Differentiation of the Cardiac Outﬂow Tract Components in ... · Differentiation of the Cardiac Outﬂow Tract Components in Alevins of the Sturgeon Acipenser naccarii (Osteichthyes,

Differentiation of the Cardiac Outflow TractComponents in Alevins of the Sturgeon Acipensernaccarii (Osteichthyes, Acipenseriformes): Implicationsfor Heart EvolutionAlejandro Guerrero,1 Jose M. Icardo,2 Ana C. Duran,1 Alejandro Gallego,1 Alberto Domezain,3Elvira Colvee,2 and Valentın Sans-Coma1*

1Department of Animal Biology, Faculty of Science, University of Malaga, 29071 Malaga, Spain2Department of Anatomy and Cell Biology, University of Cantabria, 30911 Santander, Spain3Department R&D Piscifactorıa “Sierra Nevada”, Camino de la Piscifactorıa 2, 18313 Riofrıo, Granada, Spain

ABSTRACT Previous work showed that in the adultsturgeon an intrapericardial, nonmyocardial segment isinterposed between the conus arteriosus of the heartand the ventral aorta. The present report illustrates theontogeny of this intermediate segment in Acipenser nac-carii. The sample studied consisted of 178 alevins be-tween 1 and 24 days posthatching. They were examinedusing light and electron microscopy. Our observationsindicate that the entire cardiac outflow tract displays amyocardial character during early development. Be-tween the fourth and sixth days posthatching, the distalportion of the cardiac outflow tract undergoes a pheno-typical transition, from a myocardial to a smoothmuscle-like phenotype. The length of this region withregard to the whole outflow tract increases only moder-ately during subsequent developmental stages, becom-ing more and more cellularized. The cells soon organizeinto a pattern that resembles that of the arterial wall.Elastin appears at this site by the seventh day post-hatching. Therefore, two distinct components, proximaland distal, can be recognized from the fourth day post-hatching in the cardiac outflow tract of A. naccarii. Theproximal component is the conus arteriosus, character-ized by its myocardial nature and the presence of endo-cardial cushions. The distal component transforms intothe intrapericardial, nonmyocardial segment mentionedabove, which is unequivocally of cardiac origin. We pro-pose to designate this segment the “bulbus arteriosus”because it is morphogenetically equivalent to the bulbusarteriosus of teleosts. The present findings, togetherwith data from the literature, point to the possibilitythat cells from the cardiac neural crest are involved inthe phenotypical transition that takes place at the dis-tal portion of the cardiac outflow tract, resulting in theappearance of the bulbus arteriosus. Moreover, theysuggest that the cardiac outflow tract came to be formedby a bulbus arteriosus and a conus arteriosus from anearly period of the vertebrate evolutionary story. Fi-nally, we hypothesize that the embryonic truncus ofbirds and mammals is homologous to the bulbus arteri-osus of fish. J. Morphol. 260:172–183, 2004.© 2004 Wiley-Liss, Inc.

KEY WORDS: heart; conus arteriosus; bulbus arteriosus;Acipenseriformes; Osteichthyes

It is generally accepted that the most anteriorcomponent of the primitive vertebrate heart is theconus arteriosus, a contractile chamber whose wallsconsist largely of cardiac muscle overlying an elasticfibrous coat (Gegenbauer, 1866; Santer, 1985;Zummo and Farina, 1989). A well-developed conusarteriosus is present in the adult heart of the elas-mobranchs (Gegenbauer, 1866, 1891, 1901; Stohr,1876; Kisch, 1930; Parsons, 1930; Santer, 1985;Sans-Coma et al., 1995), lungfishes (Lankester,1879; Boas, 1880a; Robertson, 1914), coelacanths(Anthony et al., 1965; Millot et al., 1978), and prim-itive actinopterygians such as the polypteriforms(Parsons, 1930), acipenseriforms (Parsons, 1930;Icardo et al., 2002a,b), and lepisosteiforms (Parsons,1930).

In the halecomorph Amia calva and in primitiveteleost species belonging to the genera Albula, Ptero-thrissus, Megalops, and Tarpon, the cardiac outflowtract (OFT) is composed of two distinct portions: aposterior, myocardial conus arteriosus and an ante-rior, nonmyocardial bulbus arteriosus (Stannius,1846; Boas, 1880b; Senior, 1907abc; Parsons, 1930;Satchell, 1991). In most teleosts, however, the conusarteriosus has classically been considered to be veryreduced in size or even absent (Smith, 1918; Santer,1985; Satchell, 1991; Farrell and Jones, 1992), a factwhich is concomitant with the remarkable develop-

Contract grant sponsor: D.G.E.S. (Ministerio de Educacion y Cul-tura); Contract grant numbers: PB98-1418-C02-01, PB98-1418-C02-02, AGL2000-0832-P4-05; Contract grant sponsor: Ministerio de Cien-cia y Tecnologıa, Spain; Contract grant number: fellowship FP99-25680733 (to A.G.).

*Correspondence to: Valentın Sans-Coma, Department of AnimalBiology, Faculty of Science, University of Malaga, 29071 Malaga,Spain. E-mail: [email protected]

DOI: 10.1002/jmor.10200

JOURNAL OF MORPHOLOGY 260:172–183 (2004)

© 2004 WILEY-LISS, INC.

ment of the bulbus arteriosus in the phylogeneti-cally advanced bony fishes. The bulbus arteriosus ofteleosts is an intrapericardial, elastic chamber thatconnects the conus arteriosus with the ventral aorta,or the ventricle with the ventral aorta when theconus arteriosus might be absent. Histologically, thebulbus arteriosus is organized into two layers thatcontain myofibroblasts, smooth muscle cells, colla-gen, and elastin (Priede, 1976; Santer, 1985; Satch-ell, 1991), distributed in species-specific patterns(Icardo et al., 1999a,b, 2000a,b).

The evolutionary origin of the bulbus arteriosushas been the subject of a still unresolved contro-versy. Several authors believe that the bulbus is abackward extension of the ventral aorta into thepericardial cavity (Boas, 1901; Bridge, 1904; Krause,1923; Grodzinski, 1938; Bertin, 1958; Parker andHaswell, 1962; Weichert and Presch, 1975; Lawson,1979). The observations of other authors (Parsons,1930; Licht and Harris, 1973; Priede, 1976) are bestunderstood if it is assumed that the bulbus arterio-sus originates as a modification of the anterior partof the conus arteriosus, i.e., that the bulbus is ofcardiac origin (Yamauchi, 1980; Farrell and Jones,1992).

