Epicardial development in lamprey supports an evolutionary origin of the

vertebrate epicardium from an ancestral pronephric external glomerulus

Manuel A. Pombal,a Rita Carmona,b Manuel Megıas,a Alejandro Ruiz,b Jose Marıa Perez-Pomares,b andRamon Munoz-Chapulib,�

aNeurolam group, Department of Functional Biology and Health Sciences, Faculty of Biology, University of Vigo, 36310 Vigo

(Spain)bDepartment of Animal Biology, Faculty of Sciences, University of Malaga, 29071 Malaga (Spain)�Author for correspondence (email: chapuli@uma.es)

SUMMARY The epicardium is the outer layer of thevertebrate heart. Both the embryonic epicardium and itsderived mesenchyme are critical to heart development,contributing to the coronary vasculature and modulating theproliferation of the ventricular myocardium. The embryonicepicardium arises from an extracardiac, originally pairedprogenitor tissue called the proepicardium, a proliferation ofcoelomic cells found at the limit between the liver and the sinusvenosus. Proepicardial cells attach to and spread over thecardiac surface giving rise to the epicardium. Invertebratehearts always lack of epicardium, and no hypothesis has beenproposed about the origin of this tissue and its proepicardialprogenitor in vertebrates. We herein describe the epicardialdevelopment in a representative of the most basal livinglineage of vertebrates, the agnathan Petromyzon marinus

(lamprey). The epicardium in lampreys develops by migrationof coelomic cells clustered in a paired structure at the roof ofthe coelomic cavity, between the pronephros and the gut.Later on, these outgrowths differentiate into the pronephricexternal glomerulus (PEG), a structure composed of capillarynetworks, mesangial cells, and podocytes. This observationis consistent with the conclusion that the primordia of themost anterior pair of PEG in agnathans have been retainedand transformed into the proepicardium in gnathostomes.Glomerular progenitor cells are highly vasculogenic andprobably allowed for the vascularization of a cardiac tubeprimarily devoid of coronary vessels. This new hypothesisaccounts for the striking epicardial expression of Wt1 andPod1, two transcription factors essential for development ofthe excretory system.

INTRODUCTION

The heart in vertebrates is constituted of three cell layers,

epicardium, myocardium, and endocardium, with a contribu-

tion of neural crest cells. Both myocardium and endocardium

derive from the precardiac mesoderm. The epicardium is

the outer layer of the vertebrate heart, and it gives rise to a

mesenchymal cell population which is critical to heart devel-

opment, as these epicardially derived cells (EPDC) contribute

to the formation of the coronary vasculature and the cardiac

interstitium (Vrancken-Peeters et al. 1999; Perez-Pomares

et al. 2002; Guadix et al. 2006). Furthermore, epicardium and

EPDC induce the proliferation of the myocardium and it has

been reported that their activity is essential for proper devel-

opment of the ventricular compact layer (Stuckmann et al.

2003; Lavine et al. 2005; Merki et al. 2005). For this reason,

anomalous epicardial development may cause severe heart

malformations (Kwee et al. 1995; Yang et al. 1995).

The embryonic epicardium arises from an extracardiac and

originally paired progenitor tissue called the proepicardium, a

proliferation of coelomic cells at the limit beetween the liver

and the cardiac sinus venosus in all the vertebrates so far

studied (Manner et al. 2001). Both proepicardial primordia

develop in fishes (Munoz-Chapuli et al. 1994) whereas only

the right one fully forms in birds (Schulte et al. 2007). In

mammals both proepicardial primordia fuse in the midline

forming a crescent that covers all the septum transversum

area (Schulte et al. 2007). In all cases, proepicardial coelomic

cells attach to the cardiac surface and spread on the myocar-

dium, giving rise to the epicardium.

In contrast to the vertebrate cardiac bauplan, the hearts

in non-vertebrate metazoans are usually formed by one or

several myoepithelial cell layers but they always lack a

epicardium. Despite this main difference, no hypothesis has

hitherto been proposed about the evolutionary origin of this

tissue and its proepicardial progenitor whose formation

is dependent on molecular and cellular mechanisms that are

poorly known (Schlueter et al. 2006).

