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Developmental Biology 277 (2005) 1 – 15
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Review
The evolutionary origin of cardiac chambers
Marcos S. Simões-Costaa, Michelle Vasconcelosa, Allysson C. Sampaioa, Roberta M. Cravoa,
Vania L. Linharesa, Tatiana Hochgreba, Chao Y. I. Yanb, Brad Davidsonc, José Xavier-Netoa,*
a
Laboratório de Genética e Cardiologia Molecular, Instituto do Coração, Hospital das Clı́nicas, Faculdade de Medicina,
Universidade de São Paulo, São Paulo-SP 05403-900, Brazil
b
Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas da Universidade de São Paulo,
São Paulo-SP 05508-900, Brazil
c
Department of Molecular and Cell Biology, Division of Genetics and Development, Center for Integrative Genomics,
University of California, Berkeley, CA 94720, United States
Received for publication 13 July 2004, revised 7 September 2004, accepted 20 September 2004
Available online 26 October 2004
Abstract
Identification of cardiac mechanisms of retinoic acid (RA) signaling, description of homologous genetic circuits in Ciona intestinalis and
consolidation of views on the secondary heart field have fundamental, but still unrecognized implications for vertebrate heart evolution.
Utilizing concepts from evolution, development, zoology, and circulatory physiology, we evaluate the strengths of animal models and
scenarios for the origin of vertebrate hearts. Analyzing chordates, lower and higher vertebrates, we propose a paradigm picturing vertebrate
hearts as advanced circulatory pumps formed by segments, chambered or not, devoted to inflow or outflow. We suggest that chambers arose
not as single units, but as components of a peristaltic pump divided by patterning events, contrasting with scenarios assuming that chambers
developed one at a time. Recognizing RA signaling as a potential mechanism patterning cardiac segments, we propose to use it as a tool to
scrutinize the phylogenetic origins of cardiac chambers within chordates. Finally, we integrate recent ideas on cardiac development such as
the ballooning and secondary/anterior heart field paradigms, showing how inflow/outflow patterning may interact with developmental
mechanisms suggested by these models.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Heart; Cardiac chambers; Evolution; Development; Retinoic acid; Inflow; Outflow; Chordates; Vertebrates
Introduction
Our understanding of cardiac development has progressed to the point where we would greatly profit from
unifying views across vertebrates. Indeed, Fishman and
Chien (1997) proposed a search for themes unifying cardiac
development in vertebrates. These themes, manifested by
phenotypic traits and underlying genes, represent modules
distinguishing vertebrate hearts from basal chordate circu-
* Corresponding author. Laboratório de Genética e Cardiologia
Molecular, Instituto do Coração, Hospital das Clı́nicas, Faculdade de
Medicina, Universidade de São Paulo, Av. Dr. Eneas C. Aguiar 44,
Cerqueira Cezar, São Paulo-SP 05403-900, Brazil. Fax: +55 11 3069 5022.
E-mail address: [email protected] (J. Xavier-Neto).
0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2004.09.026
latory pumps (Fishman and Chien, 1997; Fishman and
Olson, 1997).
Years after these reviews, we have reasons to believe that
anterior-posterior (AP) patterning may be a common theme
in vertebrate heart development. We discuss evidence for
this interpretation under a working paradigm that pictures
vertebrate hearts as pumps projected to perform double
functions of inflow and outflow. We propose that the cardiac
inflow/outflow organization is laid down in development as
an AP code. This code is presumably established by
mechanisms that include, but are not necessarily limited to
differential retinoic acid (RA) signaling, which divides the
cardiac field into precursors of sinus venosus (SV) and
atrium, as inflow chambers, and precursors of ventricle and
conus arteriosus, as outflow chambers (Rosenthal and
Xavier-Neto, 2000; Xavier-Neto et al., 2001).
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M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
RA signaling seems to be a chordate innovation. The
genes involved in RA metabolism and signaling are
conserved in their sequences and expression patterns from
urochordates to vertebrates (Nagatomo and Fujiwara, 2003).
This suggests that some elements required for partitioning
the vertebrate heart into chambers are already present in
basal chordates. Now that evidence points to RA signaling
as a major determinant in amniote cardiac AP patterning, the
time is ripe to search for the evolutionary roots of cardiac
chambers.
Our aims are fivefold: (1) identify gaps in our understanding of the origin of vertebrate cardiac chambers, (2)
evaluate strengths and weaknesses of animal models and
scenarios of chamber evolution, (3) propose new scenarios
for chamber evolution, (4) discuss whether chordates use
similar mechanisms to pattern their circulatory pumps in the
AP axis, (5) propose a testable model for RA signaling as a
factor in the origin of cardiac chambers.
For this, we will use an interdisciplinary and multispecies analysis, taking into account not only higher
vertebrates, currently dominant animal models, but also
lower chordates and protostomes. The species cited in our
analysis and their evolutionary positions are displayed for
referencing in Fig. 1.
septated ventricles are said to display hearts with five
chambers, two atria, three ventricles (Burggren, 1988;
Burggren and Johansen, 1982). However, three-dimensional reconstruction in mouse embryos indicates that the
SV is poorly developed and that the myocardium of the
outflow tract never forms anatomical chambers (Moorman
and Christoffels, 2003b), indicating that only atria and
ventricles function as true chambers in mice (Christoffels
et al., 2000).
Difficulties in ascribing chamber identity to cardiac
compartments arise because during vertebrate evolution,
cardiac segments may have regressed, merged into others, or
divided into left, right or more compartments. Besides,
chambers can be defined on either morphological (Bourne,
1980; Fange, 1972; Kardong, 2002; Randall, 1970; Romer,
1962) or functional grounds (Christoffels et al., 2000). These
anatomical and functional standards were developed to
account for variable configurations assumed by cardiac
segments, but are adequate only when emphasizing adaptations in higher vertebrates. They are far less helpful when the
goal is to define a vertebrate standard to be compared with
the pumps of animals from a bigger group such as chordates.
We propose as more heuristic the notion that vertebrate
hearts are divided into segments specialized in inflow and
outflow.
What is a heart?
Vertebrate hearts are divided into inflow and outflow
The word heart is used to denote chambered circulatory
pumps, but is also applied to any segment of the circulation
that pumps fluid. For the strict purposes of this review, we
will use the anatomical rather than the functional definition.
Thus, we consider hearts as chambered pumps containing
inflow and outflow segments invested at some point in an
animal’s lifetime with myocytes. With this definition, we
clarify the obvious differences between chambered pumps
and all other circulation driving devices, which we will refer
simply as pumps.
What is a cardiac chamber?
