&p.1:Abstract An ultrastructural study of the development ofthe sinus venosus has been carried out on seven embryosof the dogfish (Scyliorhinus caniculaL.) between 10.5and 69 mm of total length (TL). The sinus venosus ap-pears at the end of the looping process of the cardiactube, namely in the 10.5 mm embryo, when the heartreaches its adult tetracameral S-form. The endocardiumof the smallest embryo is constituted of a single layer ofcubic cells. In larger embryo, these cells progressivelyacquire a squamous appearance as well as electron-densecytoplasmic inclusions. The subendocardium is progres-sively populated by ganglion cells, Schwann cells andbundles of amyelinic fibers that can first be recognised inthe embryo of 34 mm TL. Some subendocardial mesen-chymal cells located in earlier embryos close to the en-trance of the ducts of Cuvier might be their ectomesen-chymal progenitors. The myocardium is initially consti-tuted of a single layer of cubic cells. In the embryos of19, 27 and 34 mm TL, the myocardium becomes multi-layered, and the myocardiocytes develop myofibrils ran-domly arranged throughout the sarcoplasm. In later em-bryos, the myocardiocytes are innervated and arranged inoval bundles surrounded by a basal lamina. The epicardi-um covers the sinus venosus by the retrograde migrationof the epithelium already established around the atrio-ventricular groove and, in a lesser degree, by the adhe-sion of mesothelial cells that are floating free in the peri-cardial cavity. This process has finished in the embryo of34 mm TL. The differentiation of the sinus venosus (in-cluding the endocardial and myocardial differentiation aswell as the epicardial covering) progresses in an antero-ventral-posterodorsal direction.

&kwd:Key words Ultrastructure · Heart development ·Ganglion cells · Nodal tissue · Fish&bdy:

Introduction

The four-chambered heart of the fish receives blood fromthe liver and the ducts of Cuvier and delivers it to thesystem of branchial arteries through the ventral aorta.Following the bloodstream, the first chamber of the fishheart, in its venous inlet, is the sinus venosus, which ex-tends from the septum transversum to the sinoatrial junc-tion. In homoiothermic species, the sinus venosus is atransient embryonic structure that contributes to severalparts of the adult heart, such as the sinoatrial node and,in mammals, the coronary sinus (Manasek et al. 1986).However, this sinus venosus keeps its function as achamber during the whole lifetime of the fish.

The sinus venosus is the cardiac chamber of the fishthat is least known from the anatomical and histomor-phological point of view. However, the functional roleplayed by the sinus venosus seems to be essential in thecardiac cycle. Our recent studies carried out on adultdogfish (Scyliorhinus canicula) have shown the nodaltissue-like structure of the sinusal myocardium (Ramoset al. 1996) as well as the existence, in the sinus venosusof this species, of a neuroendocrine system, probably se-creting a substance P-related peptide (Muñoz-Chápuli etal. 1994a, 1995).

The development of the sinus venosus in fish is virtu-ally unknown. No ultrastructural descriptions of thischamber in fish embryos are available in the literature.Most papers dealing with cardiac embryology of fish atthe ultrastructural level are devoted to the myocardium(Santer 1972; Rönnau 1977; Myklebust and Kryvi 1983;Kitoh and Oguri 1985). Zummo (1983) described someultrastructural features of the cardiac development inone embryonic stage of Scyliorhinus stellariswith nomention of the sinus venosus. Given this lack of infor-mation, we decided to perform an ultrastructural studyof the sinus venosus in embryos of the dogfish. The aim

C. Ramos · D. MacíasDepartment of Animal Biology, Faculty of Science,University of Málaga, E-29071 Málaga, Spain

C. Ramos (✉)Labo de Génétique Moléculaire de la Morphogenèse,Département de Biologie Moléculaire, Institut Pasteur,25, rue du Dr. Roux, F-75724 Paris, Cedex 15, FranceTel.: 33-1-40613524; Fax: 33-1-45688963;e-mail: cramos@pasteur.fr&/fn-block:

Anat Embryol (1998) 198:523–536 © Springer-Verlag 1998

O R I G I N A L A RT I C L E

&roles:Casto Ramos · David Macías

Ultrastructural study of the sinus venosusin embryos of the dogfish (Scyliorhinus canicula)

&misc:Accepted: 12 June 1998

of this paper is to show the findings obtained in ourstudy.

Materials and methods

The material studied consisted of nine dogfish embryos. Fertilizeddogfish eggs, obtained from female specimens caught severalhours before, were collected at the fishmarket and kept in indoortanks of well-aerated seawater. An external filter device kept thewater clear. Nitrite concentration, density, and pH of the waterwere monitored daily during the experiment. The water tempera-ture ranged from 11 to 21° C. The total length (TL) of the embry-os studied was 5.5, 8, 10,5, 19, 27, 34, 44.5, 53 and 69 mm. Thelatter length is close to the hatching size (about 73 mm TL).

The rate of development of dogfish embryos depends on thetemperature (T°) of the water. In our laboratory conditions, the T°of the tanks depended on the ambient T°, which was not constantduring the year. Therefore, the degree of development of dogfishembryos was assessed by their size rather than by incubation time.

The embryos were anesthetized in sea water with 0.04% trica-ine methanesulphonate (MS-222), before fixation in 1% parafor-maldehyde and 1.25% glutaraldehyde in elasmobranch buffer(16.38 g/l NaCl, 0.89 g/l KCl, 1.11 g/l, CaCI2, 0.38 g/l NaHCO3,0.06 g/l NaH2PO4, 21.60 g/l urea, pH 7.2–7.4). The embryos of5.5–10.5 mm TL were fixed whole by immersion for 2 h. The em-bryos of 19–34 mm TL were fixed for 30 min by perfusionthrough a micropipette inserted in the vitelline vein. Then, a seg-ment of the body containing the heart was immersed in the fixativefor 90 min. In the largest embryos, the hearts were removed andfixed for 2 h. Then, the pieces were washed in buffer, postfixed in1% OsO4 for 2 h, washed, dehydrated and embedded in Araldite502. Semithin and ultrathin sections were cut in a Reichert UMO-2 ultramicrotome. Semithin sections were stained with toluidineblue. Ultrathin sections were contrasted with lead citrate and ura-nyl acetate. Observations were made in a Phillips 300 and a JeolJEM-1OOCX transmission electron microscope.