We have studied the epicardial development in a repre-

sentative of the most basal living lineage of vertebrates, the

EVOLUTION & DEVELOPMENT 10:2, 210 –216 (2008)

& 2008 The Author(s)

Journal compilation & 2008 Blackwell Publishing Ltd.

210

agnathan P. marinus (lamprey). Our results have revealed

an unsuspected scenario on the evolutionary origin of the

proepicardium, relating this tissue with the primitive excretory

system of vertebrates. Surprisingly, a relationship between the

epicardium and the kidneys had been previously suspected

due to some common features in gene expression, but an

evolutionary and developmental basis accounting for this re-

lationship was lacking in the literature. The aim of this paper

is to show our findings on the epicardial development in

lampreys and to provide a novel model on the origin of the

epicardium in vertebrates.

MATERIAL AND METHODS

This study was carried out in early prolarvae of the sea lamprey

(P. marinus L.; n522) reared from artificially fertilized eggs.

Gametes were obtained from sexually mature adult lampreys

caught during their upstream migration (from late May to early

July) in the Ulla and Mino Rivers (northwest of Spain). The

methods for maintaining the embryos and prolarvae were essen-

tially the same as those described by Piavis (1971). The fertilized

eggs were transferred to the hatchery incubators and placed into

plastic trays. They were kept with circulating water and under

appropriate conditions of darkness and temperature (181C). Under

these conditions, in our broods hatching occurred at 12–13 days

postfertilization (dpf). Stages of prolarvae are indicated according

to their age in days after fertilization. All experiments were

conducted in accordance with European Community guidelines

on animal care and experimentation to minimize pain and

discomfort.

Prolarvae were fixed in Bouin’s fixative, washed in alcohol,

dehydrated in a graded series of ethanol, and embedded in paraffin.

Transverse and sagittal sections (5–8mm thick) were cut on

a Leica RM 2145 microtome (Leica Microsystems, Barcelona,

Spain) and subsequently stained with hematoxylin–eosin.

Dogfish embryos (Scyliorhinus canicula L.) were used for com-

parative purposes. Fertilized eggs were obtained from adult females

collected in the Bay of Malaga (Western Mediterranean) by

commercial trawl vessels. The eggs were kept in indoor tanks of

well-aerated seawater. Egg capsules were open at intervals and the

embryos were anesthetized in 0.04% tricaine methanesulfonate

(MS-222, Sigma-Aldrich Co., St. Louis, MO, USA) in seawater

and measured. Some embryos were fixed in 1.25% glutaraldehyde

and 1% paraformaldehyde and embedded in Araldite 502 as

described previously (Munoz-Chapuli et al. 1996). Semithin

sections (0.5mm) were obtained with an ultramicrotome and

stained with toluidine blue. Other embryos were fixed in methanol–

acetone–water (2:2:1), paraffin embedded, sectioned, and stained as

described above.

Immunolocalization of the Wilms’ tumor suppressor transcrip-

tion factor (Wt1) in avian embryos was performed as described

previously (Carmona et al. 2001). Briefly, the embryos were excised

and cryoprotected in 10%, 20%, and 30% sucrose solutions,

embedded in OCT and snap frozen in liquid nitrogen-cooled is-

opentane. Cryostat sections were collected on poly-L-lysine-coated

slides and fixed for 10min in 1:1 methanol–acetone at � 201C.

Then, the sections were rehydrated, the endogenous peroxidase

activity was quenched by incubation for 30min with 3% hydrogen

peroxide in Tris-phosphate buffered saline (TPBS). Non-specific

binding sites were saturated for 30min with 16% sheep serum, 1%

bovine serum albumin, and 0.5% Triton X-100 in TPBS (SBT).