The anatomical tradition distinguishes four original
vertebrate cardiac compartments: SV, atrium, ventricle
and conus arteriosus (Bourne, 1980; Fange, 1972; Kardong, 2002; Randall, 1970; Romer, 1962) (Fig. 2A).
However, the assignment of chamber status to these
compartments can be controversial to the point that it is
rather difficult to get the chamber count right in a given
species. Hagfish hearts are described with three chambers,
SV, atrium, ventricle (Kardong, 2002; Pough et al., 2002;
Randall, 1968), although they may possess a rudimentary
conus arteriosus (Wright et al., 1984). Lampreys are
represented with four chambers: SV, atrium, ventricle and
conus, all very well defined, conspicuous morphological
compartments (Fange, 1972). Varanid lizards with partially
Upon closer inspection, the segmental complexity of the
heart betrays a simpler organization. This is because all
vertebrate hearts are assemblies of segments devoted to two
hemodynamic functions: to receive blood upstream (inflow)
and to pump it downstream (outflow) (Fig. 2A).
This dichotomy is expressed at anatomical, developmental and genetic levels. Inflow and outflow compartments are
distinguished in the hearts of hagfishes and lampreys, the
basal extant vertebrates (Fig. 2B). These animals have Sshaped hearts in which cardiac inflow compartments, SV and
atrium, are located in a different dorso-ventral plane than the
cardiac outflow compartments, ventricle and conus arteriosus
(Fig. 2A). This arrangement is a clever adaptation to improve
mechanical efficiency and constitutes a leap forward from
linear pumps such as peristaltic vessels of cephalochordates
and urochordates (Moller and Philpott, 1973) (Fig. 2C) and
two-chambered hearts of gastropods (Ripplinger, 1957) (Fig.
2D). In these pumps, influx generates symmetric streams that
reflect on valves or constrictions, decreasing mechanical
efficiency (Fig. 2D). The hearts from vertebrates solved this
problem placing inflow chambers dorsal to outflow chambers
and creating atrio-ventricular orifices almost at right angles
with the planes of their inflow/outflow openings (Fig. 2B).
This arrangement establishes asymmetrical inflow streams
directed to atrio-ventricular orifices through a winding
circuit, rather than aimed straight at them (Fig. 2D). Sshaped hearts also eliminate the offsetting effects of outflow
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
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Fig. 1. Phylogenetic tree, consolidated from multiple sources, depicting the evolutionary relationships between the species discussed in the text. The tree has
two main branches stemming from an ancestor with bilateral symmetry. The branch in red corresponds to deuterostomes, while the branch in blue corresponds
to ecdysozoans and lophotrocozoans (protostomes). Question mark indicates that echiuran affinities are still uncertain.
chamber recoil on inflow chamber expansion when fluid is
ejected from the pump (Kilner et al., 2000) (Fig. 2D).
Developmental distinctions between cardiac inflow/outflow are apparent in fate maps where their precursors occupy
different sections in epiblast, primitive streak and cardiac
crescent (De Haan, 1963). Differences between cardiac
inflow/outflow are also reflected in the profile of genes they
express, such as tbx-5, hrt1, raldh2, couptf-II, amhc1 for
inflow, and Irx-4, mlc2-v, hrt-2 for outflow (Bruneau, 2002;
Moorman and Christoffels, 2003a; Xavier-Neto et al., 2001).
Restriction of gene expression is manifested before or after
muscle differentiation. Importantly, genes differentially
expressed almost never restrict their expression to cardiac
chambers and some, such as h-MHC and MLC-2V are
expressed in gradients along the AP axis, suggesting that
strict chamber-specific expression is rare and likely an
elaboration of a primary AP pattern (Christoffels et al., 2000).
These findings provide compelling evidence that the
segmentation of the heart reflects an underlying morphogenetic principle that divides advanced circulatory pumps into
units devoted to inflow or outflow. In vertebrates, this
morphogenetic plan includes changes in the relative position
of these units. Thus, vertebrate hearts are endowed with an
additional level of mechanical sophistication that markedly
improved circulatory efficiency.
Using our criteria, even the sophisticated hearts of
crocodilians, birds and mammals can be reduced to inflow/
outflow segments, chambered or non-chambered, that trace
their origin to hagfishes and lampreys. Thus, from this new
point of view, the apparent variety of vertebrate hearts turns to
a monotonic repetition of inflow/outflow units.
Evolutionary origin of cardiac chambers
We observe a striking discontinuity between the blueprints of pumps in hemichordates, urochordates, cephalochordates, and those of vertebrate hearts (Fig. 2C).
Particularly impressive is the apparent abrupt appearance
of four-chambered hearts in hagfishes and lampreys when
we take the cephalochordate amphioxus as reference. Pumps
in amphioxus are so rudimentary that it is considered
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Fig. 2. (A) S-shaped, vertebrate heart. Inflow chambers are dorsal, outflow chambers, ventral. Red arrows indicate flow direction (Kardong, 2002; Kilner et al.,
2000). (B) In amphibians and amniotes, atrium and sinus venosus (SV) are displaced rostrally (green arrow), obscuring the original dorso/ventral disposition.
(C) Deuterostome circulatory pumps (Brusca and Brusca, 2003; Fange, 1972; Kardong, 2002; Kriebel, 1968; Meglitsch, 1972; Moller and Philpott, 1973;
Rahr, 1979; Randall, 1970). The four-chambered vertebrate heart contrasts with the simpler peristaltic pumps of cephalochordates and urochordates.
Enteropneusts hemichordates display a vesicle squeezing blood from a central sinus. Multiple pumps of the hemal system in holothuroid echinoderms. (D)
Gastropod hearts are linear (Ripplinger, 1957). Hemolymph enters the atrium symmetrically, reflecting away from the atrioventricular valve. Hemolymph
ejection in the aorta produces ventricular recoil (dotted arrows) against the atrium, preventing efficient filling. The dorsal/ventral arrangement of inflow/
outflow chambers in vertebrate hearts produces asymmetrical streams directed to the valve. Systolic ventricular recoil is away from the atrium and assists
inflow (Kilner et al., 2000).
heartless (Moller and Philpott, 1973; Moorman and Christoffels, 2003a; Randall and Davie, 1980). In fact, four
different vessels drive circulation in amphioxus (Moller and
Philpott, 1973). Whether these vessels are homologous to
the vertebrate heart will be discussed later. What concerns
us now is the recognition of a gap in our understanding of
the events that underlie the appearance of chambers in the
hearts of hagfishes and lampreys. So far, there have been
few attempts to understand the genetic basis of this gap and
we believe this will be a fruitful area of investigation.