Results

Formation of the cardiac loop

In the 5.5 mm embryo, the heart is ventrally located be-tween the vitelline peduncle and the gills, just above thebody wall; it consists of a horizontal tube whose ends arelightly curved up. In this embryo, we can appreciate thedifferent parts of the cardiac tube that will give rise tothe four chambers, namely the conus arteriosus, the ven-tricle and, not yet demarcated between them in this em-bryo and in the next one, the atrium and the sinus veno-sus (Fig. 1A).

In the 8 mm embryo, the median part of the cardiactube, which corresponds to the ventricle, has noticeablycurved towards the right in the horizontal plane, whereasthe posterior portion, which is the precursor of the sinusvenosus and the atrium, rises and forms a right anglewith respect to the ventricle (Fig. 1B).

In the 10.5 mm embryo, the shape of the heart showsnoteworthy modifications: torsion in the sagittal planeand in clockwise direction carries the atrium and sinusvenosus above the left side of the ventricle. Thus, theatrioventricular groove is situated in the left side of theheart, with respect to the outflow tract. In this embryo,the cardiac tube adopts its definitive tetracameral S-

form, the sinus venosus being the most posterior cham-ber and the conus arteriosus the most anterior one (Fig.1C).

Embryo of 10.5 mm

Although we have studied smaller embryos, the embryoof 10.5 mm TL is the smallest one in which the sinoatrialgroove appears. This groove, located at the lateral andventral parts of the cardiac tube, allows us to distinguishbetween the atrium and the sinus venosus (Fig. 2).

The posterior part of the roof of the sinus venosus isstill united to the parietal pericardium through the dorsalmesocardium. The posterior part of the sinus venosus isin anatomical connection with the liver, which drains in-to the heart through a large suprahepatic vein located atthe left side. In later embryos two symmetrical suprahe-patic veins are always located between the liver and thesinus venosus.

The wall of the sinus venosus is constituted of twolayers of epithelial cells, the endocardium and the myo-cardium, separated by a thin layer of cardiac jelly.Groups of proepicardial cells of smooth surface can beseen free in the pericardial cavity and adhering to themyocardium, particularly at the atrioventricular groove.These cells, which will give rise to the primitive epicar-dium, are released from protrusion of the splanchnic me-sothelium located in the ventral and anterior part of theliver. The proepicardial cells are rare on the sinus veno-sus wall (Fig. 2).

The endocardial cells of the sinus venosus of this em-bryo usually have a cubic shape and they show thin cyto-plasmic projections towards the cardiac jelly (Fig. 3).Their cytoplasm contains numerous cisterns of the Golgicomplex, mainly located in a basal position. The roughendoplasmic reticulum and the polyribosomes are veryabundant in the endocardial cells (Fig. 4). Pointed (Fig.5) and ruffled cytoplasmic expansions towards the cardi-ac lumen can also be seen.

The myocardial epithelium is cubic or columnar andits cells are joined by junctional complexes (Fig. 6). Thecytoplasm contains numerous dictyosomes, often dilated,cisterns of rough endoplasmic reticulum, and abundantpolyribosomes. The myofilaments are scarce and usuallylocated in a basal position, close to the nucleus. Themyofilaments are organized into loose bundles or joinedto an amorphous, electron-dense material, which is theprecursor of the Z-lines (Fig. 7).

Embryo of 19 mm

Most endocardial cells in the sinus venosus are flattened,with an elongated nucleus (Fig. 8). However, a numberof cubic endocardial cells in the posterodorsal part of thesinus venosus still retain the primitive features describedin the 10.5 mm embryo. The cytoplasmic projections ofthe endocardium towards the lumen are scarce. The cyto-

524

525

Fig. 1A–C Process of cardiaclooping in dogfish embryos. A 5.5 mm embryo. The heart isa tube whose ends are lightlycurved up. B 8-mm embryo.Note the curve to the right ofthe median part and the“straightening up” of the poste-rior part of the cardiac tube. C 10.5 mm embryo. The cardi-ac organ has undergone notice-able morphological changes:the posterior end of the tubetwists in a clockwise directionand becomes placed on theventricle (V). In the 10.5 mmembryo, the heart has reachedits definitive four-chambered S-shape (A atrium, C conus arte-riosus, H head, SVsinus veno-sus, T tail, arrowheadgills).Bar 0.25 mm&/fig.c:

526

plasm of the endocardial cells contains numerous cis-terns of rough endoplasmic reticulum. The Golgi appara-tus is well-developed and it is constituted of groups of 3or 4 slender cisterns. Clear vesicles are very numerousthroughout the cytoplasm.

Some mesenchymal cells can be seen between the en-docardium and the myocardium of the posterior part ofthe sinus venosus, close to the entrance of the ducts ofCuvier (Fig. 9).

The myocardium is generally constituted of a singlelayer of slightly flattened cells with a cytoplasmic vol-ume greater than in the 10.5-mm embryo (Fig. 8). How-ever, several layers of muscular cells can be seen in thelateral part of the sinus venosus. The amount of fibrillarmaterial has increased compared with the 10.5-mm em-bryo. The developing myofibrils are randomly arrangedthroughout the sarcoplasm. The most differentiated onesare composed of 2 or 3 sarcomeres. The apparatus ofGolgi is conspicuous and the rough endoplasmic reticu-lum is well-developed. A few peripheral couplings andsubsarcolemmal vesicles are present in the sinusal myo-cardium of this embryo (Fig. 10). Intercellular spaces arefrequent between the myocardiocytes, which are joinedby fascia adherensand zonula occludens(Fig. 11).

The sinus venosus of this embryo is not covered bythe epicardium (Fig. 9), except for its most ventral andanterior part, close to the sinoatrial groove (Fig. 12).

Embryo of 27 mm

Most endocardial cells of the sinus venosus are flattenedand have a nucleus protruding into the cardiac lumen.The cytoplasm contains some large vacuoles, partiallyfilled with an amorphous material, and a number ofdense and moderately dense bodies (Fig. 13).