Icardo et al. (2002a,b) reported that the ventralaorta of the sturgeon Acipenser naccarii consists oftwo segments, one located within the pericardialcavity and the other above this cavity. The intraperi-cardial segment, interposed between the conus arte-riosus and the extrapericardial aortic segment, dis-plays a discontinuous internal elastic lamina liningthe endocardium, a middle layer of smooth musclecells, and an epicardial covering. The smooth musclecells are organized into an inner longitudinal layer,a middle circumferential layer, and an external lon-gitudinal layer. In contrast, the extrapericardialaortic segment shows a continuous subendocardialelastic lamina and a thick layer of circumferentiallyoriented smooth muscle cells. The outer longitudinalcell layer is less developed, while the inner longitu-dinal layer is mostly absent. In addition, a distinctintima-like layer is located between the inner elas-tica and the smooth muscle cells. These structuraldifferences between the two aortic portions indicatethat the intrapericardial segment constitutes a tran-sitional segment, the embryonic origin of whichmight differ from that of the extrapericardial ven-tral aorta (Icardo et al., 2002b).

The existence of this transitional, intrapericardialsegment between the conus arteriosus and the ex-trapericardial ventral aorta in the sturgeon heart isundoubtedly of great interest from an evolutionaryviewpoint. Knowledge of its formation might throwsome light on the controversy concerning the evolu-tionary origin of the bulbus arteriosus. In this re-gard, it should be emphasized that sturgeons belongto the chondrosteans, a primarily Paleozoic andTriassic fish assemblage that constitutes the firststage of actinopterygian evolution (Colbert, 1955;

Carroll, 1988). On this basis, we conducted a studyto illustrate the ontogeny of the cardiac OFT ofAcipenser naccarii, an autochthonous species of boththe Adriatic sea and the Iberian Peninsula (Garrido-Ramos et al., 1997) which presumably inhabited thewhole European Mediterranean basin (Domezain etal., 2003; Robles et al., 2003). The main goals were:1) to describe the development of the transitionalsegment lying between the conus arteriosus and theextrapericardial ventral aorta, and 2) to gather newdata that may contribute to a better understandingof the evolution of the heart within the lineage ofactinopterygian fish.

MATERIALS AND METHODSAnimals

The sample examined consisted of 178 alevins of Acipensernaccarii obtained from the Sierra Nevada Fishery at Riofrıo,Granada, Spain. The age of the alevins ranged from 1–24 daysposthatching (dph). They were overanesthetized in 0.04% MS222(tricaine methane sulfonate, Sigma Chemical Co., Poole, UK) andmeasured. In this context, it should be noted that the total length(TL; in mm) and the age (A; in days) of the present specimenswere highly correlated (TL � 0.843 A� 8.507; R2 � 0.977). Thehearts of 141 alevins were examined using histological, histo-chemical, and immunohistochemical techniques; another 21 werestudied by scanning electron microscopy, and the remaining 16 bymeans of semithin sections and transmission electron micros-copy.

Histological and Histochemical Techniques

The alevins were fixed by immersion in MAW fixative (meth-anol:acetone:water, 2:2:1; ratio of fixative to tissue volume �80:1) and embedded in Paraplast (Sigma). Serial sections of theheart, transversely, longitudinally, or sagittally cut at 10 �m,were stained with Delafield’s hematoxylin-eosin or Mallory’strichrome stain for a general assessment of the histological com-ponents of the heart. In addition, the orcein-HCl method wasused for the specific detection of elastin.

Immunohistochemical Techniques

Transverse, longitudinal, and sagittal sections of the heart,obtained following the protocol described above, were stainedwith monoclonal antibody MF20 (Developmental Studies Hybrid-oma Bank, University of Iowa) against a myosin heavy chainepitope, or with monoclonal anti-�-actin AC-15 (Abcam, UK)against the N-terminal of the �-isoform of actin. It should benoted that the use of monoclonal antismooth muscle �-actin(Sigma, clone 14) was unsuccessful.

The sections were dewaxed in xylene, hydrated in an ethanolseries, and washed in Tris-phosphate buffered saline (TPBS, pH7.8). Endogenous peroxidase activity was quenched by incubationwith 3% hydrogen peroxide in TPBS for 30 min. After washingwith TPBS, nonspecific binding sites were saturated for 1 h with10% sheep serum and 1% bovine serum albumin in TPBS plus0.5% Triton X-100 (SBT). Sections were washed with TPBS andthen incubated for 18 h in the primary antibody diluted in SBT.Control slides were incubated in SBT only.

After incubation, the sections were washed in TPBS (3 � 5min), incubated for 1 h at room temperature in biotin-conjugatedantimouse IgG (Sigma) diluted 1:100 in TPBS, washed again, andincubated for 1 h in ExtrAvidin conjugate (Sigma) diluted 1:150in TPBS. Peroxidase activity was developed with Sigma Fast3,3�-diaminobenzidine tablets according to the instructions of the

173CARDIAC OUTFLOW TRACT IN THE STURGEON

supplier. Finally, the sections were counterstained withDelafield’s hematoxylin.

Semithin Sections and TransmissionElectron Microscopy

Removed hearts were fixed by immersion in 3% glutaraldehydein PBS for 3 h, postfixed in osmium tetroxide, dehydrated ingraded acetone and propylene oxide, and embedded in Araldite(Fluka, Buchs, Switzerland). Semithin sections were cut with anLKB III ultratome, stained with 1% Toluidine blue, and observedwith a Zeiss III photomicroscope. Ultrathin sections were cutwith a Leica Ultracut UCT, stained with uranyl acetate and leadcitrate, and examined with a Zeiss ME 10C microscope.

We used serial semithin sections and ultrathin sections of thecardiac OFT to detect cell death. In this regard it should be notedthat similar observations in birds showed apoptosis in both themyocardium and mesenchyme (Icardo, 1990).

Scanning Electron Microscopy

Removed hearts were fixed by immersion in 3% glutaraldehydein PBS for 3 h. Then the specimens were dehydrated in gradedacetone, dried by the critical point method, and sputter-coatedwith gold. Observations were made using a Philips 501 scanningelectron microscope operated at 7.2 kV.

Nomenclature

We use the terms proximal and distal to describe the location ofthe components of the cardiac OFT with regard to the ventricle.

RESULTS

In alevins age 1–3 dph, the heart displayed asimple tubular configuration (Fig. 1a). It consisted offour chambers: the OFT, the ventricle, the atrium,and the sinus venosus, arranged in a craniocaudalsequence. The conoventricular sulcus marked theboundary between the OFT and the ventricle. Theatrioventricular sulcus separated the ventricle fromthe atrium. On the whole, the heart adopted theshape of a “C,” the convexity of which was directedtoward the right side of the body. Histologically, theheart was a uniform structure composed of two cel-lular layers, the endocardial endothelium and themyocardium, separated by cardiac jelly (Fig. 2a).The entire myocardial layer showed positive immu-noreactivity for MF20 (Fig. 3a) up to the craniallimit of the pericardial celomic cavity.