Amphioxus
Previous reviews (Fishman and Chien, 1997; Fishman
and Olson, 1997; Moorman and Christoffels, 2003a)
discussed the differences between the vertebrate heart and
the ancestor chordate pump. Attention was directed to
Branchiostoma (amphioxus or lancelet) as an extant genus
representing cephalochordates. Using amphioxus as reference, we identify vertebrate novelties such as chambers,
valves, endocardium, septae, epicardium, coronary circulation, a uniform endothelial layer and an electric system.
Since none of these features are observed in amphioxus,
there is merit in assuming this cephalochordate as possessing a proto-vertebrate circulation. However, we need
caution when using amphioxus as a background for the
evolution of the vertebrate circulation. This is because
several features of amphioxus such as its sedentary life, lack
of fins as those of the extinct cephalochordate Paleobranchiostoma, lack of a centralized circulatory system, lack of
striated muscle in their pumps, lack of a midbrain, and
absence of expression patterns already present in urochordates (Blieck, 1992; Hirakow, 1985; Kozmik et al., 1999;
Mallatt and Chen, 2003; Randall and Davie, 1980) suggest
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
that it is anatomically derived and that its body plan was
secondarily simplified (Conway Morris, 2000).
Several circulatory characteristics of amphioxus are
rudimentary. Those traits are shared with basal deuterostomes such hemichordates and holothuroid echinoderms and
even protostomes like annelids, lophophorate phoronids and
echiurans (Brusca and Brusca, 2003). Circulation in
amphioxus is closed and displays: (1) dorsal and ventral
vessels, (2) vascular channels clustered around intestine and
pharynx, (3) lack of continuous endothelial coverage, (4)
multiple contractile vessels (Fig. 3A).
The multiple pumps in amphioxus are not easily
homologized to the vertebrate heart (Carter, 1967; Jefferies,
1986; Moller and Philpott, 1973; Rahr, 1979; Randall and
Davie, 1980). They are peristaltic vessels such subintestinal,
portal and hepatic veins, as well as the endostylar artery, the
vessel that irrigates pharynx and endostyle (Fig. 3A). The
multiple pumps of amphioxus invite questions about its
circulatory physiology, development and genetics. It will be
important to define whether there is a common embryonic
origin for its pumps, whether amphioxus has a main pump
and whether any of these pumps is homologous to the
vertebrate heart.
A step towards solving these questions was taken by
characterization of expression patterns for AmphiNk2-tin,
an amphioxus NK2 gene. Holland et al. showed that the
subintestinal vessel is the first to express the gene after its
5
evagination from the visceral peritoneum. Since the
peritoneum buds off bilaterally from somitic mesoderm
and join ventral to the digestive tract, the theme of paired
mesodermal precursors migrating and fusing at the midline
to form a Nk2-expressing tube is also manifest in
amphioxus (Holland et al., 2003).
Holland et al. suggested that amphioxus pumps have a
common origin in the subintestinal vessel, which after
expanding in AP directions is split by hepatic tissues to
originate all contractile vessels (Holland et al., 2003).
Among these, the endostylar artery developed bulbulii and
may have become the main pump (Randall and Davie,
1980). Thus, we believe the subintestinal vessel is indeed a
homologue of the urochordate pump and vertebrate heart.
Another issue is the nature of the SV in amphioxus, which
is located at the confluence of major returning veins (Fig. 3A)
and resembles the vertebrate SV. However, the status of the
SVas a cardiac chamber is controversial. Whilst some authors
are cautious about its cardiac affinity (Jefferies, 1986; Rahr,
1981; Randall and Davie, 1980), others are emphatic against
it (Moller and Philpott, 1973). Controversy partially stems
from the convention of naming blood reservoirs upstream
from the vertebrate heart as bSVQ (Kardong, 2002). Moreover, the amphioxus SVapparently does not contract (Farrell,
1997; Kardong, 2002; Rahr, 1981), contrasting with the SVof
hagfishes and lampreys (Randall, 1970). Also, if we accept
that all pumps in amphioxus have the same embryological
Fig. 3. (A) The cephalochordate amphioxus (Moller and Philpott, 1973; Rahr, 1979). Circulation is closed. Red arrows identify four peristaltic vessels and flow
direction. At the conjunction of main veins lies a large blood reservoir, the SV. (B) The urochordate Ciona intestinalis (Brusca and Brusca, 2003; Kriebel,
1968). Circulation is open and centralized with a single peristaltic pump enclosed in pericardium. The double-headed red arrow indicates that peristalsis is
bidirectional. (C) The hemichordate Balanoglossus (Brusca and Brusca, 2003; Nubler-Jung and Arendt, 1996). Contraction of a vesicle squeezes the central
sinus, driving hemolymph in the reverse direction of chordates.
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M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
origin, but were separated by hepatic vasculature, then the
anatomical position of the amphioxus SV is not homologous
to its vertebrate counterpart. While the latter is the posteriormost chamber, the former is located between endostylar
artery and hepatic vein, two derivatives of the primordial
subintestinal vessel (Holland et al., 2003). Therefore,
dubbing the amphioxus blood reservoir as SV may be
misleading (Moller and Philpott, 1973).
Nonetheless, there is also support for the hypothesis that
the amphioxus SV is a cardiac chamber. It has on its favor
the similarities between the circulatory plans of vertebrates
and cephalochordates, which place the SV upstream from
the pharyngeal/gill vessels. However, based on circulatory
mechanics and phylogeny, we think that rather than being
the ancestral cardiac chamber, the SV is probably the vestige
of a heart from an ancestor, which regressed in amphioxus.
In summary, more data is needed to define whether the
SV is or is not a derivative of the subintestinal vessel. Lack
of information on such a basic issue reminds us that we still
know very little about the mechanisms responsible for the
particular circulatory design in amphioxus.
Urochordate ascidians
Ascidian circulation is open and consists of a ventral
pump linked to endothelial blood vessels and unlined
sinuses (Satoh, 1994). The rudimentary pump consists of
a single-layered myoepithelial tube surrounded by a
pericardial coelom. There are no chambers, valves or
distinctive AP polarity. However, there is a dorsal ventral
axis distinct from early stages (Brad Davidson, unpublished)
and in many ascidians the pump is bent into a v-shape, (Fig.
3B). The ascidian pump shows the unique ability to reverse
the direction of peristalsis, compensating for inefficient
blood flow through the sinuses and vessels.
Despite the key position as the most basal chordate and
the possession of a bheart-likeQ organ, little research has
been done on the ascidian pump. This was probably due to
the assumption that it was not homologous to the vertebrate
heart. This was in turn based on two premises: 1. That the
ascidian pump was a bpericardial organQ, homologous to the
vertebrate pericardial coelom and not to the heart. 2. The
ability of the ascidian pump to reverse peristalsis indicated a
fundamental difference in its groundplan.