Endogenous biotin was blocked with the avidin–biotin blocking kit

(Vector, Burlingame, CA, USA). Sections were subsequently incu-

bated overnight at 41C in polyclonal anti-human Wt1 (Sc-192,

Santa Cruz, Heidelberg, Germany), diluted 1:500 in SBT, washed,

incubated for 1h at room temperature in biotin-conjugated anti-

rabbit goat IgG (Sigma-Aldrich Co.) diluted 1:100 in SBT, washed

again, and incubated for 1h in avidin–peroxidase complex (Sigma-

Aldrich Co.) diluted 1:150 in TPBS. After washing, peroxidase

activity was developed with Sigma Fasts 3,30-diaminobenzidine

(D4168; Sigma-Aldrich Co.) tablets according to the indications of

the supplier.

RESULTS

The epicardium in lampreys develops between 17 and 26 dpf,

from a paired outgrowth of coelomic cells in the dorsal

part of the coelomic cavity, between the pronephros and

the gut (Fig. 1). Ciliated nephrostomes were always located

cephalic and caudal to these cell clusters. The coelomic

outgrowths are not yet developed in embryos of 12dpf

(Fig. 1A) while in prolarvae of 14dpf, the earliest evidences

of cell outgrowth in this area are already present (Fig. 1B).

In prolarvae of 17dpf, the right cell cluster attaches to the

dorsal side of the ventricle, close to the atrioventricular

junction (Fig. 1C). From this area, epicardial cells can be

seen apparently spreading over the ventricular surface. How-

ever, the atrium and the ventral side of the atrioventricular

groove are devoid of epicardium. In prolarvae of 20 and

23dpf, the attachment between the right outgrowth of

coelomic cells has spread over the dorsal and right surfaces

of the ventricle forming a flattened, squamous-monostratified

epithelium (Fig. 1, D–F). As a consequence, most of the ven-

tricular and atrial surfaces are lined by epicardial cells. The

contact between the right cell outgrowth and the cardiac sur-

face is disappearing by 26dpf (Fig. 1, G and H). In later

stages, there is no contact between the coelomic cell clusters,

which become the right and left pronephric external

glomerulus (PEG) (Fig. 1, I–K), and the heart surface.

Blood-containing vessels are abundant in these glomeruli

from these stages on.

For comparison purposes, Fig. 2 shows the localization

and structure of the proepicardium in embryos of a basal

gnathostome, the dogfish (S. canicula). In this species the

proepicardium is paired, and it releases free-floating cells into

the pericardial cavity that adhere to the myocardium (Munoz-

Chapuli et al. 1994). However, in earlier stages there is an

attachment between the right proepicardium and the ventric-

The origin of the vertebrate epicardium 211Pombal et al.

212 EVOLUTION & DEVELOPMENT Vol. 10, No. 2, March^April 2008

ular surface (Fig. 2, A and C). Later in development, the

proepicardium is located on the developing septum transver-

sum, between the liver and the sinus venosus, and it does not

have any contact with the heart surface. When the epicardial

development is completed, the proepicardial cells showed

signs of apoptosis (not shown).

Figure 2 also shows the similarities in the expression pat-

tern of the transcription factor Wt1 in the proepicardium and

Fig. 2. Features of proepicardial development in fish and avian embryos. (A–D). Proepicardial development in early dogfish embryos(Scyliorhinus canicula). Transverse sections showing the proepicardium as a paired outgrowth of the coelomic epithelium of the developingseptum transversum (ST). Note how the right proepicardium (arrow and RPE) contacts the surface of the ventricle (V) where epicardial cells(EP) can already be seen over the myocardium (M). The left proepicardium (arrowhead and LPE) releases cells in the coelom. These floatingcells also adhere to the myocardium (Munoz-Chapuli et al. 1994). OE, oesophagous. (E). Sagittal section of a late dogfish embryo. Theproepicardium (PE) is located on the developing septum transversum, between the liver (LI) and the sinus venosus (SV), just at the level ofthe hepatic vein (HV). An epicardium (EP) containing epicardial-derived mesenchymal cells is already developed on the ventricle. Note thedistance between the proepicardium and the pronephric/mesonephric tubules (P/MN). (F–H). Expression of the transcription factorWt1 inquail embryos. Wt1 is expressed by epithelial (arrows in H) and mesenchymal cells of the proepicardium (PE), epicardium (E) andmesonephros (MN), but not by the nephric ducts (ND). It is shown in F the attachment of the right proepicardium to the ventricle (V), closeto the atrioventricular groove (AV). Note the proximity of the proepicardium to the liver (LI).