In alevins age 4 dph, the distance between thecaudal limit of the OFT and the cranial wall of theatrium had shortened due to the ascent of theatrium. Meanwhile, the ventricle had partiallymoved towards its definitive midline position (Fig.1b). In these specimens, the cardiac OFT retained itspositive immunoreactivity against the MF20 anti-body, except for a short, distal portion, which be-came MF20-negative (Fig. 3b). Semithin sectionsrevealed that this distal portion, which had presum-ably lost its myocardial phenotype, consisted of aninner endothelial layer, an outer epithelial layer,and a middle layer of highly cellularized mesen-

chyme tissue (Fig. 2b,c). Histological changes in theouter epithelium included, successively, loss of themyocardial organization, formation of a cuboidal cellmonolayer, and transformation into a flattened epi-thelium. The transient presence of large intracellu-lar lipid droplets was characteristic of this area.These histological changes occurred between days4–5 dph, and were observed first in the left side ofthe OFT (Fig. 2c,d). The mesenchyme tissue of theMF20-negative distal portion of the OFT (Fig. 2a)was continuous with the cardiac jelly proximally,and with the extracardiac mesenchyme distally. Incontrast, the MF20-positive portion of the OFTmaintained its previous histological organization.The only change detected was the presence of asmall number of cells in the cardiac jelly space (com-pare Fig. 2a, 2b).

In alevins aged 6 dph, the cardiac chambers hadnearly occupied their definitive position (Fig. 1c).The sinus venosus and the atrium were located dor-sally with regard to the ventricle and the OFT, andthe ventricle occupied a caudal position. The MF20-negative distal portion of the OFT (Fig. 3c) showedpositive immunoreactivity against the �-actin anti-body (Fig. 3d), thereby suggesting a differentiationof mesenchyme cells into smooth muscle cells at thissite. Cells in this segment were densely arranged(Fig. 2e). Large masses of extracellular material,presumably collagen, accumulated in the extracellu-lar matrix, the density of this matrix being higherthan that of the cardiac jelly. On the other hand,�-actin-positive cells were also present in the devel-oping walls of the ventral aorta and aortic arches(Fig. 3d).

In alevins age 7–8 dph, the heart acquired thefinal anatomic configuration (Fig. 1d). Concomi-tantly, a major reorganization took place in the dis-tal portion of the OFT (Fig. 2f,g). Cells became mas-sively arranged along the longitudinal OFT axis,elastic fibers appeared in the extracellular matrix,and a distinct epicardial layer could be recognized(Fig. 2g). In addition, melanocytic cells appearedunder the epicardium of this OFT segment down tothe myocardial level (Fig. 2g). Meanwhile, proximal

Fig. 1. Development of the heart of the sturgeon, Acipensernaccarii. SEM. a: 2 dph. The heart shows a “C” shape. Bending ismore pronounced at the outflow tract (OFT) and ventricle (V)than at the atrium (A). The sinus venosus (SV) is a midlinestructure. The conoventricular and the atrioventricular sulci areindicated by arrows. b: 4 dph. The ventricle is moving toward themidline. The atrium is ascending to occupy a position to the left ofthe outflow tract. c: 6 dph. The ventricle is a midline structure.The atrium has shifted to occupy a dorsolateral position withregard to the outflow tract. The double arrow indicates the MF20-negative distal portion of the outflow tract. d: 10 dph. The hearthas acquired the adult configuration. The double arrow indicatesthe nonmyocardial segment of the cardiac outflow tract. Note thatthe relative craniocaudal length of the heart decreases as thefolding into an S-shaped structure progresses. Scale bars � 100mm.

174 A. GUERRERO ET AL.

Figure 1


and distal endocardial cushions developed in themyocardial portion of the OFT, became populated bycells, and cushion excavation began (Fig. 2e–h). His-togenesis proceeded through subsequent develop-mental stages, the distal OFT portion acquiring an

aortic-like organization (Fig. 2i). In agreement withthe histological findings, at the 7th dph, orceinstaining detected for the first time the presence ofelastin in the ventral aorta, aortic arches, and distal,nonmyocardial portion of the cardiac OFT (Fig. 3e).

Figure 2


Thereafter, the amount of elastin increased gradu-ally in all of these cardiovascular structures (Fig.3f).

From the 9th to the 24th dph, the distal portion ofthe cardiac OFT continued to be unreactive to MF20(Fig. 3g) and reactive to �-actin (Fig. 3h). Whenviewed from the outside, this nonmyocardial portionappeared as a relatively short, narrow ring con-nected to the ventral aorta at the anterior limit ofthe pericardial cavity (Fig. 1d). In contrast, the prox-imal, myocardial portion of the OFT was much moredeveloped in length and width. The boundary be-tween the two portions was roughly annular (Fig.1d).

Finally, it should be noted that we detected noapoptotic cells or macrophages in the developingcardiac OFT, either in serial semithin sections or inultrathin sections

DISCUSSIONDifferentiation of Cardiac Outflow TractComponents

MF20 antimyosin antibody staining shows thatthe entire cardiac OFT of Acipenser naccarii dis-plays a myocardial character during early develop-ment. Yet its distal portion loses MF20-positive im-munoreactivity at the 4th dph and becomes �-actin-positive by the 6th dph. This indicates that this

region undergoes a phenotypic transition, from amyocardial to a smooth muscle-like phenotype. Thelength of this region with regard to the whole car-diac OFT increases only moderately during subse-quent developmental stages, becoming more andmore cellularized. Furthermore, the cells soon orga-nize into a pattern that resembles that of the arte-rial wall. According to the present findings, the dis-tal OFT segment continues to show �-actin-positiveimmunoreactivity at least until the 24th dph. Elas-tin appears at this site by the 7th dph. From then on,the amount of elastin increases progressively.

Therefore, two distinct components, proximal anddistal, can be recognized from the 4th dph in thecardiac OFT of Acipenser naccarii. The proximalcomponent is the conus arteriosus, characterized byits myocardial nature and presence of endocardialcushions from which the conus valves derive. Thedistal component displays a nonmyocardial charac-ter and, presumably, transforms into the transi-tional (or intermediate) segment, intercalated be-tween the conus arteriosus and the ventral aorta, asdescribed by Icardo et al. (2002a,b) in adult stur-geons. The present data on the formation of thistransitional segment supports the notion of Icardo etal. (2002b) that its morphogenetic origin divergesfrom that of the ventral aorta, a fact which mayexplain the structural differences between them inthe adult heart (see Icardo et al., 2002a,b).

Several of the morphogenetic events reportedherein are similar to those observed during the de-velopment of the bulbus arteriosus of the zebrafish(Hu et al., 2000). In this teleost species, the antici-pated bulbus arteriosus displays MF20-positive im-munoreactivity, revealing similar characteristics tothose of the myocardium. At 4 weeks postfertiliza-tion, the bulbus loses its positive MF20 staining,showing a smooth muscle phenotype. According toHu et al. (2001), this positive/negative MF20 stain-ing transition during early development might beattributed to removal of OFT cardiomyocytesthrough apoptosis, or to a replacement of the smoothmuscle cells from the head mesenchyme. In the stur-geon alevins examined, we were unable to detect anysign of apoptosis in the distal portion of cardiac OFTduring the period in which it loses its primary myo-cardial condition. This diverges from the morphoge-netic mechanisms that govern the remodeling of themyocardial cardiac OFT in birds, where myocyteapoptosis plays a fundamental role (Watanabe et al.,1998).