Recent studies in the Ascidian urochordate Ciona intestinalis reversed this assumption. Orthologs of vertebrate cardiac factors such as Nkx, Gata, and Hand are expressed
within paired bilateral rudiments, which migrate ventroanteriorly and fuse along the ventral midline (Davidson and
Levine, 2003). Additionally, there is evidence for a role of the
transcription factor MESP in the initial stages of pump
specification in vertebrates and Ciona (Kitajima et al., 2000;
Satou et al., 2004). In the early Ciona embryo, the future
pump lineage is derived from two MESP-expressing blastomeres, the B7.5 cells. During gastrulation, these cells divide
into posterior-most daughters, which form the anterior-most
tail muscle cells and anterior-most daughters, the trunk ventral cells (TVCs), which form the pump and pericardial
coelom. By the end of neurulation, TVCs consist of bilateral
rudiments migrating towards the ventral trunk and expressing
Nkx, Gata, Hand and Hand-like. During tail extension the
TVCs divide in 816 cells and migrate ventrally, fusing into a
flat plaque of cells, the pump field (Davidson and Levine,
2003).
Thus, the urochordate pump, the vessels of amphioxus
and the hearts of vertebrates are united in a lineage sharing
genetic and morphogenetic mechanisms (Davidson and
Levine, 2003; Dehal et al., 2002; Hirakow, 1985; Holland
et al., 2003; Rahr, 1981).
Amphioxus and Ciona: a synthesis
Analysis of the circulation in Ciona and amphioxus
indicates common features also shared by vertebrates. All
chordates place their main pumps upstream from pharyngeal/gill vessels and, with the exception of urochordates, their
dorsal vessels carry blood posteriorly and ventral vessels
anteriorly. This contrasts with enteropneusts and protostomes such as annelids, lophophorates or echiurans in
which flow direction is reversed (Brusca and Brusca, 2003;
Nubler-Jung and Arendt, 1996) (Fig. 3C).
The centralized circulatory system of urochordates
resembles that of vertebrates, which gradually consolidated
the branchial heart as their main pump. Another interesting
parallel between urochordates and some vertebrates is the
encasing of their pumps in stiff pericardial cavities, an
efficient adaptation designed to match outflow with inflow
(Percy and Potter, 1991; Schmidt-Nielsen, 1990). However,
circulation in urochordates is open rather than closed as in
vertebrates.
Although circulation in amphioxus is closed, as in
vertebrates, a balanced view of its affinities requires analysis
of its multiple pumps and vessels at anatomical and
histological levels. Anatomical and optic microscopic
descriptions indicate overwhelming similarities between
vascular architectures of amphioxus and vertebrates, with
most major vessels in amphioxus being easily homologized
to their vertebrate counterparts (Moller and Philpott, 1973;
Rahr, 1979). However, the multiple contractile vessels in
amphioxus contrast with the main circulatory pumps of
urochordates and vertebrates. In addition, ultrastructural
studies indicate that contractile vessels in amphioxus lack
striated muscle, a continuous endothelium and are poorly
separated from connective tissues, features that link
amphioxus to basal deuterostomes (Hirakow, 1985; Rahr,
1981). Thus, any superficial analysis of the circulation in
amphioxus will generate opposing interpretations according
to the character considered.
In summary, we believe that neither amphioxus, nor
Ciona intestinalis provide, alone, all elements to infer the
ancestral vertebrate circulatory system. Ciona intestinalis
with a circulatory system centralized in its main pump will
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
likely be a much better model for the ancestral vertebrate
pump than the multiple contractile vessels of amphioxus.
Conversely, amphioxus is the closest model for the
vertebrate vascular plan.
A phylogeny for cardiac chambers
Two views guided earlier considerations of the evolutionary relationships between chordate pumps and the
origins of vertebrate hearts. Harvey (1996) suggested that
vertebrate hearts could be traced back to the ancestral
cephalochordate level (Harvey, 1996). Fishman and Chien
(1997) and Fishman and Olson (1997) speculated that the
ancestral chordate pump was a tubular, valveless organ,
similar to those of tunicates and that the peristaltic endostylar artery of amphioxus is an intermediary between the
pumps of urochordates and vertebrates. However, it is
difficult to envision how the sophisticated, pericardially
enclosed pump of urochordates is a predecessor for the
simpler, non-striated contractile vessels of amphioxus
(Hirakow, 1985) (Fig. 3B). In truth the profound changes
experienced by chordates after the split from their common
ancestor complicate the distinction of primitive from
derived characters in any single organism, thus several
scenarios can be suggested.
We propose that one way to provide an analytical
framework relating chordate pumps and a balanced view of
the origins of the vertebrate heart is to integrate pumps
with vessels. In Fig. 4 we depict phylogenies for the
vertebrate heart that take into account pump design and
whether circulatory work is concentrated in one main
pump, or distributed in several roughly equivalent pumps.
7
Scenarios thus vary depending upon whether the chordate
ancestor had a centralized circulatory system with a main,
peristaltic, urochordate-like pump (A1 and A2) or a
decentralized system with multiple, amphioxus-like pumps
(B1 and B2).
In scenario A1, the main urochordate-like pump of the
chordate ancestor was retained in urochordates and in the
cephalochordate/vertebrate ancestor. Cephalochordates,
reverted to a decentralized circulatory system, while
vertebrates elaborated on the ancestral plan to generate
inflow and outflow chambers. In A2, the urochordate-like
pump of the chordate ancestor was retained in urochordates,
but lost in the cephalochordate/vertebrate ancestor. Cephalochordates retained this decentralized circulatory plan,
while vertebrates elaborated on it. In B1, the multiple
peristaltic pumps of the chordate ancestor were substituted
by a main pump in urochordates, while the cephalochordate/
vertebrate ancestor preserved the decentralized arrangement.
The decentralized plan, with multiple pumps was maintained in cephalochordates, and elaborated further in
vertebrates. Finally, in B2 no less than four independent
events acted on chordate pumps.
Scenario A1 is parsimonious. When postulating the
ancestral chordate pump as urochordate-like, all it takes to
explain circulation design in chordates is one regression
event in cephalochordates and a refinement of the ancestral
plan in vertebrates. Scenario A2 requires that one type of
pump arose first in the chordate ancestor only to regress in
the cephalochordate/vertebrate ancestor, which laid down
the basis for an independent lineage of pumps that
developed fully only in vertebrates. Scenario B1 also
requires independent origin of centralized pumps in
Fig. 4. (A1 and A2) A main urochordate-like pump for the ancestor chordate. (B1 and B2) A decentralized circulatory system with multiple pumps for the
ancestor chordate. (A1 and B2) A central urochordate-like pump for the cephalochordate/vertebrate ancestor. (A2 and B1) A decentralized arrangement with
multiple pumps for the cephalochordate/vertebrate ancestor.