Fig. 1. Epicardial development in the lamprey Petromyzon marinus. (A) Embryo of 12 days post-fertilization (dpf). The coelomic epitheliumcovering the developing pronephros shows no signs of outgrowth towards the coelom (arrows). DT, digestive tube; H, heart; NS,nephrostome. (B) Newly hatched prolarva of 14dpf. Clusters of coelomic cells (arrows) can be seen between the nephrostomes (NS) and thedigestive tube (DT). These clusters represent the primordia of the pronephric external glomerulus (PEG). (C) Prolarva of 17dpf. The rightcell outgrowth contacts the ventricle surface (arrow) close to the limit between the ventricle (V) and the atrium (A). Note the lack ofepicardial lining in the ventral surface of the heart (arrowheads). (D–F) Prolarvae of 20 (D) and 23 (E, F) dpf. Pronephric tubules (PT) arealready well developed in the pronephros. The right PEG primordium is widely attached to the ventricle (V) (arrows, section shown in D islocated slightly ahead of the attachment site). Epicardial cells (EP) apparently spread over the cardiac wall. The left PEG primordium doesnot attach to the heart (arrowheads). (G–H) Transverse and sagittal sections of a 26dpf P. marinus prolarvae, respectively. Large PEGprimordia are indicated by arrows. The attachment of the right PEG primordium persists on the dorsal and posterior part of the ventricle(V). Note the long distance between the liver (LI) and the ventricle. C, Conus arteriosus. HV, hepatic vein. (I–J) The attachment of the rightPEG (RPEG) to the ventricle has disappeared by 29dpf. The right and the left PEG (LPEG) are shown in I and J, respectively. A, Atrium.(K) Transverse section of a 32dpf P. marinus prolarvae. Both PEG (arrows) are already well separated from the heart.

The origin of the vertebrate epicardium 213Pombal et al.

in the mesonephros of quail embryos. In both cases Wt1 is

strongly expressed in the coelomic lining and also, with differ-

ent intensity, in the adjacent mesenchyme. Wt1 expression

apparently decreases as these mesenchymal cells differentiate,

as suggested by the decrease of staining at the inner areas and

also by the lack of expression in the epithelial structures of the

mesonephros.

DISCUSSION

The proepicardium is an originally paired structure present in

all the vertebrate models so far studied. It is originally extra-

cardiac (it does not apparently form from the early primary or

secondary heart fields), but it attaches to the heart surface

and spreads lining the myocardium and giving rise to the

epicardium. No hypothesis had been hitherto made about its

origin and relationships to other embryonic systems. We

herein propose that the proepicardium is the reminiscence of

the PEG progenitor that was conserved through the evolution

essentially due to its role in providing vasculogenic cells to the

heart. The paired PEG primordia gave rise to the bilateral

proepicardia of fishes (Munoz-Chapuli et al. 1994), but the

right PEG primordium/proepicardium seems to have been

favored by the evolution, because it is the only one that at-

taches to the heart in lamprey, dogfish, and birds. Thus, the

attachment of the right PEG primordium/proepicardium to

the heart surface is highly conserved during the evolution of

vertebrates. In mammals, both primordia fuse in the midline

giving rise to a single proepicardium (Schulte et al. 2007), but

attachment to the myocardium also occurs (Nesbitt et al.

2006). The unequal contribution of the PEG primordia to the

epicardium is probably related with the asymmetrical devel-

opment of the heart, because the rightward looping of the

early cardiac tube faces the ventricular surface to the right

primordium. Thus, proepicardial asymmetry seems to be a

consequence of the cardiac asymmetry.