In Acipenser naccarii, the subendothelial space ofthe ventral aorta, and that of the nonmyocardialdistal portion of the cardiac OFT, is populated earlyin development by a considerable number of mesen-chyme cells that soon become organized into anarterial-like pattern. The morphogenetic patternclearly diverges from that followed by the cells pop-ulating the endocardial cushions, which presumablyoriginate from the cushion endocardium. This sug-

Fig. 2. Longitudinal semithin sections illustrating the histo-logical changes occurring in the distal portion of the cardiacoutflow tract of the sturgeon, Acipenser naccarii. a: 3 dph. Theouter layer of the outflow tract is composed exclusively of myo-cytes. The cell cytoplasm contains dark vitellin inclusions (arrow-heads). The arrow indicates the distal portion of the outflow tract.b: 4 dph. The distal portion of the outflow tract (arrows) is losingthe typical myocardial organization. Several cells appear in thecardiac jelly space. Vitellin inclusions have disappeared. c: Detailfrom b. Distal portion of the outflow tract; the arrows indicate itsleft and right limits. This portion is losing the myocardial char-acter. The left side is longer than the right side. Note the presenceof large intracellular lipid droplets (arrowheads). The extracellu-lar matrix is continuous with the extracardiac tissues. d: 5 dph.Distal portion of the outflow tract. In the left side (boundarymarked by arrows), the outer cells are flattened. In the right side,the outer cells are cuboidal. Note the increase in cell and extra-cellular matrix density. e: 6 dph. Note the continuity between theextracardiac mesenchyme, the distal portion of the outflow tract(arrows) and the cushion mesenchyme. The myocardium and thecushion tissue end at the same transverse level. f: 7 dph. Someextracellular fibers can histologically be recognized as elastin(arrowhead). g: 8 dph. In the distal outflow tract, many cellsappear aligned along the longitudinal axis. The epicardium (dou-ble arrows) can be recognized for the first time. The typicalgranulations of melanocytes (arrowheads) appear under the epi-cardium. h: 10 dph. Note the parallel alignment between extra-cellular fibers and long cell prolongations. This type of organiza-tion does not occur in the cushion tissue. The process of cushionexcavation is beginning. The arrowheads indicate melanocytes. i:15 dph. In the distal outflow tract, cells appear massively alignedalong the longitudinal cell axis. Note the progressive organizationwith age. CJ, cardiac jelly; E, endocardium; M, myocardium.Asterisk, cushion tissue. Scale bars � 80 �m (a,b), 20 �m (c–i).


Figure 3

gests that the mesenchyme populating the distalportion of the OFT does not originate in situ, butproceeds from another morphogenetic source.

It is well known that in birds and mammals thecranial neural crest gives rise to ectomesenchymethat contributes to the septation of the cardiac OFTand to the arterialization of the aorta, aortic arches,and pulmonary artery. In fact, the presence of cellsof neural crest origin in the OFT coincides with theloss of the myocardial character of the distal portionof the OFT and the acquisition by the mesenchymecells of a smooth muscle phenotype (Bockman et al.,1987; Nishibatake et al., 1987; Hiruma and Hira-kow, 1992; Yablonka-Reuveni et al., 1995, 1998;Bergwerff et al., 1996, 1998; Waldo et al., 1998,1999; Jiang et al., 2000).

Knowledge on the contribution of the neural crestto the development of the fish vascular system con-cerns a very limited number of teleost species. Inmembers of the genus Xiphophorus, cells proceedingfrom the neural crest are involved in the formationof the vasculature of the gill arches (Sadaghiani andVielkind, 1990). The segmental series of the pharyn-geal arches of the zebrafish is formed by two migratorycell types, namely, neural crest and paraxial meso-derm cells (Schilling and Kimmel, 1994). In this latterspecies, neural crest cells participate in structuringthe bulbus arteriosus, ventricle, atrioventricular junc-tion, and atrium (Sato and Yost, 2003). After reachingthe heart, neural crest-derived cells intermingle withthe myocardium, adopting a cardiomyocyte cell lin-eage (Li et al., 2003; Sato and Yost, 2003).

It has been demonstrated that the neural crestcells that populate the bulbus arteriosus of the ze-brafish proceed from different cellular groups of boththe medial and lateral cardiac neural crest (Sato andYost, 2003). Yet the spectrum of morphogenetic ac-tions performed by these cells is still uncertain.They were adduced as possible precursors of thechondrocytes of the cartilaginous deposits occurringin the bulbus arteriosus of adult teleosts (Blanco et

al., 2001). The melanocytes that appear in the em-bryonic cardiac OFT of the sturgeon are apparentlyneural crest-derived elements. Interestingly, thepresent observations suggest that the distal portionof the OFT becomes populated by cells proceedingfrom an extracardiac source at the time at which theswitch from a myocardial to a smooth muscle-likephenotype takes place. This points to the possibilitythat neural crest cells might participate in the re-modeling of the distal segment of the cardiac OFT, aquestion that merits further investigation.

Implications for Heart Evolution

Gegenbaur (1866) introduced the name “conus ar-teriosus” to designate the myocardial segment lo-cated between the ventricle of the elasmobranchheart and the ventral aorta. He retained the ancientterm “bulbus arteriosus” to indicate the nonmyocar-dial chamber interposed between the ventricle andthe ventral aorta, or between the conus arteriosusand the ventral aorta in actinopterygian fishes. Ge-genbaur’s nomenclature was adopted by numerousauthors. Parsons (1930), however, used the name“conus arteriosus” to designate the morphologicallyanterior portion of the primitive cardiac tube, in-cluding the nonmyocardial bulbus arteriosus of theactinopterygians. At the same time, Goodrich (1930)argued that bulbus cordis should be applied to theanterior embryonic cardiac chamber throughout thecraniata, reserving the name conus arteriosus forthe adult muscular, contractile chamber, derivedfrom the bulbus cordis of fish and amphibians. Morerecently, De la Cruz et al. (1999) claimed that theterm conus arteriosus should be reserved to indicatea region or segment present only in the embryonicheart, whereas the mature, anterior chamber of allvertebrate hearts should be called bulbus cordis.Nonetheless, the viewpoint that prevails in text-books is that the cardiac OFT of both the elasmo-branchs and primitive actinopterygians consists of amyocardial conus arteriosus, whereas in advancedactinopterygians it is mainly composed of a nonmyo-cardial bulbus arteriosus (Parker and Haswell,1962: Weichert and Presch, 1975; Lawson, 1979;Johansen and Burggren, 1980; Hildebrand, 1982;Young, 1983; Nadal, 2001; Kardong 2002). The evo-lutionary switch from a cardiac OFT formed by apatent conus arteriosus, furnished with several rowsof valves, to an OFT composed of a well-developedbulbus arteriosus and a reduced or even absent co-nus arteriosus, with a single row of valves, has beenthe subject of numerous conjectures.