The cardiac jelly is uniformly electron-lucent. How-ever, the subendocardial mesenchymal cells close to theducts of Cuvier are more numerous than in the 19 mmembryo. Between the embryos of 19 and 27 mm there isno significant increment in the number of collagen fiberscontained in the subendocardial space.

The myocardium is generally formed by 2 or 3 celllayers between the endocardium and the epicardium(Fig. 14). Some discontinuities of the myocardium allowcontact between the subendocardial and subepicardialextracellular matrices (Fig. 15). The most differentiatedmyocardial cells, which are mainly located at the anteri-or part of this cardiac chamber, show myofibrils with 5or 6 sarcomeres, as well as numerous groups of shortmyofilaments scattered throughout the sarcoplasm. Themitochondria are also more abundant in these myocardialcells. Cubic and less-developed myocardiocytes, en-dowed with bundles of short myofilaments randomly ar-ranged, are still present in the most dorsal and posteriorpart of the sinus venosus. This type of myocardial cellsis not seen in the 34-mm embryo.

The primitive epicardium covers most of the sinus ve-nosus except for its most dorsal and posterior part (Fig.15). This area not covered by the epicardium is thatwhich shows less-differentiated myocardial cells. In gen-eral, the epicardial cells show long cytoplasmic expan-sions very similar to those of the endocardial cells. Anincomplete basal lamina is present along the basal sur-face of the epicardial cells. Occasionally, some cells canbe seen apparently detaching from the epicardial liningto populate the subepicardial space. It has been mainlyobserved near the ventral part of the sinoatrial junction.

Embryo of 34 mm

We have identified some cells located in the subendocar-dium of the posterior part of the sinus venosus at the pre-cursors of the parasympathetic ganglion cells (Fig. 16).These cells are large, with a central, indented nucleusand a relatively clear cytoplasm containing a few elec-tron-dense granules. Groups of amyelinic fibers can oc-casionally be seen associated with these cells (Figs. 18,19). Figure 17 shows one cell between the endocardiumand the myocardium of the sinus venosus, very close tothe entrance of the ducts of Cuvier.

The myocardial cells are longer than those of the 27-mm embryo. At the ultrastructural level, there is an in-crease in the number of myofibrils, which are, in general,more developed a the subsarcolemmal areas than indeeper areas of the cell. The cisterns of Golgi, the roughendoplasmic reticulum and the polyribosomes are less

527

Fig. 2 Semithin sagittal section of the cardiac tube of the 10.5mmdogfish embryo. The four chambers are perceptible. There is aclose connection between the sinus venosus (SV) and the hepatictissue (H). Groups of cells with clear cytoplasm (arrowheads) canbe seen free in the pericardial cavity and adhering to the myocar-dium (M) of the ventricle (V) and of the atrium (A). These cells arethe precursors of the primitive epicardium. Note the thin layer ofcardiac jelly (asterisk) of the sinus venosus – much more thick inthe other three chambers (BWbody wall, E endocardium). ×102&/fig.c:

Fig. 3 Electron micrograph showing the endocardium of the sinusvenosus. 10.5mm embryo. Cytoplasmic projections (arrows) of anendocardial cell (E) are extended towards the cardiac jelly (aster-isk). In this embryo, the sinusal endocardium lacks a conspicuousbasal lamina. Note the basal lamina (arrowhead) of the myocardi-um (M). Bar 5 µm&/fig.c:Fig. 4 Cytoplasm of an endocardial cell of the sinus venosus.10.5mm embryo. Note the abundant polyribosomes arranged inrosettes (G Golgi apparatus, N nucleus, arrowheadscisterns ofrough endoplasmic reticulum). Bar 1.1 µm&/fig.c:Fig. 5 Luminal surface of the endocardium of the sinus venosus.10.5mm embryo. Slender cytoplasmic projections are directed to-wards the cardiac lumen (L) (M) mitochondrion, arrowheadcis-terns of the rough endoplasmic reticulum). Bar 1 µm&/fig.c:Fig. 6 Myocardium of the sinus venosus. 10.5mm embryo. Themyocardial cells are generally cubic or columnar (arrows junc-tional complexes, arrowheadprecursor material of a Z-line). Bar 1µm&/fig.c:Fig. 7 Myocardial cell of the sinus venosus. 10.5mm embryo.Observe the poorly developed sarcomeres with globular precursorsof the Z-lines (Z) (H H-band, I I-band, arrowheadsrosettes of ri-bosomes). Bar 500 nm&/fig.c:

528

abundant than in the younger embryos. However, thepresence of the sarcoplasmic reticulum and the subsarco-lemmal vesicles begins to be significant.

The epicardium in this embryo covers the completesinus venosus. At the ultrastructural level, the epicardialcells show no significant changes, but their greater thick-ness allows them to be distinguished from the endocardi-al cells.

Embryo of 44.5 mm

The endocardium of the sinus venosus of this embryo issimilar to that of the adult. The endothelial cells show aprominent nucleus, and the lateral cytoplasmic expan-sions are long and thin. The cytoplasm contains numer-ous dense and moderately dense bodies of 200–800 nmin diameter. Numerous tubules can be seen inside thesebodies (Fig. 20).

Compared to the 34-mm embryo, the subendocardialprecursors of the ganglion cells are more abundant, andthey can be found in more anterior areas. These cellsgenerally show a large, pale nucleus with a dense nucle-olus. Their cytoplasm contains a few electron-dense vesi-cles and the apparatus of Golgi and the rough endoplas-

mic reticulum are poorly developed. The amyelinic fi-bers, also with an immature appearance, are looselypacked and partially surrounded by a Schwann cell. Atthis stage of development, most amyelinic fibers lackelectron-dense vesicles (Fig. 21).

The myocardium is generally constituted by longercells than in the 34-mm embryo. Numerous intercellularspaces can be seen between these cells. The mitochon-dria are abundant throughout the sarcoplasm (Fig. 22). Inthis embryo, there is not a significant increase in thenumber of peripheral couplings and subsarcolemmal ves-icles. However, the sarcoplasmic reticulum is more de-veloped.