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M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
urochordates and vertebrates, while scenario B2 is the least
parsimonious.
Evo-devo, mechanics and the origin of cardiac chambers
A key question in the evolution of vertebrate hearts is the
order of appearance of cardiac chambers. Analysis of
circulatory pumps in extant deuterostomes suggests that
ventricles arose only in vertebrates. Consistent with this, it
has been proposed that the ventricle was the second
chamber in the heart, the one designed to provide high
blood pressures (Fishman and Chien, 1997). The nature of
the original chordate chamber was not clear in this scheme.
One possibility is that the SV is the ancestral chamber and
the ventricle, a vertebrate novelty. There are problems with
this hypothesis. It leaves the origin of the remaining
chambers, atrium and conus arteriosus, already present in
the most basal extant vertebrates, unaccounted for. Furthermore, the classification of the SV in amphioxus as a
cardiac chamber is questionable and besides, its thin walls
and sparse myocytes make it a poor first chamber candidate.
Recent data in amniotes indicate that RA is required in the
cardiac field to generate atrial phenotypes in precursors that
otherwise would differentiate into ventricular cells (Hochgreb et al., 2003). Thus, a ventricular-like cell may be the
cardiac default, instead of a sinus-like cell. Finally, outflow
segments form first in ontogenesis when fusion of the
cardiac crescent into a midline ventricular tube leaves
precursors of atria and SV still undifferentiated in the lateral
mesoderm (De la Cruz and Markwald, 1998). Therefore, the
precedence of outflow over inflow in ontogenesis is not
consistent with the SV being the ancestral chamber.
The identity of this first chamber is difficult to establish.
Hearts do not fossilize. Moreover, there is no living chordate
with such single-chambered heart. Although we cannot rule
out that such an animal existed, it is hard to conceive what
advantages a single-chambered, peristaltic device (Fishman
and Olson, 1997) offers over the peristaltic pump of urochordates or the multiple peristaltic pumps of amphioxus.
Centralizing circulatory work in a single contractile chamber
eliminates the safety factor in multiple pumps, while
providing no mechanical advantage over conventional
peristaltic tubes such as the urochordate pump.
Peristaltic tubes are inefficient because fluid energy is
dissipated by distension of segments downstream from the
contraction wave, their linearity may create stream reflections and retrograde flow can be formed unless coordination
is tight. Vertebrate chambers are efficient because their contractions are synchronized by extensive electrical connections. Proposing the single ancestral chamber as vertebratelike will lead into a trap, since a non-peristaltic chamber
contracting simultaneously will not provide directional flow
unless a valve is present. An ancestor chordate circulation
driven by such pump (Pough et al., 2002) would be fragile
because of the demand placed on valve efficiency and
besides, valves could no longer be vertebrate innovations.
Thus, we propose that cardiac chambers arose in
evolution, not as single units, but as components of a pump
divided by patterning events. Is there evidence for these
primitive chambered hearts? Two-chambered hearts are not
found among extant chordates, nor there is unambiguous
evidence in fossil records (Chen et al., 1999; Mallatt and
Chen, 2003). Two-compartment pumps are, however, found
among gastropods (Ripplinger, 1957), showing that this
circulatory arrangement is viable.
Fig. 4 predicts different embryological origins for cardiac
chambers and several sequences for their appearance. All
these scenarios depend on the status attributed to the
amphioxus SV.
Scenarios A2 and B1 depict an amphioxus-like circulation for the cephalochordate/vertebrate ancestor and
predict complicated sequences for the appearance of
chambers. If we consider the amphioxus SV a chamber,
endostylar artery precursors could have changed in vertebrates to develop conus arteriosus, ventricle and atrium, the
missing chambers in amphioxus. There are two possibilities
if we reject chamber status for the amphioxus SV. In one
view, the non-cardiac SV regressed and all four chambers
were developed de novo from endostylar artery precursors.
Alternatively, endostylar artery precursors originated conus
arteriosus, ventricle and atrium and the SV was co-opted by
an invasion of cardiac myocytes to function like a chamber.
Scenario A1 pictures an urochordate-like heart for the
cephalochordate/vertebrate ancestor and predicts that all
four chambers originated from the partition of a pump field.
This is a simpler scenario because it only requires patterning
the pump field into subdivisions that will define chambers.
This scenario makes sense only if we assume that the
amphioxus SV is not a true chamber, but rather, that there
was a complete regression to a decentralized circulation in
cephalochordates. Otherwise, we would have to propose
independent origins for the SV in cephalochordates and
vertebrates. These considerations emphasize the need to
accommodate the uncertain chamber status of the
amphioxus SV, incorporate pumps and vasculature in the
phylogenetic analysis and rate circulatory configurations
according to their hypothetical mechanical efficiencies.
In Fig. 5 we discuss scenarios that satisfy these
conditions. Scenario C rejects a chamber status for the
amphioxus SV. The ancestor chordate had a main, urochordate-like pump, accessory peristaltic pumps and an open
circulation. Urochordates consolidated the main pump and
lost accessory pumps. The cephalochordate/vertebrate
ancestor developed a closed circulation and maintained the
ancestral pump arrangement. Hence, only the amphioxus
assumed a decentralized circulatory system with multiple
peristaltic pumps. Scenario C suggests that other cephalochordates had amphioxus-like vascular systems, but a
main pump like urochordates and vertebrates. Vertebrates
improved the ancestral pump project, reduced the need for
accessory organs and consolidated the heart as the only
pump.
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
9
Fig. 5. Alternative scenarios for cardiac chambers depending on the status attributed to the amphioxus SV. (C) Cardiac chambers appeared in vertebrates. (D)
Cardiac chambers appeared in the cephalochordate/vertebrate ancestor.
Scenario D considers the amphioxus SV as a chamber.
Predictions for the ancestor chordate and for changes in
urochordates are the same as in C. Scenario D differs from C
by postulating that the cephalochordate/vertebrate ancestor
already had at least 2 cardiac chambers. This pump project
regressed specifically in amphioxus, but was retained or
improved in other cephalochordates. Vertebrates improved
on the ancestor plan and developed their four-chambered
hearts. This suggests that the amphioxus SV is a vestigial
chamber of a sophisticated pump already present in the
cephalochordate/vertebrate ancestor. This explains away the
uncomfortable notion that the weakly contracting SV was
the first chamber in evolution.
One attractive feature of scenarios C and D is the
suggestion that the chordate ancestor had an open vascular
system akin to holothuroid echinoderms and hemichordates,
consistent with its position as a basal deuterostome.
