca. 35 days) and arterial valves (ca. 35 days),
constituted 0.1–0.15 ml throughout development and 0.17 mlat
three months after hatching. By 20 days in the corn snake, one-
third through development, the ventricle and atria constituted ca.
80% and ca. 15% respectively of the total myocardial volume
which is essentially the proportions of the 3 months heart (Fig. 3).
Halfway through development in anoles, st12/19, the proportions
of the ventricle and atria (72,6% and 15,8% respectively)
components approximates that of the adult (80,5% and 18,6%
respectively, Fig. 3).
The hearts mature towards the adult shape throughout
development. This is particularly evident in corn snakes, where
the heart becomes more elongate than the anole heart (Fig. 4). At
2 days the corn snake heart is 1.2 mm long (caudo-cranially) and
Figure 5. Ventricular morphology of the corn snake. Looping of the heart tube leaves a fold (white asterisk and orange dot), the bulbo-
ventricular fold, on the border of the ventricle and the myocardial outflow tract (mot) (2–10 days). Later, and associated with the ventricularization of
the mot, this fold constitutes the muscular ridge (14 days and later), with a free standing part (orange dot) and a complete part (white asterisk).
Opposite the muscular ridge, the bulbuslamelle forms (+). The early ventricle has multiple parallel trabecular sheets (white arrows) of equal height but
as the ventricle grows (at 20 and 42 days) only one sheet retains a short distance to the atrioventricular canal (indicated by the broken blue line)
which may then be annotated as the vertical septum. Inserts show sectioning plane and angle of inspection.
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1 mm wide (from left to right). By 20 days, where the atrial and
ventricular compartments have the proportional volume of the
adult (Fig. 3), the heart is still stockier than the heart at 42 days,
which is more than twice as long as wide, 4,5 mm to 1,9 mm,
respectively (Fig. 4). From 2 days to 42 days the corn snake
ventricle expands less than 2 fold from left to right and dorso-
ventrally, but expands more than 4 fold caudo-cranially (Fig. 4).
Appearance of structures
The appearance of structures is listed chronologically for the
corn snake, the sailfin lizard and the anoles in Table 1. We deem
that development in the corn snake of 2 to 14 days and in the
anoles of stages 5 to 9 correspond to 3 to 6 days of chicken
development (Hamburger-Hamilton stages 18–28 [37]) and
Carnegie stages 14 to 18 of human development. By Hamburg-
er-Hamilton stage 28, the developing chicken heart has an
external resemblance to that of corn snake day 14 and anole st9/
19 but internally the ventricular septum, which does not develop
in squamates, is already well-defined. In the following sections the
development of each compartment will be treated.
Inflow to the heart
There is initially a very short distance from the entry of the
systemic veins through the pericardial wall to the (right) atrium,
but it increases many fold during development. Accordingly,
vessels form and elongate in between and they are referred to as
the sinus venosus when they acquire cardiac musculature. The
sinus venosus initially (2 days) consists of the left and right
Figure 6. Bulboauricularlamella. Smooth-walled ventricular myocardium, the bulboauricularlamella, is formed early in development (A–C, corn
snake) and remains in the adult heart (D–E, anole). A. The bulboauricularlamella, ventral and dorsal (red dotted lines), are contiguous with the
atrioventricular canal (contains mesenchyme, shown in yellow). Inserts show plane of sectioning, angle of inspection and the sectioned plane. B–C.
Once formed, the bulboauricularlamella remains a constant feature of the ventricle. D. Dorsal halve of the heart of an adult green anole
(atrioventricular valve in yellow). D’. Same view as in D with all structures made transparent except the smooth-surfaced myocardium of the ventricle,
the majority of which is the dorsal bulboauricularlamelle. E. Ventral halve of the same heart, slightly rotated, showing the ventral
bulboauricularlamelle and how it blends, without a boundary, into the muscular ridge (askerisk). +, bulbuslamelle; l(r)a, left(right) atrium; orange
dot; bulbo-ventricular fold/free-standing part of the muscular ridge; pv, pulmonary vein; ven, ventricle.
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(anterior) sinus horns. Later, ca. 12–16 days, a third vessel appears
which stems from the liver and is confluent with the sinus horns
immediately upstream of the right atrium. Video recordings show
sequential contractions in the sinus venosus, the atria and the
ventricle (Movies S1-6) and sinuatrial valves are present at this
point. The sinuatrial valves are myocardial, contrary to the
mesenchymal valves of the atrioventricular canal and arteries. The
cardiac musculature encloses the vessels throughout development
and only in the left sinus horn does the myocardial enclosure
become discontinuous by 42 days.