A pair of PEG is present in lamprey and amphibian larvae

(Kluge and Fischer 1990), and they are composed of capillary

networks, mesangial cells, and coelomic-derived podocytes.

Rudimentary PEGs have also been described in the chick

embryo, although their functionality is doubtful (Hiruma and

Nakamura 2003). However, PEGs are functional in both

lamprey and amphibian larvae, thus retaining the original

pattern of the vertebrate excretory system, i.e., a blood-filter-

ing glomerulus which releases the filtrate into the coelomic

cavity, where it is aspirated by the ciliated funnels of the

pronephric ducts. The glomerular filtrate was, in amniote

vertebrates, released into the nephrocele. Consequently, the

PEG disappeared from adult vertebrates.

Proepicardium had never been related with the excretory

system, and in fact it is anatomically associated with the liver

and/or the septum transversum in all the vertebrate models

hitherto studied. Therefore, an open question is how did the

PEG primordium become associated with the liver/septum

domain?We think that the disappearance of the most anterior

part of the pronephros, the progressive enlargement of the

liver, and especially the expansion and looping of the cardiac

inflow tract account for this new localization of the pro-

epicardium (Fig. 3). The enlargement of the cardiac inflow

tract was probably concomitant with a looping of the whole

area, thus accounting for the dorsal location of the atrium

and the new position of the proepicardium caudal to the

ventricle. In fact, in lamprey embryos and prolarvae the liver

is far away from the heart, the atrium is located laterally to

the ventricle and a defined sinus venosus is not present at the

time of epicardial development (Figs. 1 and 3). On the other

hand, the evolutionary expansion of the cardiac inflow tract is

probably recapitulated during the development of amniote

vertebrates. A mesenchymal population which does not ex-

press Nkx2.5 (a marker of the myocardial progenitors) and

expresses instead podoplanin (a podocytic marker), the T-box

gene Tbx18 and Wt1 incorporates to the venous pole of

the embryonic heart (Christoffels et al. 2006; Gittenberger-

de Groot et al. 2007; unpublished observations). It is impor-

tant to remark that Tbx18 is strongly expressed in the pro-

epicardium and genital ridges (Haenig and Kispert 2004). In

addition, the liver growth towards the dorsal and right side,

intermingling with the septum transversum mesenchyme,

Fig. 3. Comparison between the anatomical arrangement of thepronephric external glomerulus (PEG) in agnathans and the pro-epicardium (PE) of gnathostomes. The different localization of theproepicardium in gnathostomes is probably due to the disappear-ance of the most anterior part of the pronephros, the enlargementof the liver, the incorporation of venous areas to the cardiac inflowtract giving rise to the sinus venosus and a further cardiac rotationleaving the atrium dorsal and the proepicardium caudal to theventricle. In this way the primitive PEG primordium became as-sociated to the liver and septum transversum.

214 EVOLUTION & DEVELOPMENT Vol. 10, No. 2, March^April 2008

probably uncoupled the PEG progenitor from the primitive

nephrogenic area. Thus, the anatomical association of the

proepicardium with the liver and the septum transversum

seems to be purely contingent.

A main support of our hypothesis is provided by the

strong epicardial expression of Wt1 and Pod1, two transcrip-

tion factors essential for genitourinary development. (Moore

et al. 1999; Cui et al. 2003). The Wilms’ tumor suppressor

transcription factor Wt1 is essential for development of

kidneys and gonads, two organs which do not develop in

Wt1-deficient mouse embryos (Kreidberg et al. 1993). In these

embryos, the epicardial development is defective, showing

premature and anomalous differentiation (endothelial

differentiation is particularly impaired) as well as reduced

proliferation (Wagner et al. 2005; J. M. Perez-Pomares et al.

unpublished observations). On the other hand, the bHLH

transcription factor epicardin/Pod1 is strongly expressed in

podocytes, epicardial, and epicardial-derived cells. Although

Pod1-null embryos show glomerular defects, spleen agenesis

and hypoplasic lungs and gonads (Quaggin et al. 1999; Lu

et al. 2000; Cui et al. 2003), no epicardial defects have been

reported.