Depiction of a closed, centralized circulatory system with
a main pump in the cephalochordate/vertebrate ancestor
throws light into important issues: 1) It reconciles the
existence of a centralized circulatory system in urochordates
and vertebrates as an ancestral chordate character. 2) It
emphasizes progress towards a proto-vertebrate vascular
plan from a less complex, open urochordate-like vasculature. 3) It obviates the need to suggest the rudimentary
endostylar artery as an intermediate between the urochordate pump and the vertebrate heart. 4) It suggests that the
portal pump in hagfishes is homologous to a pump
presumably present in the liver circulation of the cephalochordate/vertebrate ancestor and amphioxus.
Scenario D suggests that chambers appeared among
deuterostomes right after the split from urochordates. In this
10
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
view the circulatory pump of the cephalochordate/vertebrate
ancestor already had multiple chambers. Thus, the abrupt
appearance of the four cardiac chambers in vertebrates may
be a misrepresentation resulting from an incomplete fossil
record and/or the absence of intermediate forms in extant
animals.
their own RA. Hence, these elements support the view that
inflow/outflow divisions are organized as AP sections in
two steps, first by diffusion of RA and later by a wave of its
synthetic enzyme (Fig. 6).
The two-step model and the origin of cardiac chambers
RA divides the amniote heart into inflow and outflow:
the two-step model
We suggested that chambers arose in evolution through
patterning of cardiac precursors by signaling events. In fact,
work from different groups converged to show that RA
signaling is a serious candidate for one such event (Chazaud
et al., 1999; Kostetskii et al., 1999; Niederreither et al.,
1999; Xavier-Neto et al., 1999).
RA signaling divides the cardiac field in the AP axis
creating domains containing precursors of sino-atrial or
ventricular tissue (Hochgreb et al., 2003). This binary division
foretells the apportionment of adult heart tissue into inflow/
outflow chambers and can be identified as an early AP pattern
by expression of several markers (Xavier-Neto et al., 2001).
The work supporting the role of RA in cardiac AP
patterning was reviewed (Xavier-Neto et al., 2001). Briefly,
RA induces the sino-atrial phenotype in posterior cardiac
precursors, while anterior precursors must be protected from
RA, albeit transiently, to differentiate into ventricular cells
(Hochgreb et al., 2003).
Recently, we identified the mechanisms that convey the
RA signal to amniote cardiac precursors at the times of
commitment to AP fates (Hochgreb et al., 2003). RA
reaches cardiac cells by two modes: diffusion and activation
of its synthesis in the heart field. These depend on
expression patterns of RALDH2 (Aldh1A2), the key
retinaldehyde dehydrogenase and a predictor of cardiac
RA synthesis (Moss et al., 1998).
Diffusion of RA from posterior mesoderm seems to be
the main mechanism for specification of cardiac AP fates.
At early gastrulation stages several hundred micrometers
separate the chicken cardiac field from RALDH2-expressing mesodermal cells. This distance is progressively
reduced, such that at headfold stages the anterior limit of
RALDH2 expression in the lateral mesoderm abuts the
posterior limit of the cardiac field. Thus, RA diffusing from
posterior paraxial and lateral mesoderm is thought to specify
posterior cardiac precursors to an inflow fate (Fig. 6).
Determination of cardiac AP fates occurs only between
stages HH7–HH8 and may require concentrations of RA
higher than those provided by diffusion. Indeed, it is likely
that a special mechanism evolved to guarantee increased RA
concentrations in the posterior cardiac field at the time
inflow fates are sealed. This increase is presumably set up
by a caudorostral wave of RALDH2 that appears in the
cardiac field at HH7–HH8. This caudorostral wave endows
posterior cardiac precursors with the capacity to produce
A conservative view of cardiac segmentation is that
double assurance mechanisms operate in cardiac AP
patterning. As such, separate mechanisms may act on a
neutral myocardial default to specify inflow and outflow
fates (Hochgreb et al., 2003).
Evidence in zebrafish supports the idea that the cardiac
default is a neutral myocyte and that separate events specify
atrial and ventricular phenotypes. Zebrafish vmhc1 and
amhc1 already mark separate cardiac populations at the time
amhc1 is activated (Berdougo et al., 2003). Moreover,
recent results suggest that the nodal pathway may be
involved in ventricular specification (Keegan et al., 2004).
However, one prediction of the two-step model is that the
myocardium default may approximate the ventricular
phenotype (Hochgreb et al., 2003). This is supported by
evidence from RA-insufficiency studies (Chazaud et al.,
1999; Heine et al., 1985; Niederreither et al., 1999; XavierNeto et al., 1999) and by experiments showing that while
combinations of morphogens induce vmhc1-expressing
cardiac tissue, the atrial marker amhc1 is never induced
(Lopez-Sanchez et al., 2002). Furthermore, this hypothesis
is also consistent with the fact that, at early chicken heart
tube stages, even presumptive atrial precursors express
vmhc1 before turning on amhc1 (Yutzey et al., 1994).1
Assuming the cardiac default as a ventricular-like cell has
some advantages. If we think of all basal chordate pumps as
formed by archetypal ventricular-like cells we can suggest
insights for two issues of cardiac evolution and development.
The first is the nature of ventricular determinants. These
factors have not yet been characterized. It is possible that
these determinants may have, in a sense, already been found
but not fully recognized. We suggest that these agents may be
the factors associated with expression of the standard cardiac
phenotype. Thus, the ground state of a chordate pump cell
may have been similar to a ventricular myocyte, a phenotype
modified in evolution to originate chamber cell variants.
The second is the evolutionary transition from a peristaltic
vessel to a contractile chamber. Cardiac chambers might have
originated from further differentiation and morphogenetic reorganization of ventricular-like cells, rather than from
1
Contrast between patterns of vmhc1 and amch1 expression in
chicken and zebrafish may relate to slight differences in the timing of
muscle differentiation versus anterior-posterior patterning. In chicken
inflow precursors, cardiac muscle differentiation (whose readout is vmhc1
expression) precedes posterior patterning (whose readout is amhc1
expression), while in zebrafish inflow precursors may become committed
to a posterior fate even before cardiac muscle differentiation, thus bypassing
an early phase of vmhc1 expression.