The future pulmonary vein is indicated by 7 days by a
mesenchymal cushion associated with the dorsal wall of the
atrium and from 10 days onward a lumen can be seen within the
cushion. Unlike mammals, no myocardium develops around the
pulmonary vein. By 7 days, sinuatrial valves have started to form,
protruding into the atrial lumen.
The atria
Initially, the atrium is a smooth-walled chamber encompassing
a single lumen. The lateral walls of the atrium seemingly outgrow
the roof and a deep sulcus is eventually created between the left
and right atrium (Fig. 4). Internally, trabeculations (commonly
called pectinate muscles or musculi pectinati) appear by 10 days, the
most prominent of which is the septum spurium of the right atrium
which is continuous with the sinuatrial valves. The atrial septum
has also started to form at 10 days but is not completed by
42 days. The atrial septum develops slightly to the left of the body
midline such that a small cul-de-sac is formed to the right atrium
to the left of the deepest part of the sulcus of the atrial roof.
Eventually, by 42 days, the atrial walls are entirely trabeculated
except immediately above the atrioventricular canal, i.e. the atrial
floor, or vestibule.
The atrioventricular canal initially is a free standing tube, but
becomes progressively enclosed by ventricular myocardium
(Fig. 1A–B, 2E). Already at 2 days of development, the entire
atrioventricular canal is lined with mesenchyme, which is
particularly thick dorsally and ventrally and thus forming two
cushions with a single narrow lumen centrally. Laterally the
mesenchyme has disappeared by 10 days and only the two
cushions remain. By 16 days the two cushions have fused whereby
separate left and right inflows to the ventricle are created. A lateral
cushion is seen transiently on the right side of the atrioventricular
canal around 20 days but not much remains by 3 months. At no
point is the atrioventricular canal myocardium interrupted by
fibro-fatty tissue from the atrioventricular sulcus (as seen in
mammalian and avian development).
Figure 7. Ventricularization of the myocardial outflow tract of the corn snake. A. At 2 days the myocardial outflow tract (mot) constitutes
most of the inner curvature of the heart (broken line) and the luminal length is 1180 mm. B. The myocardial outflow tract shortens to 470 mmby
12 days. C. By 42 days the myocardial outflow tract is shorter still (200 mm) and the inner curvature (arrow) is no more than a small piece of
myocardium, the bulboauricularsporn of Greil [15], between the atrioventricular canal (avc) and the aortae (ao). a, atrium; la, left atrium; ra, right
atrium.
doi:10.1371/journal.pone.0063651.g007
Figure 8. The relative masses of the atrium(atria) and ventricle(s) in vertebrates. Values based own data, the species studied here are
marked by a red asterisk, or adopted from [92,126–133].
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The ventricle
The ventricle develops in between a left-sided atrioventricular
canal and a right-sided conus orifice. Trabeculations develop in
the entire ventricle, except towards the atria and in between the
atrioventricular canal and the conus orifice and this smooth-walled
part is referred to as the inner curvature. The early trabeculations
(2 days) are congregated into multiple trabecular sheets that are
parallel to the saggital plane (Fig. 5). Already by 10 days, however,
most of the ventricular trabeculations appear as a spongy
meshwork in between the sheets that remain centrally and the
outer ventricular surface. The sheets are retained throughout
development but only one or a few remain at a short distance to
the atrioventricular canal from ca. 20 days onwards. If a single
sheet is identified this can constitute the vertical septum with the
cavum arteriosum to its left and the cavum venosum on its right
(Fig. 5).
Formation of the cavum pulmonale probably occurs as a
consequence of the ventricularization of the conus and by 14 days
a shallow cavum pulmonale appears that will become deeper as
the ventricle continues to elongate. The early ventricle therefore
appears to consist of the cavum arteriosum and cavum venosum
and later, from ca. 14 days onward, the cavum pulmonale
develops (Fig. 5). Concomitantly with the deepening of the cavum
pumonale, the muscular ridge also elongates in caudo-cranial
direction. In earlier stages, the muscular ridge appears as a (bulbo-
ventricular) fold in between the early ventricle and the myocardial
outflow tract (Fig. 5).
From about 10 days onward smooth-walled myocardium
associated with the atrioventricular cushions appears to project
into the ventricle from the atrioventricular canal ventrally and
dorsally and these are called the bulboauricularlamella (Fig. 6)
[15]. In the 3D pdfs they are shown to extend to the inner
curvature. To the right of the inner curvature smooth-walled
myocardium is then regarded as the bulbuslamelle if it is
continuous with the dorsal bulboauricularlamelle and the muscu-
lar ridge if it is continuous with the ventral bulboauricularlamelle.