The reduction in size of the conus arteriosus inteleosts has been implicitly assumed as a conse-quence of the progressive development of the bulbusarteriosus. The disappearance of the conus arterio-sus in the adult heart of several teleost species hasbeen attributed to its incorporation into the proxi-mal end of the bulbus arteriosus or, much more

Fig. 3. Longitudinal (a,f) and sagittal (b–e,g,h) sections of thedeveloping cardiac outflow tract (OFT) of the sturgeon, Acipensernaccarii. a–c,g: MF20 immunostaining counterstained with he-matoxylin; d,h: �-Actin immunostaining counterstained with he-matoxylin; e,f: Orcein-HCl. a: 2 dph. The entire outer layer of theOFT shows positive immunoreactivity for MF20. b: 4 dph. Thedistal portion (arrows) of the OFT is MF20-negative, whereas theproximal portion (arrowheads) retains its MF20-positivity. c,d: 6dph. The distal portion (arrows) of the OFT is MF20-negative (c)and �-actin-positive (d), whereas the proximal portion (arrow-heads) is MF20-positive (c) and �-actin-negative (d). e (7 dph)and f (14 dph): Elastin fibers are present in the aortic wall and inthe cardiac OFT down to the base of the endocardial cushions(black stars). g,h: 9 dph. The distal portion (arrows) of the OFT isMF20-negative (g) and �-actin-positive (h), whereas the proximalportion (arrowheads) is MF20-positive (g) and �-actin-negative(h). The white stars indicate the posterior aortic arches. Thewhite arrowheads point to the epicardial layer. Ao, aorta; PC,pericardial cavity. Scale bars � 100 �m (a–d); 50 �m (e,f).


frequently, to its condensation into the ventricle(Smith, 1918).

Boas (1901) hypothesized that, evolutionarily, thebulbus originated from the proximal part of the ven-tral aorta, which later extended backwards to re-place the conus arteriosus of the elasmobranchs.This hypothesis relied on the fact that the histologyof the bulbus is undoubtedly more arterial than car-diac in nature. Some years later, Senior (1909)showed that in the shad, Alosa sapidissima, thebulbus develops within the pericardial cavity. None-theless, Grodzinski (1938) pointed out that the bul-bus cannot be regarded as a cardiac chamber, be-cause it lacks cardiac muscle. This view was adoptedby numerous subsequent authors (Bridge, 1904;Krause, 1923; Grodzinski, 1938; Bertin, 1958;Parker and Haswell, 1962; Weichert and Presch,1975; Lawson, 1979).

Parsons (1930) disputed Boas’ hypothesis, consid-ering that all segments of the cardiac tube withinthe pericardial cavity belong morphologically to theheart. He stated that the bulbus arteriosus of teleostfish is simply part of the original conus arteriosus,i.e., the preventricular portion of the primitive car-diac tube. The histological and biochemical studiesof the bulbus arteriosus carried out by Licht andHarris (1973) in the carp, Cyprinus carpio, illus-trated that the bulbus is basically distinct from theventral aorta, a fact that led to the assumption thatthe bulbus arteriosus is an intrinsic part of the te-leost heart derived, probably, from the primitiveconus arteriosus (Priede, 1976; Yamauchi, 1980;Satchell, 1991; Farrell and Jones, 1992).

The present findings in Acipenser naccarii sub-stantiate that the intrapericardial segment lying be-tween the conus arteriosus and ventral aorta, de-scribed by Icardo et al. (2002a,b) as a transitionalsegment, is not a backward extension of the ventralaorta, but derives from embryonic cardiac OFT. Ourfindings, together with those reported by Hu et al.(2000), strongly suggest that the transitional seg-ment of the sturgeon and the bulbus arteriosus ofteleosts are homologous structures. This conclusionrelies on the fact that the two structures originatefrom the distal portion of the cardiac tube followingsimilar morphogenetic steps, namely: a first stepcharacterized by the expression of a myocardial phe-notype, a second step consisting of the loss of themyocardial phenotype, and a third, longer, histoge-netic step, in which each of these structures acquiresits definitive tissue organization.

It is currently accepted that both the embryonicand adult heart of elasmobranchs is composed of fivemyocardial compartments: the sinus venosus,atrium, atrioventricular canal, ventricle, and conusarteriosus (Gallego et al., 1998; Franco et al., 2002).However, Parsons (1930) reported that in severalelasmobranch species, the muscular portion of theconus arteriosus in the adult heart does not as a ruleextend as far as the cranial pericardial boundary. In

such cases the distal portion of the conus consists ofa tissue resembling that of the wall of the ventralaorta beyond the pericardial cavity. To our knowl-edge, no further attention has been paid to thisnoncontractile, distal portion of the elasmobranchconus arteriosus, either from the morphological orfrom the embryological viewpoint. Yet from Parsons’descriptions it can be inferred that this nonmyocar-dial portion is morphologically equivalent to thetransitional segment of the adult sturgeon heart.

From the preceding data we conclude that a truebulbus arteriosus exists in primitive actinoptery-gian fishes, i.e., the sturgeons; it corresponds to thesecondarily nonmyocardial, distal portion of the car-diac OFT that Icardo et al. (2002b) referred to byusing the term “intrapericardial transitional seg-ment”. Moreover, we presume that a bulbus arteri-osus also exists in the elasmobranchs. The anatom-ical configuration of the cardiac OFT of the mostprimitive living craniate chordates, i.e., the hag-fishes and lampreys, differs between authors. Someof them consider that the cardiac OFT of the adultcyclostomes (Kent, 1978; Randall and Davie, 1980;Nadal, 2001; Kardong, 1998) and ammocoete larva(Daniel, 1934) is composed of a contractile conusarteriosus. Others (Fontaine, 1958; Santer, 1985)state that it consists of a noncontractile bulbus ar-teriosus, containing smooth muscle cells and elasticfibers (Yamauchi, 1980).

In our opinion, the bulbus appeared in an as-yetundetermined, early period of the craniata (verte-brate) evolutionary story. Thus, the cardiac OFTcame to be formed by two components: 1) a second-arily nonmyocardial, distal component, the bulbusarteriosus; and 2) a myocardial, proximal compo-nent, the conus arteriosus, which supports the en-docardial cushions that give rise to the cardiac OFTvalves. Within the lineage of teleosts, the bulbusarteriosus evolved into a well-developed chamber,splitting into a wide range of structural variants(Icardo et al., 1999a,b, 2000a,b).

The exact significance of the bulbus arteriosus inelasmobranchs and primitive actinopterygians re-mains uncertain. In teleosts, it is classically ac-cepted that the bulbus acts as a passive elastic res-ervoir or “Windkessel” (Von Skramlick, 1935) duringthe cardiac cycle, with particular importance inmaintaining the ventral aorta blood flow during ven-tricular diastole (Johansen, 1962; Priede, 1976;Satchell, 1991; Bushnell et al., 1992; Jones et al.,1993). Yet recent work has shown that the teleostbulbus may exhibit a wider variety of physiologicaland behavioral patterns than previously believed(Icardo et al., 1999a,b, 2000a,b). In contrast, thepossible significance of the conus arteriosus in te-leosts has been disregarded. However, it has re-cently been reported that the gilthead seabream,Sparus auratus, a phylogenetically advanced teleostspecies, possesses a distinct conus arteriosus formedby a compact, well-vascularized myocardium, which


seems to be actively implicated in the performanceof the conus valves (Schib et al., 2002). The discrim-ination between the conal and ventricular myocar-dia and the histomorphological characterization ofthe conus in this species was made possible by em-ploying a combination of different microscopic tech-niques. This suggests that the existence of a conusarteriosus in other teleosts might have been over-looked because of the use of inappropriate methods.Therefore, we consider that the assumed disappear-ance of the conus in teleosts is a question thatshould be reviewed.