We have observed in this embryo the first nerve ter-minals in the sinus venosus. They are filled with abun-dant, clear vesicles of about 50 nm in diameter and somelarger electron-dense vesicles. The terminals form synap-tic contacts with the myocardiocytes (Fig. 23) and alsowith the ganglion cells.

In relation to the earlier embryo, the cytoplasmic ex-pansions of the epicardial cells have thickened, while thenuclei have acquired a more or less rectangular shape. Acomplete basal lamina separates the epicardial cells fromthe subepicardial space where the mesenchymal cellshave become more abundant. These cells are frequentlystars-shaped, and they show contacts (but no junctioncomplexes) with the myocardial cells. The subepicardialcells are rich in cisterns of rough endoplasmic reticulum.The presence of collagen fibers close to their membranessuggests that they are fibroblasts (Fig. 22).

The extracellular matrix of both the subendocardialand the subepicardial spaces shows large electron-lucentareas. The collagen is relatively scarce but it begins to beorganized in bundles of transverse and longitudinal fi-bers that are more abundant in the subepicardium (Fig.22).

Embryo of 53 mm

As described above, the endocardial cells have alreadyacquired the adult features, and no changes are recordedin this embryo. The subendocardium still shows elec-tron-lucent areas with very little collagen.

Compared to the 44.5-mm embryo, the amyelinic fi-bers are more numerous and they are tightly packed inlarger bundles, partially or totally surrounded by themembrane of a Schwann cell (Fig. 24). The ganglioncells are more abundant and they show a slightly greaterdegree of differentiation, with a well-developed Golgiapparatus and more abundant rough endoplasmic reticu-lum. The ganglion cells form, together with the amyelin-ic fibers, nervous bundles that bulge into the lumen ofthe sinus venosus.

The myocardium is organized in bundles of 4 or 5cells in a fashion similar to that described for the nextembryo.

The epicardial cells of the sinus venosus are larger inrelation to the earlier embryo. Their nuclei have ap-

529

Fig. 8 Sinus venosus of the 19mm embryo. Most endocardialcells (E) are flattened (M myocardium, asteriskcardiac jelly). Bar3 µm&/fig.c:Fig. 9 Semithin transverse section of the posterior part of the si-nus venosus. 19mm embryo. At this level, the sinus venosus (SV)Lacks epicardium and is composed of myocardium (M) and endo-cardium (E). The epicardium (EP) is already present on the dorsalpart of the ventricle (V), [AO aorta, DT digestive tube (transientlyclosed), arrows mesenchymal cells between the endocardium andthe myocardium, asteriskentrance of the right duct of Cuvier].×158&/fig.c:

Fig. 10 Myocardial cell of the sinus venosus. 19mm embryo.Note the random orientation of the immature myofibrils (MF) andthe irregular aspect of the Z-lines (Z) (arrow peripheral coupling,arrowheadsubsarcolemmal vesicle, open arrowsarcoplasmic re-ticulum). Bar 500 nm&/fig.c:

Fig. 11 Myocardial cell of the sinus venosus. 19mm embryo.Two myocardial cells are connected by fascia adherens(FA) andzonula occludens(ZO). Bar 500 nm&/fig.c:

Fig. 12 Semithin transverse section of the anterior part of the si-nus venosus. 19mm embryo. The epicardium (arrows) covers theventral part of the sinus venosus (SV) as well as the dorsal part ofthe ventricle (V) (A atrium, arrowheadflattened proepicardial cellon the dorsal part of the sinus venosus). ×224&/fig.c:

Fig. 13 Endocardium of the sinus venosus. 27mm embryo. Thecytoplasm of this endocardial cell shows a conspicuous Golgi ap-paratus (G), some large vacuoles (arrow) and a few electrodensebodies (arrowheads). Bar 1 µm&/fig.c:Fig. 14 Sinus venosus wall of the 27mm embryo. The endocardi-al (E) and epicardial cells (EP) cover the myocardium (M) on itsinner and outer surfaces, respectively. Observe the gaps (stars) inthe myocardium (arrowheadfibers of collagen in the subendocar-dium). Bar 3 µm&/fig.c:Fig. 15 Semithin transverse section of the sinus venosus. 27mmembryo. At this level, the epicardium (EP) covers all the sinus ve-nosus (SV) (arrow myocardial discontinuity). ×206&/fig.c:

proached each other, occasionally giving the cells the cu-bic shape of the adult epicardium. Interdigitations be-tween the cytoplasmic membranes of adjacent epicardialcells have appeared, and some microvilli are alreadypresent on the apical surface (Figs. 25, 26).

The amount of collagen in the sinus venosus has no-ticeably increased in this embryo, particularly at the sub-epicardial space. The collagen is organized in bundles oflongitudinal, transverse and oblique fibers, which areusually surrounded by thin cytoplasmic processes of fi-broblasts (Fig. 26).

Embryo of 69 mm

Compared to the 53-mm embryo, there is a substantialincrease in the number of ganglion cells, which are often

530

Fig. 16 Semithin transverse section of the sinus venosus. 34mmembryo. The first ganglion cells (arrow) can be seen just belowthe endocardium of the posterior part of the sinus venosus (SV),near the entrance of the ducts of Cuvier. ×310&/fig.c:

Fig. 17 34mm embryo. Electron micrograph showing a cell (as-terisk) between the endocardium (E) and the myocardium (M) ofthe sinus venosus, near the entrance of the ducts of Cuvier (arrowscytoplasmic processes, arrowheadbasal lamina). Bar 2 µm&/fig.c:Fig. 18 Subendocardium of the sinus venosus. 34mm embryo (Eendocardium, M myocardium, arrow bundle of amyelinic fibers,arrowhead junctional complex, asterisksganglion cells). Bar 3µm&/fig.c:Fig. 19 Enlargement of Fig. 18. The amyelinic fibers (arrows)contain clear vesicles and a few electrodense vesicles (asteriskganglion cell). Bar 1 µm&/fig.c:

gathered in groups of 4 or 5 just under the endocardialendothelium. We can describe them as true ganglioncells on the basis of their ultrastructural features. Theelectron-dense vesicles, the cisterns of Golgi and thoseof the rough endoplasmic reticulum are more numerousthan in the precursors of earlier embryos (Fig. 27). Thereis also a clear increment in the number of amyelinic fi-bers carrying electron-dense vesicles are well as in thenumber of nerve bundles entering the subendocardialspace (Fig. 28).