The myocardial outflow tract
At 2 days the myocardial outflow tract is thin-walled but
nevertheless has little lumen because it is almost entirely filled with
cushion material (the myocardial outflow tract may also be
referred to as the conus arteriosus or the bulbus cordis). Its luminal
length is more than 1000 mm as it curves from the extreme right of
the ventricle to the left and then cranially (Fig. 7). By 10 days the
conal length is halved to ca. 500 mm and from 35 days onwards it
constitutes ca. 200 mm. Also by 10 days, at the distal end of the
myocardial outflow tract, the single lumen is bifurcated by a
mesenchymal protrusion into a (future) pulmonary channel and an
aortic channel. Immediately distal hereof, the aortic channel is
bifurcated by a second but smaller protrusion into a dorsal (future
right aorta) and a ventral channel (future left aorta). The relative
Figure 9. The atrioventricular valve complex of the formed lizard heart. In reptiles, the atrioventricular valve complex is dominated by large
medial leaflets (B), albeit lateral cushions can be found (arrows, in B–C). 12 mm section of the adult heart of a green anole stained with picro-sirius red
(collagen red, myocardium orange), a, atria; as, atrial septum; la, left atrium; lav, left atrioventricular valve leaflet; ra, right atrium; rav, right
atrioventricular valve leaflet; ven, ventricle.
doi:10.1371/journal.pone.0063651.g009
Figure 10. 3D reconstruction of the ventricular base of near-
hatching
Anolis sagrei
(st19). Using the esophagus and trachea to
determine the body midline, it can be seen that the atrioventricular
canal remains on the left side. asterisk, complete part of the muscular
ridge; avv, atrioventricular valve; cp, cavum pulmonale; cv, cavum
venosum; orange dot, free-standing part of the muscular ridge.
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position of the three channels is maintained from 10 days on but
the region of the bifurcation is further approximated to the
ventricle as the myocardial outflow tract shortens. Concomitant
with the ventricularization of the myocardial outflow tract, the
myocardium between the atrioventricular canal and the aortic
base shortens and thickens and has been coined the bulboaur-
icularsporn by Greil [15] (Fig. 7).
Discussion
Cardiac development in squamates
Heart tube formation and looping has not been treated here and
takes place prior to egg laying in the corn snake, the anole lizard
and the Philippine sailfin lizard. These processes were described
and visualized in the turtle Chelydra serpentina, where a straight heart
tube is formed on the embryonic midline ca. 9 days post egg laying
[38]. Looping has been described for the lizard Lacerta agilis [15]
and the snake Natrix natrix [17,18] (for heart development in
crocodilians, see [39] and [40,41]).
In using nomenclature of the fully formed heart on embryonic
structures, it is important to appreciate that the formation of the
heart is a highly dynamic process where many cells are added from
extra-cardial precursor pools [42,43]. Studies on cell fate and
lineage tracing in chicken and mouse show that cells from the early
outflow tract form the right ventricle and part of the ventricular
septum, whereas the atrioventricular canal contributes to the
entire left ventricular free wall [26,30,44,45]. Amazingly, the
myocardium of the early ventricle therefore contributes to little
more than the left surface of the fully formed ventricular septum
[30]. Similar studies remain to be made on reptiles.
The association of specific ventricular cava with an atrial inflow
or an arterial outflow relates to distinct phases in formation of the
ventricle; the early trabeculated ventricle, from ca. 2 days onward,
Figure 11. Comparative ventricular development. A. In early cardiac development, all amniote hearts show a bulbo-ventricular fold (orange
dot) on the border of the trabeculated ventricle and the myocardial outflow tract. The atrioventricular canal (blue circle) is exclusively to the left of the
fold. B. In non-crocodilian reptiles, the early design is maintained in later development. In amniotes with full ventricular septation (indicated by
broken line between two orange dots) the atrioventricular canal expands to the right (note that compared to the stages in A, stages E6 and CS18 are
ca. 5% further in gestation, whereas 20 days is ca. 15% further). Miniatures show sectioning plane and angle of inspection. CS, Carnegie stage; E,
embryonic day.
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is associated with the atrioventricular canal and gives rise to the
cavum arteriosum and most of the cavum venosum. Only about
14 days later does the cavum pulmonale start to form by
ventricularization of the myocardial outflow tract. Also, it is only
by the ventricularization of the myocardial outflow tract that the
aortic base becomes associated with the cavum venosum. A small
bit of heart tube, the inner curvature of the ventricle, always
remains between the right aortic base and the atrioventricular
canal (the bulboauricularsporn of Greil, [15]). Thus, the position
of the atrioventricular canal to the left of the right-sided
myocardial outflow tract is maintained from the earliest stages of
chamber formation to the fully formed heart.