The next questions that should be asked arewhether the existence of these two segments withinthe heart OFT is exclusive to fishes, and whetherthey are present in other vertebrate taxa. In birdsand mammals the nomenclature used to describethe developing OFT diverges widely between au-thors (see Pexieder, 1995, for an extensive review ofthe literature). However, it is generally acceptedthat the embryonic OFT consists of two components,a proximal conus and a distal truncus. The twosegments are invested by myocardium, developwithin the pericardial cavity, and become furnishedwith opposing endocardial cushions or ridges(Kramer, 1942; Shaner, 1962; Van Mierop et al.,1963; Icardo et al., 1990; Icardo and Manasek, 1991;Manner, 2000). Division of the OFT involves fusionof apposing cushions and the development of theaortopulmonary septum. The latter is a horseshoe-shaped structure whose two limbs penetrate thetruncal cushions down to the semilunar valve level(Icardo, 1985). As division of the truncus takesplace, the truncus loses its myocardial nature and islater transformed into the proximal parts of theaorta and the pulmonary artery. The conus retainsits myocardial investment, provides support for thedeveloping arterial valves, and ends up as part ofthe ventricular outflows (see Icardo et al., 1990, andIcardo and Manasek, 1991, for reviews). The mech-anisms involved in myocardial regression of thetruncus are not yet clear, but apoptosis (Hurle et al.,1977), dedifferentiation (Arguello et al., 1978), andtranslocation of the entire septation complex to-wards the ventricle (Thompson et al., 1983) havebeen suggested (see Icardo, 1990, for a review).Thus, we believe that the embryonic truncus of birdsand mammals is homologous to the fish bulbus ar-teriosus. This segment is myocardial in nature ini-tially, loses its myocardial investment later, andfinally adopts an arterial or arterial-like structure.In addition, the initial segment of the arterialtrunks in humans is invested by the serous pericar-dium and, therefore, contained within the pericar-dial cavity. On the other hand, the embryonic conusof birds and mammals appears to be homologous tothe fish conus arteriosus. This segment maintainsits myocardial nature, supports the outflow valves,and constitutes the ventricular outflow. Whether theembryonic conus remains as a distinct segment in

the adult heart or blends with the ventricular mass,and whether the entire OFT is septated or not, are,most probably, minor questions. It seems reasonablethat, once a particular structure has appeared inevolution, and demonstrates a real survival or ad-aptative advantage, it should be maintainedthrough successive evolutionary steps. Thus, itobeys the principle of universality (see Opitz andClark, 2000). It is also reasonable that specific mod-ifications are necessary to adapt that particularstructure to meet changing physiological require-ments. For instance, the relation between OFT sep-tation and the beginning of air breathing is obvious.The OFT of the vertebrate heart appears to consti-tute a morphogenetic unit which becomes phyloge-netically adapted to other parts of the heart and tothe rest of the body in order to accomplish specificfunctions. This fits well with recent findings indicat-ing that the myocardium of the conotruncus arisesfrom a secondary heart field cranial to the initialarea of fusion of the heart primordia (Waldo et al.,2001).

LITERATURE CITED

Anthony J, Millot J, Robineau D. 1965. Le coeur et l’aorte ven-trale de Latimeria chalumnae (Poisson, Coelacanthidae). CRAcad Sci 261:223–226.

Arguello C, De la Cruz MV, Sanchez C. 1978. Ultrastructural andexperimental evidence of myocardial cell dedifferentiation intoconnective tissue cells in embryonic chick heart. J Mol CellCardiol 10:307–315.

Bergwerff M, DeRuiter MC, Poelmann RE, Gittenberger-DeGroot AC. 1996. Onset of elastogenesis and downregulation ofsmooth muscle actin as distinguishing phenomena in arterydifferentation in the chick embryo. Anat Embryol 194:545–557.

Bergwerff M, Verberne ME, DeRuiter MC, Poelmann RE,Gittenberger-De Groot AC. 1998. Neural crest cell contributionto the developing circulatory system. Implications for vascularmorphology? Circ Res 82:221–231.

Bertin L. 1958. Appareil circulatoire. In: Grasse PP, editor. Traitede Zoologie, vol. XIII, fasc 2. Paris: Mason. p 1399–1458.

Blanco C, Lopez D, De Andres AV, Schib JL, Gallego A, DuranAC, Sans-Coma V. 2001. Cartilage in the bulbus arteriosus ofteleostean fishes. Neth J Zool 51:361–370.

Boas JEV. 1880a. Uber Hertz und Arterienbogen bei Ceratodusund Protopterus. Morphol Jahrb 6:321–354.

Boas JEV. 1880b. Uber den Conus arteriosus bei Butirinus undbei anderen Knochenfischen. Morphol Jahrb 6:527–534.

Boas JEV. 1901. Lehrbuch der Zoologie. Jena: Gustav Fischer.Bockman DE, Redmond ME, Waldo K, Davis H, Kirby ML. 1987.

Effect of neural crest ablation on development of the heart andarch arteries in the chick. Am J Anat 180:332–341.

Bridge TW. 1904. Fishes (exclusive of the systematic account ofteleostei). In: Harmer SF, Shipley AE, editors. The Cambridgenatural history, vol. 7. London: Macmillan. p 141–537.

Bushnell PG, Jones DR, Farrell AP. 1992. The arterial system. In:Hoar WS, Randall DJ, Farrell AP, editors. Fish physiology, vol.12. The cardiovascular system. Part A. San Diego: AcademicPress. p 89–120.

Carroll RL. 1988. Vertebrate paleontology. New York: WH Free-man.

Colbert EH. 1955. Evolution of vertebrates. New York: JohnWiley & Sons.

Daniel JF. 1934. The circulation of blood in ammocoetes. UnivCalif Berkeley Publ Zool 39:311–339.


De la Cruz MV, Moreno-Rodrıguez R, Angelini P. 1999. Phylog-eny of coronary arteries. In: Angelini P, editor. Coronary arteryanomalies. Philadelphia: Lippincott Williams & Wilkins. p 1–9.

Domezain A, Soriguer MC, Domezain J, Hernando JA. 2003.Acipenser naccarii Bonaparte 1836. Historia de un des-conocimiento. IX Congreso Nac Acuicultura. Cadiz, Espana.

Farrell AP, Jones DR. 1992. The heart. In: Hoar WS, Randall DJ,Farrell AP, editors. Fish physiology, vol. 12. The cardiovascularsystem. Part A. San Diego: Academic Press. p 1–87.

