16642016

Formation of Cartilage in the Heart of the Spanish

Terrapin,

Mauremys leprosa

(Reptilia, Chelonia)

David Lo´pez,

Ana C. Dura´n,

A. Victoria de Andre´s,

Alejandro Guerrero,

Manuel Blasco,

and

Valentı´n Sans-Coma

_*

1_{Department of Animal Biology, Faculty of Science, University of Ma´laga, 29071 Ma´laga, Spain}

2_{Department of Morphological Sciences and Animal Biology, Faculty of Science, University of Extremadura,} 06071 Badajoz, Spain

ABSTRACT Cartilaginous deposits are regularly present in the heart of several reptilian, avian, and mammalian species. The formation of these extraskeletal cartilages has been studied in birds and mammals, but not in reptiles. The aim here was to elucidate this question in the Spanish ter-rapin. Hearts from 23 embryos belonging to Yntema (1968) developmental stages 17 to 26 and eight terrapins age 3 months to 10 years were examined using histological, histo-chemical, and immunohistochemical techniques. In the heart of the Spanish terrapin (Mauremys leprosa), chondro-genesis can start during embryonic life. Cartilaginous tissue develops from a mesenchymal cellular condensation that extends along the aorticopulmonary septum and the incipi-ent pars fibrosa of the vincipi-entricular horizontal septum. This cellular condensation, which is smooth muscle␣-actin (SM␣ -actin)-negative and type II collagen-negative during stages 17 to 22, acts as a prechondrogenic condensation. In stage 23, production of type II collagen begins in the central core of the condensation and gradually spreads toward its periph-ery. The type II collagen-positive (chondrogenic) cellular con-densation remains devoid of perichondrium prior to birth. Thereafter, it converts into hyaline cartilage that extends along the proximal part of the aorticopulmonary septum and the pars fibrosa of the horizontal septum. Our findings are consistent with the assumption that, as in birds and mam-mals, the precursors of the cardiac chondrocytes in cheloni-ans are neural crest-derived cells of nonmuscular nature. In addition, they point to the possibility that cells from the neural crest populate the embryonic pars fibrosa of the hor-izontal septum, thereby contributing to its alignment with the aorticopulmonary septum. In the present species, a sec-ond cartilaginous deposit of a hyaline nature extends along the sinus wall of the right semilunar valve of the right aorta, penetrating the fibrous cushion that constitutes the proxi-mal support of the corresponding valve leaflet. This cartilage develops after birth, between the third and eighteenth month of life; its morphogenetic origin is unclear. The carti-laginous foci occurring in hearts of Spanish terrapin appear to act as pivots resisting mechanical tensions generated dur-ing the cardiac cycle. In the specimens examined there was no sign of replacement of the cardiac cartilages by bone tissue. J. Morphol. 258:97–105, 2003. © 2003 Wiley-Liss, Inc.

KEY WORDS: cartilage; heart, embryo; reptilia; chelonia

Cartilaginous deposits are regularly present in

the cardiac fibrous skeleton of several reptilian

spe-cies (see Matumoto, 1938; Kashyap, 1950; White,

1956,1959; Webb, 1979; Young, 1994, for original

descriptions and extensive reviews of the literature).

This early work is concerned with the histological

features, shape, size, location, and function of the

cartilages occurring in adult animals. The hyaline

cartilaginous deposits vary in shape and size. In

turtles, rhynchocephalians, lizards, and snakes the

deposits are usually located in portions of the

car-diac outflow tract, such as the aorticopulmonary and

interaortic septa. In crocodiles the cartilage appears

in the interventricular septum and in the semilunar

and atrioventricular valves.

The morphogenesis of cardiac cartilage in reptiles

is unknown. Up to now, this process has only been

studied in birds (Stiefel, 1926; Matumoto, 1938;

Tsusaki et al., 1956; Sumida et al., 1989; Lo´pez et

al., 2000) and mammals (Lo´pez et al., 2001). The

results reported suggest that in both groups the

precursors of the cardiac chondrocytes are cells

orig-inating from the neural crest. In birds, the

develop-ment of cardiac cartilage takes place during

embry-onic life and is initiated with the formation of

mesenchymal cell condensations, whereas in the

mammalian heart chondrogenesis starts after birth

without formation of conspicuous prechondrogenic

condensations.

The present work was designed to illustrate the

formation of cartilaginous tissue in the heart of the

Spanish terrapin,

Mauremys leprosa

, a living

repre-sentative of the anapsid reptiles. The ultimate goals

of our study were 1) to gain new insight into the

ontogeny of the cardiac cartilages in amniotes from

Contract grant sponsor: D.G.E.S. (Ministerio de Ciencia y Tecnolo-gı´a) to AG; Contract grant number: PB98-1418-C02-01. Alejandro Guerrero is the recipient of fellowship FP99-25680733 (Ministerio de Ciencia y Tecnologı´a, Spain) to (GS).