Chamber formation is similar in vertebrates
It has long been recognized that very early stages of reptilian
cardiogenesis resemble other vertebrates with the formation of a
heart tube that subsequently loops [12,38,41,46,47]. Subsequently,
atrial and ventricular chamber formation proceeds as ballooning
due to moderate-high proliferation on the outer curvature of the
heart tube. The flanking myocardium, the smooth-walled inner
curvature and myocardial outflow tract, remains heart tube-like,
and is characterized by very low proliferation [48–51](Fig. S9).
Such differentiation reflects an underlying molecular patterning
that conveys differences in contractile and electrical properties and
is governed by transcription factors [23,52–55]. This concept of
chamber growth opposes the segmental model, where chambers
are thought to develop within distinct modules on the heart tube
and the early heart is thought to contain all the precursors of the
formed heart [43,50,53]. On the contrary, cells from extracardiac
precursor pools are being added to the heart at the venous and
arterial poles and, amazingly, in mouse, the early chambered heart
contains little more than the precursor for the left surface of the
ventricular septum [30,44]. The developmental program of
mammals and birds, then, is distinguished by a great thickening
of the compact wall of the ventricles [11,50,56].
The sinus venosus is maintained in the formed hearts of
ectotherms
The sinus venosus is arguably not a chamber as it does not form
by ballooning [43], like the atria and ventricle, but we nonetheless
categorize it as a chamber because it contains myocardium.
Interestingly, the sinus venosus is retained throughout develop-
ment as revealed with myocardial stains. Thus, the sinus venosus is
much bigger than commonly perceived, namely the area where
the three caval veins are confluent, and in fact includes the caval
veins [5,12,14,57]. Consistently, the ‘caval veins’ all contract prior
to the atria (Movies S4-6) as also shown with electrocardiography
[58,59]. Also, in amphibians, the sinus venosus is an expanded
cavity [60–64]. In fishes collectively, the sinus venosus can be fairly
large, e.g. in hagfish, or may be much reduced, e.g. in zebrafish
[65–67].
Atrialization of venous myocardium does not occur in
reptiles
Mammalian development sees incorporation (or atrialization) of
the sinus venosus into the right atrium, which renders the dorsal
wall smooth and the early atrium remains as the appendage
characterized by pectinate/trabeculated musculature (e.g.[68]).
The left atrium of mammals also acquires a smooth dorsal wall by
incorporation of mediastinal, or pulmonary myocardium [68,69].
The extent of atrialization of systemic and pulmonary venous
myocardium varies a lot between mammalian species [70,71]. It
has been stated that parts of the sinus venosus is atrialized in
reptiles [12] but the large size of this compartment and the
absence of a smooth dorsal wall in the right atrium suggests
otherwise. We never saw a myocardial sleeve around the
Figure 12. Size of the atrioventricular junction in vertebrates. Values are the cross sectional areas of the atrioventricular orifice(s) to the
ventricular base (in %) measured on published and unpublished images in ImageJ (1.44d) using the ‘Polygon selections’ tool. In most cases, the state
of contraction was not known. Horizontal bars are averages. In fishes, only systemic venous blood returns to the heart and the atrioventricular
junction is small. With pulmonary ventilation, the heart receives both systemic and pulmonary venous blood (ectothermic tetrapods) and the
atrioventricular junction more than doubles in size. In animals with full ventricular septation the atrioventricular junction is much larger (crocodilians,
birds and mammals) regardless of ectothermic or endothermic metabolism. Published images were from [14,15,70,71,87,112,134–143].
doi:10.1371/journal.pone.0063651.g012
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pulmonary veins in the corn snake or in anole lizards. The left
atrium remains trabeculated in its entirety.
Comparative anatomy of the atria
The atrium, or atria, of ectothermic vertebrates is very
voluminous compared to those of endotherms and contributes
more to ventricular filling than in endotherms [10,72]. Surpris-
ingly, the atrium/atria of all vertebrates constitutes close to 20% of
the ventricular mass despite the difference in importance for
ventricular filling. There is a slight tendency for smaller atria in
endotherms, but this could equally well be attributed to species-
dependent variation (Fig. 8). At least in some fishes, there is
substantial passive filling of the ventricle [73,74].
The formed heart of all amniotes contains two atria separated
by a complete septum, but only in mammals is the atrial septum
formed by two septa. The two septa are the septum primum and,
associated with the atrialization of the sinus venosus, the septum
secundum. The septum primum is formed first and has a free edge
towards the atrioventricular canal that expresses the transcription
repressor Tbx3 [75,76]. This expression pattern is also found in
chicken and we have recently shown it to be conserved since
reptiles [23]. We conclude that a septum secundum does not form
Figure 13. The bulboauricularlamella are a feature of most vertebrate hearts. In most adult ectotherms smooth-surfaced myocardium, the
bulboauricularlamelle (bal), is interspersed between the atrioventricular canal and the ventricular trabeculations. A similar design is seen in embryonic
hearts of mammals and birds. orange dot, bulboventricular fold or free-standing part of the muscular ridge. The image of the zebrafish heart is
adopted and modified from [65].