The myocardium is composed of large bundles of ovalcells. These bundles are completely surrounded by anouter basal lamina (Fig. 29). Nerve terminals located be-tween the myocardiocytes, and making synaptic contactswith them, are more numerous than in the younger em-bryos.

The collagen fibers are thicker and more numerous,particularly in the subepicardium, but there are still areas

531

Fig. 20 Endocardium of the sinus venosus. 44.5mm embryo. Themoderately dense bodies and dense bodies are numerous (arrows).Note the microtubules contained in these bodies (L cardiac lumen,R cisterns of the rough endoplasmic reticulum, arrowheadvacu-oles). Bar 1 µm&/fig.c:

Fig. 21 Subendocardium of the sinus venosus. 44.5mm embryo.Transverese section of a bundle of immature amyelinic fibers (ar-rows). They are loosely packed and partially covered by aSchwann cell (S). Bar 1 µm&/fig.c:

Fig. 22 Sinus venosus wall of the 44.5mm embryo. The myocar-dial cell (M) contain long and well-differentiated myofibrils andnumerous mitochondria. The endocardial cells (E) show very thincytoplasmic processes (arrow). Some fibroblasts (F) are present inthe subepicardium (asterisk) (EP epicardium, arrowheadsbundlesof collagen). Bar 5 µm&/fig.c:

Fig. 23 Myocardial innervation of the sinus venosus. 44.5mmembryo. Nerve terminals are in close contact with a myocardio-cyte (M). The clear vesicles are abundant, and there also are a fewelectrodense vesicles (arrowheads) (arrow synaptic region). Bar500 nm&/fig.c:

532

of amorphous extracellular matrix devoid of fibrous ele-ments.

Discussion

Our study in a primitive gnathostome show that the pri-mordial cardiac organ is a tube located ventrally. Thisevent therefore occurs in all vertebrates, despite morethan 400 millions of years of divergent evolution. Fur-thermore, as in tetrapods, the cardiac tube of the dogfishloops to the right and, from an anatomical point of view,it is only at the end of the looping process that the hearttube becomes a clearly differentiated tetracameral organ.Our study in S. caniculaalso show that throughout loopformation the heart is a very simple structure, composedof two monolayers of endocardium and myocardium sep-arated by a thick layer of cardiac jelly, in accordancewith the findings described in higher vertebrates (re-viewed by Icardo et al. 1990a).

Development of the endocardium and subendocardium

The endocardium of the early cardiac tube in tetrapods isconstituted of closely packed, cubic endothelial cellswith a smooth apical surface (Markwald et al. 1975, inthe rat embryo; Lemanski and Fitzharris 1989, in the ax-olotl). Our findings in the youngest dogfish embryosstudied (until 10.5 mm TL) agree with these descrip-tions. Furthermore, we have shown how the endocardium

of the posterior and dorsal part of the sinus venosus re-tains these primitive features for a longer time (until 27mm TL). In older dogfish embryos, the endothelial cellsacquire long and slender cytoplasmic processes. Thisevent has also been recorded in rodent embryos (Mark-wald et al. 1975; Virágh and Challice 1981).

Zummo (1983) reported in the ventricle of embryosof Scyliorhinus stellaris(9 cm TL, i.e. close to hatchingsize) two types of endocardial cells: endothelial-like andgranulated cells. In the sinus venosus of the dogfish wehave found only one type of endocardial cell, providedwith numerous dense and moderately dense bodies.These structures appear approximately at the secondthird of the embryonic stage and persist during the life-time in this species.

Saetersdal et al. (1974) distinguished in the cytoplasmof the endocardial cells of the adult Gadiadae electron-dense bodies, and classified them into two types accord-ing to their affinity to the osmium. According to theseauthors, those of the first type, called “dense bodies”, areintensively osmiophilic, and therefore very electron-dense. Those of the other type are moderately dense,contain a granulofibrillar matrix and are called “moder-ately dense bodies”. These organelles are characteristicof the endocardial cells of fish (Saetersdal et al. 1974;Lemanski et al. 1975; Berge 1979), and their size, num-ber and ultrastructure greatly vary between species andbetween cardiac chambers (Leknes 1980, 1981a, b,1983; Yamauchi 1980; Benjamin et al. 1983).

In a scanning microscopy study of the endocardium inseveral embryos of higher vertebrates, Pexieder (1981)reported the presence of cell microappendages such asmicrovilli, ruffles, filopodia, lamellipodia and cytoseg-resomes. According to this author, the number of thesestructures is variable and depends on the developmentalstage, the area studied and the species. We have also ob-served, in the sinus venosus of the 10.5-mm dogfish em-bryo, some cytoplasmic projections and ruffles towardsthe lumen.

According to our results, the first identifiable precur-sors of the ganglion cells can be seen in the posteriorpart of the sinus venosus of the 34-mm embryo, close tothe entrance of the ducts of Cuvier. In later embryos,these ganglionar precursors show an increasing numberof electron-dense granules and rough endoplasmic retic-ulum cisterns, and they can be found in more anterior ar-eas of the sinus venosus. On the other hand, the progres-sive accumulation of amyelinic fibers in the subendocar-dial space gives rise to the formation of nerve bundlesthat bulge into the lumen. These nerve bundles and theassociated ganglion cells constitute a poorly-known neu-rosecretory system in the adult dogfish (Saetersdal et al.1975; Muñoz-Chápuli et al. 1994a, 1995).