Two signaling systems, mediated by retinoic acid (RA) and

the activin receptor ALK2, respectively, are also essential for

both epicardial/proepicardial development (Lavine et al. 2005;

Merki et al. 2005; Olivey et al. 2006) and PEG differentiation

(Osafune et al. 2002). The myocardial signals that promote

myocardial proliferation are RA-dependent (Lavine et al.

2005), and proepicardial apoptosis has been described in

RXRa-null embryos (Jenkins et al. 2005). On the other hand,

the proepicardial epithelial–mesenchymal transition is in-

duced by TGFb1 and TGFb2 through ALK2 activation

(Olivey et al. 2006). These molecular mechanisms parallel the

role of RA and activin for the induction of the pronephric

glomus and tubules in Xenopus (Osafune et al. 2002).

Finally, a striking feature of the proepicardium can also be

explained by its evolutionary relationship with the prone-

phros. The presence of blood island-like structures in the

epicardium of the mammalian embryos is known since long

time ago (Komiyama et al. 1987; Hirakow 1992). However,

the heart is not considered a true hematopoietic organ.

Recently, some evidence has been provided about hemato-

poietic cells in the heart (basically erythropoietic) derived

from proepicardial-derived progenitors (Kattan et al. 2004;

Tomanek et al. 2006; Wilting et al. 2007). This fact can be

now explained by the pronephric origin of the proepicardium,

since the pronephros is known to be a main hematopoietic site

in fish and amphibians (Carpenter and Turpen 1979; Willett

et al. 1999).

The evolutionary conservation of the cell transfer from the

PEG primordium to the heart illustrates the importance of

these cells for heart evolution. We think that the primary

contribution made by glomerular cells to the cardiac devel-

opment probably was to supply the heart with vascular cells.

The embryonic heart is primarily devoid of blood vessels,

which limits the thickness of the myocardial wall and, con-

sequently, its performance. Glomerular progenitor cells trans-

ferred to the heart and bearing a high vasculogenic potential

might represent the first evolutionary step to accomplish a

complete myocardial vascularization through an epicardial-

derived coronary vascular bed. In this way, the vertebrate

heart could increase its thickness and its performance. Sec-

ondarily the epicardial and epicardial-derived cells probably

acquired a signaling role, producing factors actively inducing

the myocardial proliferation.

We can finally speculate about the relationships between

the proepicardium/heart connection and a striking feature of

the hemichordates, the heart–kidney complex. This structure

is located in the proboscis, above the stomochordFa mouth

diverticle-, and it is constituted by a pulsatile blood sinus

associated to an excretory glomerulus. The apparently dorsal

location of the heart in hemichordates historically ruled out

an evolutionary correspondence with the vertebrate heart.

However, recent studies on gene expression in the hemichor-

date Saccoglossus kowalevskii have shown that the dorsoven-

tral axis of this species is reversed respect to that of vertebrates

(Lowe et al. 2006). This surprising finding opens the possi-

bility that the heart–kidney complex of hemichordates can be

related with the heart–pronephros connection observed in

lamprey embryos and prolarvae. In this case, the pro-

epicardium could be the last vestige of an ancestral link be-

tween the primitive heart and the primitive excretory system

of deuterostomes.

We believe that the recognition of the proepicardium as an

evolutionary derivative of the PEG will provide new avenues

on the molecular mechanisms involved in its development and

the impact of the epicardium on cardiac morphogenesis.

AcknowledgmentsWe thank David Macıas for supplying dogfish sections. This workhas been supported by the European Community’s Sixth FrameworkProgramme contract (‘‘HeartRepair’’) LSHM-CT-2005-018630 andby grants BFU05-00483, SAF2006-26666E, 07V1A12, andBFU2006-14127.

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