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in reptiles. Instead, in reptiles and birds the atrial septum has
multiple perforations that are closed around the time of hatching
[1,77]. The most prominent trabeculation of the atria is the right
atrial septum spurium (or suspensory ligament, [5,14]), which is
continuous with the left and right leaflets of the sinuatrial valve
and can be found in back in sharks (the co-called dorsal
commissure, [78]).
The insulating plane is only found in endotherms
In embryogenesis, all vertebrates have a myocardial atrioven-
tricular canal. Only in mammals and birds does the so-called
insulating plane of fibro-fatty tissue ingress into the myocardium of
the atrioventricular canal so that a single communication remains,
the atrioventricular bundle [79]. Insulation between the atria and
ventricle(s) is therefore primarily by the molecular patterning of
the atrioventricular canal, and, secondarily, by the insulating plane
[23,55,80]. Interestingly, in crocodiles only the ventral halve of the
atrioventricular canal is interrupted by an insulating plane,
whereas the dorsal halve remains myocardial. Crocodiles have a
full ventricular septum and an atrioventricular valve apparatus like
birds [81].
The atrioventricular valve configuration is evolutionarily
old
The mesenchyme of the atrioventricular canal is strikingly
similar in amniotes with a large cushion dorsally and ventrally
[1,53]. This suggests that the fully formed left and right
atrioventricular valve of the reptilian heart are homologous to
the septal leaflet of the right-sided tricuspid valve and the aortic
leaflet of the left-sided mitral valve of the mammalian heart. The
reptilian heart also develops, to a variable degree, lateral cushions,
again similar to other amniotes, and these may be retained in the
formed heart and are most likely functionally redundant (Fig. 9).
This configuration of four cushions further resembles the tetra-
cuspid atrioventricular valve of many amphibians and fishes
[1,66,82].
Figure 14. Evolutionary fates of the bulbuslamelle. A. Phylogenetic tree to show the show the evolution of selected groups of amniotes. B.
Ventricles inspected from the right with the ventricular wall made transparent so that the similarity in design between the bulbuslamelle (BL) and the
myocardial right atrioventricular valve (V) can be appreciated. The V of birds and monotreme mammals is positioned in the heart where the
bulbuslamelle (BL) of the reptile heart is situated. In pythons, the BL is strongly developed and participates in separating the left and right sides of the
ventricle. The image of the echidna heart is modified from [70]. B’. Scanning electron microscopic image of the human heart at 11 weeks of
development with the wall of the right ventricle removed (modified from [90]). In the human heart, and eutherian mammals in general, the
anterosuperior leaflet (asl) of the tricuspid valve is muscular until late in development. C. Cranial view of the ventricular base showing the position of
the BL in the ‘basal’ condition (corn snake), in the contributing to separate the left and right sides of the python ventricle and as atrioventricular valve
(ostrich) (the ventricle of the corn snake was deformed during fixation, as seen by indentations on the dorsal surface). ap, anterior papillary muscle;
smt, septomarginal trabeculation; svc, supraventricular crest; stars, tricuspid gully complex. PA, pulmonary artery.
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Anatomy of the atrioventricular junction relates to
septation
Once the squamate atrioventricular canal has settled on the left
of the body midline, it remains there throughout development
(Fig. 10). It does not undergo the right-ward expansion and shift
that occurs in animals with full ventricular septation (crocodilians,
birds and mammals; Fig. 11) [15,83,84]. We therefore measured
the size of the atrioventricular junction in formed hearts of
vertebrates from published images and our own material; defined
as the cross sectional area of the atrioventricular orifice(s) relative
to the cross sectional area of the ventricular base, the atrioven-
tricular junction constitutes less than 5% in fishes which have
systemic circulation only, it is ca. 10% in ectothermic vertebrates
which have both systemic and pulmonary circulation, and ca. 20%
in the animals with full ventricular septation (Fig. 12).
Ventricular trabeculations may not relate to septation
The earliest ventricular trabeculations in reptiles are aggregated
into parallel sheets and a similar design can be seen in embryonic
hearts of all vertebrates and in formed hearts of most fish and
amphibians and reptiles [17,22,53,70,72,85–88]. The vertical
septum of the formed non-crocodilian ventricle is among these
sheets and because it is positioned immediately caudal to the
atrioventricular valves, it has repeatedly been hypothesized to
constitute an important part of the complete ventricular septum of
mammals and birds (cf. [3]). It is after the initiation of septation,
however, that the center of the mammalian and avian atrioven-
tricular canal is positioned immediately above the forming septum
by expansion and shift to the right (Fig. 11). Also, in mammals and
birds, the crest of the ventricular septum expresses Bmp2 and Tbx3
and a similar crest has not been found in lizards [23].