Fontaine M. 1958. Classe des Cyclostomes. Formes actuelles.Super-ordres des Petromyzonoidea et des Myxinoidea. In:Grasse PP, editor. Traite de Zoologie, vol. XIII, fasc 1. Paris:Mason. p 13–172.

Franco D, Gallego A, Habets PEMH, Sans-Coma V, MoormanAFM. 2002. Species-specific differences of myosin content in thedeveloping cardiac chambers of fish, birds, and mammals. AnatRec 268:27–37.

Gallego A, Franco A, Habets PEHM, Munoz-Chapuli R, GauvryL, Lamers WH, Moorman AFM. 1998. A differential myosincontent is established in the segmented heart. A comparativestudy in lower and higher vertebrates. Proc Working GroupDevelopmental Anat Pathol, ESC, Malaga, Spain. Abstr 10.

Garrido-Ramos MA, Soriguer MC, De la Herran R, Jamilena M,Ruiz Rejon C, Domezain A, Hernando JA, Ruiz Rejon M. 1997.Morphometric and genetic analysis as proof of the existence oftwo sturgeon species in the Guadalquivir river. Mar Biol 129:33–39.

Gegenbauer C. 1866. Zur vergleichenden Anatomie des Herzens.I. Uber den Bulbus arteriosus der Fische. Jena Z Naturwiss2:365–383.

Gegenbauer C. 1891. Uber den Conus arteriosus der Fische. Mor-phol Jahrb 17:596–610.

Gegenbauer C. 1901. Vergleichende Anatomie der Wirbelthieremit Berucksichtigung der Wirbellosen, vol. 2. Leipzig: WilhelmEngelman.

Goodrich ES. 1930. Studies on the structure and development ofvertebrates. London: Macmillan.

Grodzinski Z. 1938. Das Blutgefa�system der Fische. In: BronnHG, editor. Klassen und Ordnungen des Tierreichs, vol. 6.Leipzig: C.F Winter. p 1–77.

Hildebrand M. 1982. Analysis of vertebrate structure, 2nd ed.New York: John Wiley & Sons.

Hiruma T, Hirakow R. 1992. Histogenesis of tunica media of thechick aorta. Acta Anat Nippon 67:749–761.

Hu N, Sedmera D, Yost HJ, Clark EB. 2000. Structure andfunction of the developing zebrafish heart. Anat Rec 260:148–157.

Hu N, Yost HJ, Clark EB. 2001. Cardiac morphology and bloodpressure in the adult zebrafish. Anat Rec 264:1–12.

Hurle JM, Lafarga M, Ojeda JL. 1977. Cytological and cytochem-ical studies of the necrotic area of the bulbus of the chickembryo heart: phagocytosis by developing myocardial cells. JEmbryol Exp Morphol 41:161–173.

Icardo JM. 1985. Distribution of fibronectin during the morpho-genesis of the truncus. Anat Embryol 171:193–200.

Icardo JM. 1990. Development of the outflow tract. A study inhearts with situs solitus and situs inversus. Ann NY Acad Sci588:26–40.

Icardo JM, Manasek FJ. 1991. Cardiogenesis: developmentalmechanisms and embryology. In: Fozzard HA, Haber E, Jen-nings RB, Katz AM, Morgan HE, editors. The heart and cardio-vascular system. Scientific foundations, 2nd ed. Vol. 2. NewYork; Raven Press. p 1563–1586.

Icardo JM, Fernandez-Teran MA, Ojeda JL. 1990. Late heartembryology. The making of an organ. In: Meisami E, TimirasPM, editors. Handbook of human growth and developmentalbiology, vol. III, Part B. Boca Raton, FL: CRC Press. p 25–49.

Icardo JM, Colvee E, Cerra MC, Tota B. 1999a. Bulbus arteriosusof Atlantic teleosts. I. The white-blooded Chionodraco hamatus.Anat Rec 254:396–407.

Icardo JM, Colvee E, Cerra MC, Tota B. 1999b. Bulbus arteriosusof Atlantic teleosts. II. The red-blooded Trematomus bernacchii.Anat Rec 256:116–126.

Icardo JM, Colvee E, Cerra MC, Tota B. 2000a. Light and electronmicroscopy of the bulbus arteriosus of the European eel (An-guilla anguilla). Cells Tissues Organs 157:184–198.

Icardo JM, Colvee E, Cerra MC, Tota B. 2000b. Light and electronmicroscopy of the bulbus arteriosus. J Fish Biol 57(Suppl A):121–135.

Icardo JM, Colvee E, Cerra MC, Tota B. 2002a. Structure of theconus arteriosus of the sturgeon (Acipenser naccarii) heart. I.The conus valves and the subendocardium. Anat Rec 267:17–27.

Icardo JM, Colvee E, Cerra MC, Tota B. 2002b. The structure ofthe conus arteriosus of the sturgeon (Acipenser naccarii) heart.II. The myocardium, the subepicardium and the conus-aortatransition. Anat Rec 268:388–398.

Jiang X, Rotwitch DH, Soriano P, McMahon AP, Sucov HM. 2000.Fate of the mammalian neural crest. Development 127:1607–1616.

Johansen K. 1962. Cardiac output and pulsatile aortic flow in theteleost Gadus morhua. Comp Biochem Physiol 7:169–174.

Johansen K, Burggren W. 1980. Cardiovascular function in thelower vertebrates. In: Bourne GH, editor. Hearts and heart-likeorgans, vol. 1. New York: Academic Press. p 61–117.

Jones DR, Brill RW, Bushnell PG. 1993. Ventricular and arterialdynamics of anesthetized and swimming tuna. J Exp Biol 182:97–112.

Kardong KV. 2002. Vertebrates. Comparative anatomy, function,evolution. New York: McGraw-Hill.

Kent GC. 1978. Comparative anatomy of vertebrates, 4th ed. St.Louis: CV Mosby.

Kramer TC. 1942. The partition of the truncus and conus and theformation of the membranous portion of the interventricularseptum in the human heart. Am J Anat 71:343–370.

Kisch B. 1930. Die Zentralwuste im Conus arteriosus derSelachier. Anat Anz 70:462–470.

Krause R. 1923. Mikroskopische Anatomie der Wirbeltiere. IV.Teleostier, Plagiostomen, Zyklostomen und Leptokardier. Ber-lin: Walter de Gruyter.

Lankester ER. 1879. On the hearts of Ceratodus, Protopterus andChimaera. Trans Zool Soc Lond 10:493–506.

Lawson R. 1979. The comparative anatomy of the circulatorysystem. In: Wake MH, editor. Hyman’s comparative vertebrateanatomy, 3rd ed. Chicago: University of Chicago Press. p 448–554.

Li YX, Zdanowicz M, Young L, Kumiski D, Leatherbury L, KirbyM. 2003. Cardiac neural crest in zebrafish embryos contributesto myocardial cell lineage and early heart function. Dev Dyn226:540–550.

Licht JH, Harris WS. 1973. The structure, composition and elas-tic properties of the teleost bulbus arteriosus in the carp, Cyp-rinus carpio. Comp Biochem Physiol 46:699–708.