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
11
Fig. 6. (A) Double in situ hybridization (ISH) for RALDH2, the key RA synthetic enzyme (orange) and for GATA-4, a marker for the chick cardiac field
(purple). (B) Double ISH for RALDH2 (orange) and for Tbx-5, a marker for the mouse cardiac field. (C) Overlay of RALDH2 (orange) and GATA-4 ISHs
from HH8 chick embryos illustrates the topological relationship between RA signaling and the cardiac field during irreversible commitment to inflow and
outflow fates. Note the caudorostral wave of RALDH2 engulfing the posterior half of the cardiac field. (D) Double ISH for RALDH2 (orange) and for Tbx-5
(purple) after determination of inflow and at outflow fates in the mouse. The caudorostral wave RALDH2 overlaps the posterior, unfused cardiac field. (E, F)
The two-step model for specification (E) and determination (F) of inflow (posterior) and outflow (anterior) identities in the vertebrate cardiac field. Purple,
cardiac field. Orange, RALDH2 expression domains. RA diffusing from posterior mesoderm specifies posterior cardiac precursors to inflow (sino-atrial) fates,
while outflow (ventricular and truncal) fates are specified by very small concentrations of the retinoid or by its absence (Step 1). Determination of inflow/
outflow identities occurs later when a caudorostral wave of RALDH2 expression in the anterior lateral mesoderm sharply increases RA concentrations in
posterior cardiac precursors (Step 2). (G) Late tail bud-stage ISH for Ci-RALDH2 in Ciona intestinalis. (H) RALDH2 is initially expressed in the anterior tail
muscle cells and the pump lineage TVCs. (I) Larval-stage ISH for Ci-RALDH2. (J) Larval-stage ISH for Ci-SercaA, a marker for cardiac lineage in Ciona. (K)
The proximity between Ci-RALDH2 expression and the cardiac field resembles phase one of the two-step model.
sequential addition of genetic modules for chambers made
from entirely new myocytes. This is consistent with the
notion that chambers may have arisen not one at a time
(Bourne, 1980), but as two, perhaps more units, created
simultaneously by patterning events such as RA signaling.
This notion, however, is not incompatible with the alternative
idea of a neutral myocyte default that was gradually shaped
by multiple mechanisms into a chambered heart. Hence, by
avoiding the need to postulate an unlikely intermediate such
as the single-chambered heart, the two-step model offers a
natural transition from peristaltic pumps of basal chordates to
four-chambered hearts of vertebrates.
Origin of cardiac chambers in a framework of RA
signaling
The two-step model suggests a framework to approach
the phylogeny of cardiac chambers. Following the paradigms of chicken and mice it is reasonable to assume that
inflow chambers are determined only when the caudorostral
wave of RALDH2 reaches posterior cardiac precursors,
while outflow chambers are indirectly defined by lack of
RALDH2 in anterior precursors. Thus, patterning of a pump
field into chambers would be a matter of changing topology
between myogenic factors and a RA synthetic enzyme. If
these relationships hold, it will be interesting to determine
whether acquisition of cardiac chambers in chordates
correlates with emergence of the RALDH2 caudorostral
wave as a developmental mechanism.
Is AP patterning by RA signaling conserved in chordate
pumps?
Conservation in vertebrates and cephalochordates
Studies in chicken and mice demonstrated similar
mechanisms of cardiac inflow/outflow patterning by RA
(Hochgreb et al., 2003). Although these mechanisms could
have been developed independently in birds and mammals,
it is likely that they were present in the ancestral amniote or
even in amphibians or fishes.
The case for RA determining the inflow/outflow organization of the amphibian heart is unclear. What is established
in amphibians as well as in fishes and birds, is that high RA
concentrations block cardiac differentiation (Drysdale et al.,
1997; Stainer and Fishman, 1992; Osmond et al., 1991).
RA mechanisms operate in fish cardiac AP patterning.
One of the first demonstrations of the effects of RA in
cardiac chamber morphogenesis was in the zebrafish. RA
deleted cardiac chambers in a dose-dependent fashion, first
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M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
in outflow chambers, only affecting inflow chambers at the
highest concentrations (Stainier and Fishman, 1992). This
observation foretold the present view that RA has multiple
effects on cardiac patterning. RA’s ability to block cardiac
differentiation (Keegan et al., 2003) may reflect an ancient
mechanism for delimiting morphogenetic fields of chordate
pumps, while the graded sensitivity may relate to a later
ontogenetic and/or phylogenetic role in AP patterning.
The lateral mesoderm in zebrafish expresses RALDH2 as
in chicken and mice, but we do not know whether these
patterns are as dynamic as the amniote caudalrostral wave.
The fact that two zebrafish RALDH2 mutants, neckless
(Begemann et al., 2001) and no-fin (Grandel et al., 2002),
display cardiac defects, suggests that cardiac AP patterning
by RA is ancestral and continues to shape vertebrate hearts.
The evidence in amphioxus implicates RA in AP
patterning. RA treatment reduces pharynx size and abrogates
mouth/gill slit development, posteriorizing the embryo.
Conversely, treatment with a RA pan-antagonist expands
the pharynx posteriorly and enlarges the mouth (Escriva et al.,
2002; Holland and Holland, 1996). These results are similar
to data from urochordates (Hinman and Degnan, 1998;
Katsuyama and Saiga, 1998), suggesting that RA signaling in
basal chordates complies with the vertebrate scheme. However, there are no reports linking RA signaling to development of the subintestinal vessel. Thus, we do not know
whether RA patterns amphioxus peristaltic pumps.
Conservation in urochordates
RALDH2 expression in Ciona demonstrates an intriguing spatial relationship between RA signaling and the pump
field. Detailed in situ analysis indicates that Ci-RALDH2 is
initially expressed in all of the progeny of B7.5, the anterior
tail muscle cells and the pump lineage TVCs (Davidson,
unpublished). At the late tailbud stage, RALDH2 expression
in TVCs is eliminated, corresponding to the up-regulation of
Nkx, Hand, and Hand-like.
This indicates that RALDH2 expression delineates the
posterior border of the emerging pump field as in fishes,
amphibians and birds, and that there may be a reciprocal
interaction in which pump specification programs downregulate RALDH2 (Fig. 6). Indeed, Hinman and Degnan
(1998) demonstrated in the urochordate Herdmania curvata
that RA suppresses pump development during embryonic
and early larval stages (Hinman and Degnan, 1998). Thus,
the early Ciona pump field seems to correspond to the initial
RA-free ventricular precursors in vertebrates. Whether there
is a secondary interaction between RA and the differentiated
Ciona pump remains to be determined.
At later stages the relationship between the differentiating pump and RALDH2 becomes dynamic. Unpublished
observations in Ciona larvae and young juveniles indicate
that there is a caudorostral wave of RALDH2 (Brad
Davidson, unpublished). However, this wave apparently
skips over the heart, progressing from posterior to anterior
neighboring structures. Thus, the caudorostral wave is also
present in urochordates, although its role in pump development is not clear.
Inflow/outflow, ballooning and secondary/anterior heart
fields
The last years witnessed the elaboration of multiple ideas
on cardiogenesis such as the modular view, the ballooning
model, the concept of a secondary heart field and, here, the
inflow/outflow paradigm (Abu-Issa et al., 2004; Christoffels
et al., 2000; Fishman and Chien, 1997; Fishman and Olson,
1997; Kelly and Buckingham, 2002).