Furthermore, the ventricular septum has a left-right gradient of
Tbx5 expression [89]. In reptiles, the cranial part of the cavum
pulmonale does not express Tbx5 and the left-right gradient is thus
established over the muscular ridge, rather than the vertical
septum (Fig. 1A–B, [4]). It is thus arguable that the vertical septum
does not relate to the ventricular septum.
The bulboauricularlamella are a common feature of
vertebrate ventricles
The atrioventricular canal is initially a free standing tube, but
becomes enclosed by ventricular myocardium and acquires a
funnel-like appearance (it is also known as the atrioventricular
funnel or ‘Trichter’ in German). In reptiles, the atrioventricular
canal myocardium is contiguous with smooth-surfaced ventricular
myocardium referred to as bulboauricularlamella [15]. The
bulboauricularlamella can also be recognized in the embryonic
hearts of mammals and birds as ‘gullies’ that contribute to
atrioventricular valve formation (Fig. 13) [84,90,91]. Fishes and
amphibians, also develop bulboauricularlamella [70]. In mouse it
has been shown that the atrioventricular myocardium contributes
much to the left ventricle [30] and we speculate that the
bulboauricularlamella constitute traces of this process.
Only in amniotes is the myocardial outflow tract
ventricularized
In contrast to amniotes, the myocardial outflow tract of fishes
and amphibians is retained in the adult formed heart. It is
prominent in cartilaginous fish, where it constitutes ca. 10–20% of
the entire cardiac mass, in sarcopterygii fish like lungfishes, and
basal actinopterygii fish, such as sturgeons as well as most
amphibians [66,92–95]. In fishes in general, the myocardial
outflow tract contains one to eight rows of valves in the transverse
plane, but only in lungfishes have the multiple valves fused to two
longitudinal ridges [1,66,96,97,97–101]. In amphibians, a spiral
valve of connective tissue may divide the myocardial outflow tract
into a systemic and a pulmocutaneous channel. The ventricular-
ization of the myocardial outflow tract is thus a defining event in
amniotes [102] and the developmental regression, ca. 1 mm in
length, has also been measured in man [103,104] and chicken
[26]. It has long been held that the myocardial outflow tract
contributes by ventricularization to the full ventricular septum in
mammals, birds and crocodilies [1,3,12,15,105–109]. Yet, to the
best of our knowledge there is no explanation as to why the
myocardial outflow tract ventricularizes in reptiles, representing
the ancestral amniote condition. In the derived condition of
mammals and birds, the ventricularization essentially creates the
right ventricle and is probably driven by push from cells being
added distally to the outflow tract from extracardiac precursors
[26,42,44,51]. In zebrafish development, cells are also added to
the arterial pole, but the myocardial outflow myocardium never
grows to the proportion of the heart seen in amniotes (cf. the
miniatures in Fig. 1) and a septation like the muscular ridge is not
manifested [110,111].
Structures derived from the ventricularized myocardial
outflow tract
In reptiles, the myocardial outflow tract is partially divided by a
muscular protrusion, which is continuous with the muscular ridge
caudally and the arterial aorticopulmonary septum cranially.
Therefore, as the muscular outflow tract becomes ventricularized
this protrusion blends seamlessly into the muscular ridge and is
considered a part of it. It has been suggested that the protrusion is
homologous to the spiral valve of the amphibian heart and the so-
called conal septum of the mammalian heart that separates the
lumen of the right ventricular outflow tract from the aorta [3,83].
Also, the complete part and the free-standing part of the muscular
ridge is said to resemble the trabecula septomarginalis (incomplete
part) and moderator band (complete part) of the mammalian and
avian right ventricle [15,21,70,105,107,112]. Consistently, a small
remnant of the myocardial outflow tract can be found ventrally in
the reptile heart, at the pulmonary arterial base, like the right
ventricular outflow tract (or conus, or infundibulum) of the
mammalian right ventricle [71,113–116]. It remains an intriguing
question how the heart tube-like outflow tract myocardium
acquires a ventricular phenotype upon being incorporated into
the ventricle.
The bulbuslamelle may contribute to the right
atrioventricular valve complex
It is perplexing that birds and monotreme mammals have a
mural muscular flap valve at the right atrioventricular junction,
because they evolved independently from reptilian ancestors
where a similar structure is not easily recognized (Fig. 14)
[46,70,117–120]. Only crocodiles also have a right muscular flap
valve and they are grouped as archosaurs together with dinosaurs
and birds [121–123]. Marsupial and eutherian mammals do not
have a right muscular flap valve, yet, in atrioventricular valve
development, the valvular complexes are partly muscular [90,91].