The origin of the above-mentioned neural elementswas not evident in our embryos. However, the presenceof subendocardial mesenchymal cells between the endo-cardium and the myocardium of the sinus venosus of the19-mm embryo, close to the entrance of the ducts of Cu-vier, is remarkable. We find it improbable that these cells

533

Fig. 24 Subendocardial neural elements of the sinus venosus.53mm embryo. Bundles of amyelinic fibers (arrows) are sur-rounded by the cytoplasmic processes of a Schwann cell (S). Thespaces between the amyelinic fibers have reduced or disappeared(compare with Fig. 21). Some fibers contain a few electrodensegranules (arrowheads). Bar 2 µm&/fig.c:Fig. 25 Epicardium of the sinus venosus. 53mm embryo. Twoepicardial cells are joined by deep membranal interdigitations (as-terisk). The cytoplasm is highly vacuolized (D desmosome, arrowbasal lamina, arrowheadglycocalyx on a microvillus). Bar 500nm&/fig.c:

Fig. 26 Epicardium and subepicardium of the sinus venosus.53mm embryo. Note the thickness of the epicardial cells (EP).Bundles of collagen fibers (C) fill the subepicardium (F fibro-blasts, arrow microvilli). Bar 500 nm&/fig.c:

Fig. 27 Neural elements in the subendocardium of the sinus veno-sus. 69mm embryo. The electron-dense granules (arrowheads) arevery numerous throughout the cytoplasm of the ganglion cells(GC) (S satellite cell, arrow cisterns of rough endoplasmic reticu-lum). Bar 1 µm&/fig.c:Fig. 28 Neural elements in the subendocardium of the sinus veno-sus. 69mm embryo. The number of electron-dense granules withinthe amyelinic fibers has significantly increased with respect to theearlier embryos (compare Figs. 21, 24) (S Schwann cell arrow-headmembrane of the Schwann cell). Bar 1 µm&/fig.c:Fig. 29 Myocardium of the sinus venosus. 69mm embryo. Themyocardial cells are arranged in bundles of 4 or 5 elements sur-rounded by a basal lamina (arrowhead). The mitochondria are nu-merous. Bar 1 µm&/fig.c:

have arisen from the areas of endocardial transformationlocated at the atrioventricular junction and the outflowtract, since no mesenchymal cells could be detected inthe large space between these areas and the ducts of Cu-vier. Furthermore, the mesenchymal cells are just begin-ning to appear in the atrioventricular canal of the 19-mmembryo (Muñoz-Chápuli et al. 1994b). Therefore, wethink that the subendocardial cells of the posterior anddorsal part of the sinus venosus might be ectomesen-chyme from the neural crest entering the heart along thewall of the ducts of Cuvier. These cells are probably theprogenitors of the neural elements of the dogfish heart(ganglion and Schwann cells), virtually restricted to thesinus venosus, although a few ganglion cells have beenreported in the atrium (Muñoz-Chápuli et al. 1994a).

Development of the myocardium

Several descriptions of the development of the myocar-dial cells show how the newly formed myofibrils appearin random directions in all the cardiac chambers (Huang1967; Manasek 1968; Markwald 1973; Rönnau 1977;Hirakow and Sugi 1990; Padrós i Bover 1994). Accord-ing to our results, it also occurs in the myocardiocytes ofthe dogfish embryos, although this feature remains in thesinus venous of the dogfish during the whole of its life-time (Ramos et al. 1996). Moreover, we have observed inthis species myofibrils with varying degrees of differen-tiation in myocardiocytes of the same embryo, in accor-dance with the descriptions of Manasek (1970), Hirumaand Hirakow (1985) and Shimada et al. (1990). Also, ac-cording to our results, the myocardium of the dogfish isdifferentiating in a craniocaudal and ventrodorsal direc-tion, in agreement with the results obtained in highervertebrates (Moorman and Lamers 1994).

Our study has shown that the most differentiatedmyofibrils, whose appearance coincides with the phaseof lengthening of the cells, are located just under the sar-colemma. Less-developed myofibrils can be observedthroughout the sarcoplasm. Santer (1972), Lemanski(1973) and Myklebust and Kryvi (1983) described in thehearts of the plaice, axolotl and dogfish, respectively,how sarcomerogenesis occurs preferentially at the sub-sarcolemmal region, in agreement with our results. In the53-mm dogfish embryo, the myocardiocytes begin to or-ganize in bundles of broad oval cells of clear cytoplasm.This fact, together with the random arrangement of themyofibrils and the strong innervation confers on thesinusal myocardium of the adult dogfish a particular as-pect that distinguishes it from the atrial or ventricularmyocardium. Furthermore, the sinusal myocardiocytesshow remarkable resemblances to the nodal cells of birdsand mammals and can be considered, from a morpholog-ical basis, as the pace-maker of the dogfish heart (Ramoset al. 1996).

The lineage of the nodal cells in higher vertebrateshas been the subject of some controversy. Calvet (1990)does not support the idea of an extrinsic origin of the

nodal cells of the rat. This author states that the nodalcells and the atrial cells of this species belong to differ-ent cell lineages, probably with a closely common origin,although they follow divergent pathways of differentia-tion. From the evidence obtained from the dogfish em-bryos, we think that the sinusal myocardiocytes, whichprobably constitute the nodal tissue of the adult dogfish(Ramos et al. 1996), originate from the specific differen-tiation of the myocardium posterior to the sinoatrialjunction. Therefore, our results support the hypothesis ofa very primitive origin, from a phylogenetic point ofview, of the nodal tissue, whose subsequent evolutioncan be strongly related to that of the sinus venosus.

Development of the epicardium and subepicardium

The extracardiac origin of the epicardium in vertebratesis now a well-established fact (Ho and Shimada 1978;Shimada and Ho 1980; Shimada et al. 1981; Virágh andChallice 1981; Komiyama et al. 1987; Hirakow 1992;Männer 1992; Virágh et al. 1993; Vrancken Peeters et al.1995). In lower vertebrates, the formation of the epicar-dium seems to involve three main processes: (1) Prolifer-ation of mesothelial cells (proepicardium) in the ventralpart of the liver-cardiac border; (2) dispersion of the me-sothelial cells from their original place; (3) adhesion ofthese cells to the myocardial surface, where they dissem-inate (Fransen and Lemanski 1990; Muñoz-Chápuli et al.1994b, 1997). In general, this scheme agrees with theavailable descriptions of the process in embryos of birdsand mammals, and it might represent the general patternof the formation of the epicardium in vertebrates.