Manner J. 2000. Cardiac looping in the chick embryo: a morpho-logical review with special reference to terminological and bio-mechanical aspects of the looping process. Anat Rec 259:248–262.

Millot J, Anthony J, Robineau D. 1978. Anatomie de Latimeriachalumnae. Tome 3. Paris: Centre National de la Recherche.

Nadal J. 2001. Vertebrados. Origen, organizacion, diversidad ybiologıa. Barcelona: Ediciones de la Universitat de Barcelona yEdiciones Omega.

Nishibatake M, Kirby ML, Van Mierop L. 1987. Pathogenesis ofpersistent truncus arteriosus and dextroposed aorta in thechick embryo after neural crest ablation. Circulation 75:255–264.

Opitz JM, Clark EB. 2000. Heart development: an introduction.Am J Med Genet 97:238–247.

Parker TJ, Haswell WA. 1962. A textbook of Zoology, 7th ed.Revised by Marshall AJ. London: Macmillan.

Parsons CW. 1930. The conus arteriosus of fishes. Q J Microsc Sci73:145–176.

Pexieder T. 1995. Conotruncus and its septation at the advent ofthe molecular biology era. In: Clark EB, Markwald RR, TakaoA, editors. Developmental mechanisms of heart disease. NewYork: Futura. p 227–247.


Priede IG. 1976. Functional morphology of the bulbus arteriosusof rainbow trout (Salmo gairdneri Richardson). J Fish Biol9:209–216.

Randall DJ, Davie PS. 1980. The hearts of Urochordates andCephalochordates. In: Bourne GH, editor. Hearts and heart-likeorgans, vol. 1. New York: Academic Press. p 41–59.

Robertson J. 1914. The development of the heart and vascularsystem of Lepidosiren paradoxa. Q J Microsc Sci 59:53–132.

Robles F, De la Herran R, Garrido-Ramos MA, Ruiz-Rejon C,Martınez-Wespın E, Lorente JA, Ruiz-Rejon M. 2003. Identifi-cacion de los esturiones del Guadalquivir utilizando cincomarcadores moleculares y tecnicas forenses. IX Congreso NacAcuicultura. Cadiz, Espana.

Sadaghiani B, Vielkind JR. 1990. Neural crest development inXiphophorus fishes: scanning electron and light microscopicstudies. Development 110:197–209.

Sans-Coma V, Gallego A, Munoz-Chapuli R, De Andres AV, Du-ran AC, Fernandez B. 1995. Anatomy and histology of thecardiac conal valves of the adult Dogfish (Scyliorhinus can-icula). Anat Rec 241:496–504.

Santer RM. 1985. Morphology and innervation of the fish heart.Adv Anat Embryol Cell Biol 89:1–102.

Satchell GH. 1991. Physiology and form of fish circulation. Cam-bridge, UK: Cambridge University Press.

Sato M, Yost HJ. 2003. Cardiac neural crest contributes to car-diomyogenesis in zebrafish. Dev Biol 257:127–139.

Schib JL, Icardo JM, Duran AC, Guerrero A, Lopez D, Colvee A, DeAndres AV, Sans-Coma V. 2002. The conus arterious of the adultgilthead seabream (Sparus auratus). J Anat 201:395–404.

Schilling TF, Kimmel CB. 1994. Segment and cell type lineagerestrictions during pharyngeal arch development in the ze-brafish embryo. Development 120:483–489.

Senior HD. 1907a. The conus arteriosus in Tarpon atlanticus(Cuvier and Valenciennes). Biol Bull 12:146–151.

Senior HD. 1907b. Teleosts with a conus having more than onerow of valves. Anat Rec 4:83–84.

Senior HD. 1907c. Note on the conus of Megalops cyprinoides(Broussonet). Biol Bull 12:378–379

Senior HD. 1909. The development of the heart in shad (Alosasapidissima Wilson). Am J Anat 9:211–262.

Shaner RF. 1962. Anomalies of the heart bulbus. J Pediatr 61:233–241.

Smith WC. 1918. On the process of disappearance of the conusarteriosus in Teleosts. Anat Rec 15:65–71.

Stannius H. 1846. Bemerkungen uber das Verhaltniss derGanoiden zu den Clupeiden, insbesondere zu Butirinus. Ros-tock: Stiller.

Stohr P. 1876. Ueber den Klappenapparat im Conus arteriosusder Selachier und Ganoiden. Morph Jb 2:197–228.

Thompson RP, Wong Y-MH, Fitzharris TP. 1983. A computergraphic study of cardiac truncal septation. Anat Rec 206:207–214.

Van Mierop LHS, Alley RD, Kausel HW, Stranahan A. 1963.Pathogenesis of transposition complexes. I. Embryology of theventricles and great arteries. Am J Cardiol 12:216–225.

Von Skramlick E. 1935. Uber den Kreislauf bei den Fischen.Ergbn Biol 11:1–130.

Waldo K, Miyagawa-Tomita S, Kumiski D, Kirby M. 1998. Car-diac neural crest cells provide insight into septation of thecardiac outflow tract: aortic sac to ventricular septal closure.Dev Biol 196:129–144.

Waldo K, Lo CW, Kirby M. 1999. Connexin 43 expression reflectsneural crest patterns during cardiovascular development. DevBiol 208:307–323.

Waldo KL, Kumiski DH, Wallis KT, Stadt HA, Hutson MR, PlattDH, Kirby ML. 2001. Conotruncal myocardium arises from asecondary heart field. Development 128:3179–3188.

Watanabe M, Choudhry A, Berlan M, Singal A, Siwik E, Mohr S,Fisher SA. 1998. Developmental remodeling and shortening ofthe cardiac outflow tract involves myocyte programmed celldeath. Development 125:3809–3820.

Weichert CK, Presch W. 1975. Elements of Chordate anatomy.New York: McGraw-Hill.

Yablonka-Reuveni Z, Schwartz SM, Christ B. 1995. Developmentof chicken aortic smooth muscle: expression of cytoskeletal andbasement membrane proteins defines two distinct cell pheno-types emerging from a common lineage. Cell Mol Biol Res41:241–249.

Yablonka-Reuveni Z, Christ B, Benson JM. 1998. Transitions incell organization and in expression of contractile and extracel-lular matrix proteins during development of chicken aorticsmooth muscle: evidence for a complex spatial and temporaldifferentation program. Anat Embryol 197:421–437.

Yamauchi A. 1980. Fine structure of the fish heart. In: BourneGH, editor. Hearts and heart-like organs, vol. 1. New York:Academic Press. p 119–148.

Young JZ. 1983. The life of vertebrates. Oxford: Clarendon Press.Zummo G, Farina F. 1989. Ultrastructure of the conus arteriosus

of Scylirohinus stellaris. J Exp Zool (Suppl) 2:158–164.


Documents

Differentiation of the Cardiac Outﬂow Tract Components in ... · Differentiation of the Cardiac Outﬂow Tract Components in Alevins of the Sturgeon Acipenser naccarii (Osteichthyes,