And persisting muscularity of the valve may lead to the congenital
malformation of Ebstein’s anomaly [84,124]. Common to
crocodiles, birds and mammals is a full ventricular septation,
which, as shown in Figure 9, is associated with a right-ward
expansion of the atrioventricular canal. The right margin of the
atrioventricular canal then approaches the position of the
bulbuslamelle as found in the reptile heart and the bulbuslamelle
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may therefore contribute to the formation of the myocardial right
atrioventricular valve (Figs. 1, 9, 11) [22,84].
Summary
The developing reptile heart is characterized by fast growth of
the chambers and slow growth of the flanking segments, exactly
like in mammals and birds. In reptiles, a myocardial sinus venosus
is retained in the formed heart, whereas the sinus venosus of
mammals atrializes to the right atrium to form a vestibule in the
dorsal right atrial wall. Associated herewith, is the absence
(reptiles) and presence (mammals) of an atrial septum secundum.
A myocardial sleeve is absent from the reptilian pulmonary vein
and there is no vestibule in the left atrium. The atrioventricular
canal of reptiles is not interrupted by an insulating plane, as in
mammals and birds. Also, the atrioventricular canal remains on
the cardiac left, whereas it expands and migrates to the right in
mammals and birds (and crocodilians) associated with the
formation of the ventricular septum.
Glossary
aortico-pulmonary septum: the arterial septum of the truncus
arteriosus that separates the pulmonary arterial lumen from the
aortic lumina.
bulboauricularlamelle, dorsal: smooth-surfaced ventricular
myocardium, situated in the inner-most dorsal base of the
ventricle in between the atrioventricular canal and the arteries.
bulboauricularlamelle, ventral: smooth-surfaced ventricular
myocardium, situated in the inner-most ventral base of the
ventricle in between the atrioventricular canal and the arteries.
bulboauricularsporn: thickened myocardium in between the
atrioventricular canal and the right aorta. Derived from the inner
curvature of the embryonic heart tube. Synonyms; bolvo
ventricular spur [125], conoauricular flange [103].
bulbo-ventricular fold: the inner curvature of the transition from
ventricle to myocardial outflow tract. Synonyms; bolvo ventricular
spur, conoventicular flange [103].
bulbuslamelle: smooth-surfaced ventricular myocardium juxta-
posed to the muscular ridge. In pythons and varanid lizards, it
separates the cavum pulmonale and the cavum venosum during
ventricular systole. Defined here as the smooth-surfaced structure
to the right of the bulboauricularsporn, and therefore the
atrioventricular canal, that holds the dorsal valves of the aortae.
Derived from the ventricularization of the myocardial outflow
tract. Synonyms; bulbar flange.
cardiac shunt, left-to-right: the circulation of pulmonary venous
blood (oxygen-rich) to the pulmonary circulation.
cardiac shunt, right-to-left: the circulation of systemic venous
blood (oxygen-poor) to the systemic circulation.
cavum arteriosum: the left-sided chamber of the ventricle that
receives blood (oxygen-rich) from the left atrium; i.e. the ‘systemic
side’ of the ventricle.
cavum dorsale: the combined cavum arteriosum and cavum
venosum, i.e. the cavities to the left of the muscular ridge and the
bulbuslamelle.
cavum pulmonale: the right-sided chamber of the ventricle that
receives blood (oxygen-poor) from the right atrium in diastole; i.e.
part of the ‘pulmonary side’ of the ventricle. Synonyms; cavum
ventrale.
cavum venosum: the central chamber of the ventricle siyuated
to the right of the vertical septum and to the left of the muscular
ridge and the bulbuslamelle. It receives (oxygen-poor) blood from
the right atrium in diastole and is traversed by blood (oxygen-rich)
from cavum arteriosum in systole; i.e. part of the ‘pulmonary side’
of the ventricle.
cavum ventrale: synonym for the cavum pulmonale.
muscular ridge: the smooth-surfaced myocardial ridge-like
structure of the ventricle to the right of the atrioventricular canal
that is continuous with the aortico-pulmonary septum and holds
the ventral valves of the aortae and the dorsal valve of the
pulmonary artery. Synonyms; horizontal septum, Muskelleiste,
cloison helı
¨coidale, ventricular septum, septum interventriculare (a
detailed literature review was made by Webb et al., 1974).
myocardial outflow tract: heart tube myocardium downstream
of the ventricle. Synonyms; conus arteriosus, bulbus arteriosus,
conus cordis, bulbus cordis.
septum spurium: prominent trabeculation of the roof of the
right atrium associated with the sinuatrial valves. Synonyms;
suspensory ligament, dorsal commissure.
spannmuskel: trabecular sheets in the ventricle that are caudal
to the bulbuslamelle and continuous with it.
truncus arteriosus: the arterial trunk proximal to the ventricle
containing the pulmonary artery and the aortae. Synonyms;
bulbus arteriosus.
vertical septum: a sheet (or sheets according to some authors) of
spongy myocardium in the ventricle situated immediately under
the atrioventricular canal. The atrioventricular valves ‘plunge’
unto it during diastole.