In the dogfish, the proepicardium is formed by cellsoriginating from two lateral mesothelial anlangen, theright and left. These anlangen are located first at the ven-trolateral part of the liver and thereafter at the lateral partof the transverse septum. In the dogfish embryos, cellsdetaching from the proepicardium adhere to the surfaceof the heart and develop into epicardial cells. They firstlyensheath the atrioventricular groove as well as the dorsaland lateral aspects of the ventricle, and the ventral andlateral aspects of the atrium (Muñoz-Chápuli et al.1997).

According to our observations, we think that the epi-cardium covers the sinus venosus of the dogfish embryoby two different mechanisms, namely by the retrogrademigration (i.e. in the direction of the transverse septum)of the epithelium already established around the atrio-ventricular groove and, to a lesser degree, by the adhe-sion of mesothelial cells that are floating freely in thepericardial cavity.

The ultrastructure of the epicardial cells of the sinusvenosus in the dogfish embryos is similar to that de-scribed in embryos of several vertebrates (Virágh andChallice 1981; Zummo 1983; Kuhn and Liebherr 1988;Fransen and Lemanski 1990; Tidball 1992; Virágh et al.1993). We have described how the initially squamousepicardial cells show a progressive thickening through-

534

out their development, acquiring a cubic shape. On theother hand, pronounced interdigitations between the cellmembranes appear in the later embryos.

Virágh and Challice (1981) stated that the subepicar-dial mesenchyme derives from the transverse septum andparticipates in the formation of fibroblasts, macrophagesand probably smooth muscle cells. Other authors havesuggested that the cells of the subepicardial space origi-nate from the delamination of the outer epithelial layer inhigher vertebrates (Icardo et al. 1990b; Virágh et al.1990; Virágh et al. 1993) as well as in the dogfish(Muñoz-Chápuli et al. 1996). In the 27-mm embryo, wehave reported the presence of cells that seem to be de-taching from the primitive epicardium and populating thesubepicardium. This phenomenon seems to be restrictedto the most anterior and ventral part of the sinus venosus,close to the atrioventricular junction. From our results,we suggest also that at least some of the mesenchymalcells of the subepicardium differentiate as fibroblasts andcontribute to the production of the abundant fibrous ex-tracellular matrix that exists in the subepicardium of theadult. The differentiation of subepicardial mesenchymalcells in vascular elements has been described in otherparts of the dogfish heart, mainly at the atrioventriculargroove (Muñoz-Chápuli et al. 1996).

&p.2:Acknowledgements This study was supported by grant PB95-0475 from the DGICYT (Ministerio de Educación y Ciencia,Spain). David Macías is the recipient of a fellowship from the Jun-ta de Andalucía, Spain.

References

Benjamin M, Norman D, Santer RM, Scarborough D (1983) His-tological, histochemical and ultrastructural studies in the bul-bus arteriosus of the sticklebacks Gasterosteus aculeatusandPungitius pungitius(Pisces: Teleostei). J Zool 200:325–346

Berge PI (1979) The cardiac ultrastructure of Chimaera monstrosaL. (Elasmobranchii: Holocephali). Cell Tissue Res 201:181–195

Calvet S (1990) Estudi ultraestructural del desenvolupament delnode sino-atrial en Rattus norvegicus.PhD Thesis, Universityof Barcelona

Fransen ME, Lemanski LF (1990) Epicardial development in theAxolotl, Ambystoma mexicanum.Anat Rec 226:228–236

Hirakow R (1992) Epicardial formation in staged human embryos.Acta Anat Nippon 67:616–622

Hirakow R, Sugi Y (1990) Intercellular junction and cytoskeletalorganization in embryonic chick myocardial cells. In: ClarkEB, Takao A (eds) Developmental cardiology: morphogenesisand function. Futura, Mount Kisco, NY, pp 95–113

Hiruma T, Hirakow R (1985) An ultrastructural topographicalstudy on myofibrillogenesis in the heart of the chick embryoduring pulsation onset period. Anat Embryol 172:325–329

Ho E, Shimada Y (1978) Formation of the epicardium studiedwith the scanning electron microscope. Dev Biol 66:579–585

Huang CY (1967) Electron microscopic study of the developmentof heart muscle of the frog, Rana pipiens.J Ultrastruct Res20:211–226

Icardo JM, Fernández-Terán MA, Ojeda JL (1990a) Early cardiacstructure and developmental biology. In: Meisami E, TimirasPS (eds) Handbook of human growth and developmental biol-ogy, vol 3 (B). CRC Press, Boca Raton, pp 3–24

Icardo JM, Fernández-Terán MA, Ojeda JL (1990b) Late heartembryology. The making of an organ. In: Meisami E, Timiras

PS (eds) Handbook of human growth and developmental biol-ogy, vol 3 (B). CRC Press, Boca Raton, pp 25–49

Kitoh K, Oguri M (1985) Differentiation of the compact layer inthe heart ventricle of the rainbow trout. Bull Jpn Soc Soc Fish51:539–542

Komiyama M, Ito K, Shimada Y (1987) Origin and developmentof the epicardium in the mouse embryo. Anat Embryol176:183–189

Kuhn HJ, Liebherr G (1988) The early development of the epicar-dium in Tupaia belangeri.Anat Embryol 177:225–234

Leknes IL (1980) Ultrastructure of atrial endocardium and myo-cardium in three species of Gadidae (Teleostei). Cell TissueRes 210:1–10

Leknes IL (1981a) On the ultrastructure of the endothelium in thebulbus arteriosus of three teleostean species. J Submicrosc Cy-tol 13:41–46

Leknes IL (1981b) The structure of the sinus venosus and sino-atrial junction in teleosts. Zool Jahrb Anat 106:400–404

Leknes IL (1983) Ultrastructural and ultrahistochemical studies onventricular endocardial cells in teleosts (Pisces). J Zool201:507-513

Lemanski LF (1973) Heart development in the Mexican salaman-der, Ambystoma mexicanum.II. Ultrastructure. Am J Anat136:487–525

Lemanski LF, Fitzharris TP (1989) Analysis of the endocardiumand cardiac jelly in truncal development in the cardiac lethalmutant axolotl Ambystoma mexicanum.J Morphol 200:123–130