*Correspondence to: Valentı´n Sans-Coma, Department of Animal Biology, Faculty of Science, University of Ma´laga, 29071 Ma´laga, Spain. E-mail: [email protected]

DOI: 10.1002/jmor.10134

a comparative viewpoint, and 2) to gather new data

on the possible morpho-functional significance of

such cartilages. The study was carried out in

embry-onic and adult hearts from Spanish terrapins, which

were examined using histological, histochemical,

and immunohistochemical techniques.

MATERIALS AND METHODS

Animals

Fertilized eggs were obtained from adult females of the Spanish terrapin,Mauremys leprosa(Schweigger, 1812), collected in the environs of Badajoz, Extremadura, Spain. The animals were kept in terraria, where food was given as required. Oviposition was induced by injecting oxytocin into females (1 IU/100 g of body weight). The eggs were kept in a rocking incubator at 28°C in a 90% humid environment. At these conditions, the average time of incubation is about 79 days. Twenty-three embryos were collected between 22 and 79 days of incubation. The formation of cartilag-inous tissue in embryonic hearts was correlated to the develop-mental stages established by Yntema (1968) in the common snap-ping turtle,Chelydra serpentina. It should be noted that these stages have previously been used successfully for the Spanish terrapin (Kalman et al., 1997). Table 1 shows the distribution of the embryos examined, according to the developmental stages.

In addition, the hearts from eight specimens, ages 3 months to 10 years, were studied: 3 months (n_⫽1), 18 months (n_⫽1), 4 years (n⫽2), 5 years (n⫽1), 6 years (n⫽1), and 10 years (n⫽ 2). These specimens had been collected in the environs of Badajoz and used in previous anatomical studies. Their respective ages were estimated according to the number of growth lines present in the horny scutes of the carapace (Castanet and Cheylan, 1979). All the animals were collected and sacrificed with permission (VS 99/198; AFG/afg) of the Regional Government of Extrema-dura, Spain, and were handled in accordance with the Spanish Regulations for the Protection of Experimental Animals (Real Decreto 223/1988).

The hearts of 22 embryos, two young animals (3 and 18 months), and three adults (4, 5, and 10 years old) were examined by histological, histochemical, and immunohistochemical tech-niques for light microscopy. The heart of the remaining embryo (developmental stage Y25) and the outflow tract of the three remaining adults were stained in toto with Alcian blue.

Histological and Histochemical Techniques

Whole embryos or isolated hearts were fixed by immersion in Bouin’s fluid (ratio of fixative to tissue volume⫽80:1) and em-bedded in Paraplast (Sigma Chemical Co., UK). Serial sections, cut transversely, longitudinally, or obliquely at 10 ␮m, were

stained with hematoxylin-eosin or Mallory’s trichrome stain for a general assessment of the histological components of the heart. Other procedures used were Weigert’s resorcin-fuchsin for stain-ing elastin and the von Kossa technique (Kiernan, 1990) for the specific detection of calcium deposits in cardiac cartilages of adult animals. Sections were examined with a light microscope and photographed with a photomicrographic attachment.

Immunohistochemical Techniques

Whole embryos or isolated hearts were fixed by immersion in Bouin’s fluid, embedded in Paraplast, as described above, and cut transversely or longitudinally at 10_␮m. Sections were stained with monoclonal antibody CIIC1 (Developmental Studies Hybrid-oma Bank, University of Iowa, Ames, Iowa) recognizing type II collagen, or with monoclonal anti-SM␣-actin (Sigma, clone 1A4). The use of the immunohistochemical technique for the detec-tion of type II collagen relied on the fact that synthesis of type II collagen is considered cartilage-characteristic (Miller and Matu-kas, 1969; Miller, 1976; Kosher, 1983; Hall and Miyake, 1992,1995), even though type II collagen is also produced by a limited number of nonchondrogenic cell types (see Kosher, 1983, and Swiderski et al., 1994, for extensive reviews of the literature). The anti-SM␣-actin was utilized bearing in mind that in the chick and quail the use of this antibody was conclusive in deciding the nonmuscular nature of the neural-crest derived cells which are believed to be the precursors of the cardiac chondrocytes (Lo´pez et al., 2000).