Christoffels et al., 2000 proposed that only atrial and
ventricular precursors in the ventral primitive cardiac tube
proliferate rapidly and grow out (balloon), becoming
highly contractile and densely connected, while the
remaining myocardium displays a phenotype akin to the
primitive heart, functionally more suitable as sphincters or
conduits.
Another major advance in cardiac development was the
recognition that not all cardiac precursors are contained in
the primitive tube formed by fusion of the cardiac crescent,
the primary heart field (De la Cruz and Markwald, 1998).
This recognition originated the concept of a secondary/
anterior heart field represented in pharyngeal mesoderm,
from which cells contribute to segments from the right
ventricle to the truncus in mice, or from the conus to the
truncus in chicken (Kelly et al., 2001; Mjaatvedt et al.,
2001; Waldo et al., 2001).
Clearly, none of these different views are, per se,
sufficient to explain how cardiac segments evolved and
were set up in all vertebrates. Therefore, to identify the truly
common mechanisms we have to weigh the relative
contributions of the diverse developmental processes that
build the heart in lower and higher vertebrates, the different
techniques utilized and their limitations, as well as the
different developmental stages considered.
Even after careful consideration of all above factors it
was still difficult to reconcile some aspects of these
various views. However, recent evidence suggests that
the so-called secondary/anterior heart field derives from a
cell population that is located medially and dorsally to the
cardiac crescent and thus, contiguous to the primary
cardiac field (Cai et al., 2003). This secondary cardiac
cell precursor pool expresses the LIM homeodomain
transcription factor isl-1and contributes to outflow as well
as inflow populations that reach the heart after fusion of
the cardiac crescent (Cai et al., 2003; Meilhac et al., 2004).
Thus, at early stages, cells that will form the secondary
cardiac field may actually be part of a complex single heart
field (Abu-Issa et al., 2004).
These new findings are powerful enough to reconcile
and integrate ballooning, secondary/anterior cardiac field
and inflow/outflow ideas. By virtually sharing the same
M.S. Simões-Costa et al. / Developmental Biology 277 (2005) 1–15
embryonic position at late gastrulation stages, cells from
the cardiac crescent and from the isl-1-expressing population, must be subjected to the same dynamic AP
patterning by RA, which at this phase, seals inflow/
outflow fates of the myocardium. After AP patterning,
migration of anterior, RA-free, isl-1-expressing cells, may
form the pharyngeal precursor pool that contributes to
outflow segments, while posterior isl-1-expressing cells
may remain in place and contribute to inflow segments.
After AP and dorsal-ventral patterning, segments of the
heart tube such as atria and ventricles in higher vertebrates
(Christoffels et al., 2000), but perhaps sinus venosus and
conus/bulbus arteriosus as well in lower vertebrates, may
activate morphogenetic programs that will organize cells
into anatomic cardiac chambers (Fange, 1972; Kardong,
2002; Randall, 1970; Romer, 1962). Thus, inflow and
outflow patterning of the heart field may set the stage for
ballooning, or additional mechanisms, to form, in a
species-specific fashion, chambers from the four original
vertebrate cardiac compartments, SV, atrium, ventricle and
conus arteriosus.
Conclusion
We reviewed evidence pointing to vertebrate hearts as
composites of inflow/outflow segments presaged by a
division of precursors in the AP axis by mechanisms
including, but not necessarily limited to, RA signaling.
Looking at hearts from this prism allows us to abstract
from the complexities of vertebrate design and define a
standard to be compared with the circulatory pumps of
the other extant chordates. Even after this simplification,
the outcome of these comparisons is recognition of the
substantial differences in groundplans separating the
peristaltic organs of the latter from the chambered devices
of the former.
Analyzing the differences in pump design in chordates it
becomes apparent that cephalochordates and urochordates
had enough time to diverge and develop specializations that
have made it difficult to recognize in them the characteristics of the ancestral chordate pump. Integrating several
lines of evidence we propose, as more parsimonious, the
phylogenetic scenarios portraying the ancestor chordate
circulation as open and driven by a main, pre-pharyngeal,
and other accessory pumps. According to our views, cells
akin to ventricular myocytes formed the main, prepharyngeal pump. At some point in evolution the precursors
of these cells were patterned in the AP axis, generating all
or a subset of chamber-specific myocytes. We suggest that
this event made possible a later morphogenetic reorganization that created at once, two, perhaps more, cardiac
chambers.
Using this conceptual background we suggested novel
evolutionary relationships between chordate pumps and
concluded that the status attributed to the amphioxus SV is a
13
crucial problem in the evolutionary origin of cardiac
chambers. If the amphioxus SV is not a cardiac chamber
then these structures appeared only in vertebrates. Alternatively, determining the amphioxus SV as a cardiac
chamber will push evolution of cardiac chambers to the
cephalochordate/vertebrate ancestor. Resolving the affinities
of the amphioxus SV will require that we establish its
molecular signature.
By assuming RA signaling as one evolutionary mechanism that creates cardiac inflow/outflow divisions, we
suggest an experimental program to test ideas proposed
here. First, it will be necessary to establish whether the two
RALDH2 expression patterns associated with the steps to
commitment to cardiac inflow/outflow fates in amniotes are
also present in other vertebrates and chordates as mechanisms of pump AP patterning. If this holds true, we will
have calibrated an investigation tool to scrutinize the
phylogenetic origins of cardiac chambers within chordates.
The conservation of these RALDH2 expression patterns
in Ciona intestinalis was surprising since urochordate
pumps lack overt AP asymmetries. RALDH2 expression
posterior to the pump field may reflect an ancient role for
RA in restricting the emerging pump field. However, the
presence of a caudorostral wave raises intriguing possibilities. Perhaps the ascidian pump is more complex than
apparent, using RALDH2 to establish dorsal/ventral asymmetries possibly related to the vertebrate atrium and
ventricle. Alternatively, the ascidian pump may derive from
a more complex organ, maintaining a dynamic RALDH2
pattern despite the loss of AP complexity. The urochordate
RALDH2 caudorostral wave may fulfill non-cardiac
requirements that were subsequently recruited in higher
chordates to pattern a newly evolved chambered heart.
Testing these hypotheses will require a thorough evaluation
of RALDH2 patterns and the effects of RA signaling in
chordate pump development.
Acknowledgments
We are indebted to Antoon Moorman for many suggestions and criticisms. We also thank Marianne BronnerFraser and Klaus Hartfelder for reviewing the manuscript.
This work was partially supported by FAPESP grants 0100009-0, 03/09998-2, 06555-2, 02/11340-2, 02/13652-1,
CNPq 478843/01 and CAPES.
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