Lemanski LF, Fitts EP, Marx BS (1975) Fine structure of the heartin the Japanese medaka, Oryzias latipes.J Ultrastruct Res53:37–65

Manasek FJ (1968) Embryonic development of the heart. I. A lightand electron microscopic study of myocardial development inthe early chick embryo. J Morphol 125:329–366

Manasek FJ (1970) Histogenesis of the embryonic myocardium.Am J Cardiol 25:149–168

Manasek FJ, Icardo JM, Nakamura A, Sweeney L (1986) Cardio-genesis: developmental mechanisms and embryology. In:Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE(eds) The heart and cardiovascular system. Raven Press, NewYork, pp 965–985

Männer J (1992) The development of pericardial villi in the chickembryo. Anat Embryol 186:379–385

Markwald RR (1973) Distribution and relationship of precursor Z-material to organizing myofibrillar bundles in embryonic ratand hamster ventricular myocytes. J Mol Cell Cardiol5:341–350

Markwald RR, Fitzharris TP, Adams Smith WN (1975) Structuralanalysis of endocardial cytodifferentiation. Dev Biol 42:160–180

Moorman AFM, Lamers W (1994) Molecular anatomy of the de-veloping heart. Trends Cardiovasc Med 4:257–264

Muñoz-Chápuli R, De Andrés A, Ramos C (1994a) Tachykinin-like immunoreactivity in the sinus venous of the dogfish(Scyliorhinus canicula). Cell Tissue Res 278:171–175

Muñoz-Chápuli R, Macías D, Ramos C, De Andrés A, Gallego A,Navarro P (1994b) Heart development in the dogfish(Scyliorhinus canicula). A model for the study of the basicprocesses of vertebrate cardiogenesis. Cardioscience 5:245–253

Muñoz-Chápuli R, Gallego A, Macías D, Ramos C, De Andrés A(1995) Endocardial pores in the sinus venosus and atrium ofthe dogfish. J Fish Biol 47:769–774

Muñoz-Chápuli R, Macías D, Ramos C, Gallego A, De Andrés A(1996) Development of the subepicardial mesenchyme andearly coronary vessels in the dogfish (Scyliorhinus canicula). JExp Zool 275:95–111

Muñoz-Chápuli R, Macías D, Ramos C, Fernández B, Sans-ComaV (1997) Development of the epicardium in the dogfish(Scyliorhinus canicula). Acta Zool (Stockholm) 78:39–46

Myklebust R, Kryvi H (1983) Myofibril development in the myo-cardial cells of the shark Scyliorhinus canicula.Comp Bio-chem Physiol [A] 76:465–469

535

Padrós i Bover F (1994) Aspectos histológicos y ultraestructuralesdel desarrollo larvario del rodaballo, Scophthalmus maximus(Linnaeus, 1788), y estudio histopathológico de las alteraci-ones observadas en cultivo. PhD thesis, University of Barcelo-na

Pexieder T (1981) Prenatal development of the endocardium: a re-view. Scanning Electron Microsc 2:223–253

Ramos C, Muñoz-Chápuli R, Navarro P (1996) Ultrastructuralstudy of the myocardium of the sinus venosus and sinoatrialvalve in the dogfish (Scyliorhinus canicula). J Zool 238:611–621

Rönnau KC (1977) Myogenesis and contraction in the early em-bryonic heart of the rainbow trout. Cell Tissue Res 180:123–132

Saetersdal TS, Justesen N-P, Krohnstad AW (1974) Ultrastructureand innervation of the teleostean atrium. J Mol Cell Cardiol6:415–437

Saetersdal TS, Sorensen E, Myklebust R (1975) Granule-contain-ing cells and fibers in the sinus venosus of elasmobranchs.Cell Tissue Res 163:471–490

Santer RM (1972) An electron microscopical study of the develop-ment of the teleost heart. Z Anat Entwicklungsgesch139:93–105

Shimada Y, Ho E (1980) Scanning electron microscopy of the em-bryonic chick heart: formation of the epicardium and surfacestructure of the four heterotopic cells that constitute the em-bryonic heart. In: Van Praag R, Takao A (eds) Etiology andmorphogenesis of congenital heart disease. Futura, MountKisco, NY, pp 63–80

Shimada Y, Ho E, Toyota N (1981) Epicardial covering over myo-cardial wall in the chicken embryo as seen with the scanningelectron microscope. Scanning Electron Microsc 2:275–280

Shimada Y, Komiyama M, Terai M, Maruyama K (1990) Earlyphases of myofibril assembly in embryonic chick cardiac myo-cytes in vitro. In: Clark EB, Takao A (eds) Developmental car-diology: morphogenesis and function. Futura, Mount Kisco,NY, pp 63–77

Tidball JG (1992) Distribution of collagens and fibronectin in thesubepicardium during avian cardiac development. Anat Em-bryol 185:155–162

Virágh S, Challice CE (1981) The origin of the epicardium and theembryonic myocardial circulation in the mouse. Anat Rec201:157–168

Virágh S, Kálmán F, Gittenberger-de Groot AC, Poelman RE,Moorman AFM (1990) Angiogenesis and hematopoiesis in theepicardium of the vertebrate embryo heart. In: Bockman DE,Kirby ML (eds) Embryonic origins of defective heart develop-ment. Ann N Y Acad Sci 588:455–458

Virágh S, Gittenberger-de Groot AC, Poelmann RE, Kálmán F(1993) Early development of quail heart epicardium and asso-ciated vascular and glandular structures. Anat Embryol188:381–393

Vrancken Peeters MP, Mentink MMT, Poelmann RE, Gittenberg-er-de Groot AC (1995) Cytokeratins as a marker for epicardi-cal formation in the quail embryo. Anat Embryol 191:503–508

Yamauchi A (1980) Fine structure of the fish heart. In: Bourne GH(ed) Hearts and heart-like organs, vol 1. Academic Press, NewYork, pp 119–148

Zummo G (1983) Ultrastructure features of the embryonic heart ofthe elasmobranch Scyllium stellare.Comp Biochem Physiol[A] 76:459–464

536