Supporting Information
Figure S1 3D models of the heart of the corn snake
(Pantherophis guttatus), 2 to 16 days post laying.
(PDF)
Figure S2 3D models of the heart of the corn snake
(Pantherophis guttatus), 20 days post laying to
3 months.
(PDF)
Figure S3 3D models of the heart of the anole lizard.
(PDF)
Figure S4 3D models of the heart of the embryonic
chicken (4 and 6 days post laying).
(PDF)
Figure S5 3D models of the heart of embryonic man
(Carnegie stages 14 and 18).
(PDF)
Figure S6 3D model of the heart of the adult Burmese
python (Python molurus).
(PDF)
Figure S7 3D model of the heart of the adult ostrich
(Struthio camelus).
(PDF)
Figure S8 Growth of cardiac compartments. The atrial
and ventricular compartments showed exponential growth,
whereas there was little change in the myocardial volume of the
atrioventricular canal and the myocardial outflow tract.
(TIF)
Figure S9 Proliferation of the hearts in two specimens
of anole lizard, as assessed by BrdU incorporation. A.A
7mm section of the st5 specimen, close to the transverse plane,
showing myocardium (blue), nuclei (green) and BrdU positive
Development of Squamate Hearts
PLOS ONE | www.plosone.org 16 June 2013 | Volume 8 | Issue 6 | e63651

nuclei (orange). B.A7mm section of the st12 specimen, close to
the horizontal plane, showing myocardium (blue), nuclei (green)
and BrdU positive nuclei (orange). C–D. Reconstructions of the
myocardium upon which is projected the fraction of BrdU positive
nuclei as described in [50]. The color-scale bar indicates BrdU
incorporation in zero (0) to every fourth nuclei (0.25). E. Internal
view of the ventral halve of the st5 specimen showing relatively
high proliferation in the ballooning atria (la, left atrium; ra, right
atrium) and ventricle (ven). F. Internal view of the ventral halve of
the st12 specimen. At this stage the cardiac compartments almost
have the proportions of the fully formed heart and proliferation is
lower than in the st5 specimen, albeit the outer curvature of the
ventricle still shows some proliferation.
(TIF)
Movie S1 Seen from the right, the beating heart of a
corn snake embryo 10 days after egg laying. mot,
myocardial outflow tract.
(AVI)
Movie S2 Seen ventrally, the beating heart of a corn
snake embryo 10 days after egg laying. mot, myocardial
outflow tract.
(AVI)
Movie S3 Seen from the left, the beating heart of a corn
snake embryo 10 days after egg laying. mot, myocardial
outflow tract.
(AVI)
Movie S4 Seen from the right, the beating heart of a
corn snake embryo 20 days after egg laying. mot,
myocardial outflow tract.
(AVI)
Movie S5 Seen ventrally, the beating heart of a corn
snake embryo 20 days after egg laying. mot, myocardial
outflow tract.
(AVI)
Movie S6 Seen from the left, the beating heart of a corn
snake embryo 20 days after egg laying. mot, myocardial
outflow tract.
(AVI)
Acknowledgments
Givskud Zoo, Denmark, kindly donated the carcass of the euthanized
ostrich. Michael Pedersen and Peter Agger of the MR Center, Skejby
Hospital, Denmark, provided the NMR-scans which the 3D models of the
hearts of the Burmese python and the ostrich are based on. Jaco Hagoort
much improved the 3D models. Diergaarde Blijdorp (Rotterdam, the
Netherlands) kindly donated fertilized eggs of Pantherophis and Hydrosaurus.
Yara Y Oostveen kindly photographed the histological sections that Fig. 10
is based on.
Author Contributions
Conceived and designed the experiments: BJ GvdB R-JO AFMM.
Performed the experiments: BJ GvdB RvdD. Analyzed the data: BJ GvdB
RvdD R-JO TW AFMM. Wrote the paper: BJ GvdB RvdD R-JO TW
AFMM.
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PLOS ONE | www.plosone.org 19 June 2013 | Volume 8 | Issue 6 | e63651