The sections were dewaxed in xylene, hydrated in an ethanol series, and washed in Tris-phosphate buffered saline (TPBS, pH 7.8). For the detection of type II collagen, the tissues were then digested for 15–30 min at 37°C with 0.5% papain in PB (pH 4.7). Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in TPBS for 30 min. After washing with TPBS, nonspecific binding sites were saturated for 30 min with 10% sheep serum and 1% bovine serum albumin in TPBS (SB) for staining with CIIC1, or with the same solution plus 0.5% Triton X-100 (SBT) for staining with anti-SM␣-actin. Sections were then incubated overnight at 4°C in the primary antibody diluted in SB when staining with CIIC1 or in SBT when staining with anti-SM_␣-actin. Control slides were incubated in SB or SBT only. After incubation, the sections were washed in TPBS (3⫻5 min), incubated for 1 h at room temperature in biotin conjugated anti-mouse IgG (Sigma) diluted 1:100 in TPBS, washed again, and incubated for 1 h in ExtrAvidin_威conjugate (Sigma) diluted 1:150 in TPBS. Peroxidase activity was developed with Sigma Fast威 3,3_⬘-diaminobenzidine tablets according to the indications of the supplier. In several cases the sections were counterstained with hematoxylin or hematoxylin-eosin.

Alcian Blue In Toto Staining Technique

The heart was removed, transferred to Ringer’s solution, and dissected to expose the cardiac outflow tract. The specimens were fixed by immersion in 5% trichloroacetic acid (ratio of fixative to tissue volume_⫽80:1) and stained with Alcian blue 8 GX (Gurr), according to the technique described by Ojeda et al. (1970), then rinsed with 70% aqueous ethanol.

Nomenclature

The terms proximal and distal are used to describe the location of the cardiac outflow tract components with regard to the ven-tricle, while anterior and posterior are employed to indicate the situation of the ventricular components with regard to the an-teroposterior axis of the heart.

RESULTS

The ventricle of the chelonian heart is divided into

a large cavum dorsale and a smaller cavum ventrale,

stages of Yntema (1968)

dps n di

17 1 22

18 1 25

19 2 29

20 2 32

21 2 34

22 2 37

23 3 38–47

24 4 48–56

25 5 58–70

26 1 70–80

Abbreviations: di, days of incubation; dps, developmental stages; n, number of specimens examined.

or cavum pulmonale, by an incomplete horizontal

septum, also called muscular ridge, Muskelleiste, or

interventricular septum (Greil, 1903; Goodrich,

1919,1930; Leene and Vorstmann, 1930; Kashyap,

1959; Guibe´, 1970; Webb et al., 1974; Holmes, 1975;

Webb, 1979; Van Mierop and Kutsche, 1981,1985).

In several species a variably developed vertical

sep-tum usually subdivides the cavum dorsale into the

cavum venosum and cavum arteriosum. In the

Spanish terrapin, the horizontal septum is the

ma-jor septal structure within the ventricle. The vertical

septum is so poorly developed as to be practically

indistinguishable from other muscular trabeculae.

As in other turtles, the horizontal septum consists of

two portions: a posterior (apical) complete portion

and an anterior incomplete portion. The posterior

portion is of a muscular nature and separates the

cavum dorsale from the cavum ventrale. The

ante-rior portion assumes a subhorizontal position; its

dorsolateral margin is free, allowing communication

between the dorsal and ventral chambers. The free

margin is covered by a thick layer of fibrous tissue,

which we call the pars fibrosa of the horizontal

sep-tum. This pars fibrosa connects with the

aorticopul-monary septum, a fact that needs to be emphasized,

since the largest cartilaginous focus observed in the

heart of the Spanish terrapin develops inside these

two anatomical elements of the cardiac outflow tract

(Fig. 1).

Embryological Findings

In the earliest embryo examined (stage Y17), the

aorticopulmonary and interaortic septa were fully

developed. In this specimen, as well as in the

em-bryos of developmental stages 18 to 22, a

conspicu-ous condensation of mesenchymal cells extended

along the central part of the aorticopulmonary

sep-tum, penetrating the incipient pars fibrosa of the

horizontal septum (Figs. 1, 2A,B).

The portion of the cellular condensation

corre-sponding to the aorticopulmonary septum was SM

␣

-actin-negative and type II collagen-negative. In the

Y22 embryos, the condensation clearly stood out

against the surrounding medial tissue of the great

arteries, which had acquired an organization of

SM

␣

-actin-positive lamellar cells and SM

␣

-actin-negative interlamellar cells (Fig. 3).

The portion of the cellular condensation located in

the anticipated pars fibrosa of the ventricular

hori-zontal septum also displayed a type II

collagen-negative extracellular matrix. Yet some scattered

SM

␣

-actin-positive cells were present in this part of

the condensation (Figs. 1, 4). The highest level of

SM

␣

-actin immunoreactivity was recorded in

em-bryos belonging to stage 22 at the caudal end of the

condensation, that is, at the boundary between the

anticipated pars fibrosa and the myocardial tissue of

the horizontal septum (Figs. 1, 5). The SM

␣

-actin

expression quickly disappeared from this site in

sub-sequent developmental stages (Y23–Y24).

Two of the four Y23 embryos showed a condition

similar to that of the preceding developmental

stages. In the other two, type II collagen was present

in the extracellular matrix of the central core of the

cellular condensation. This occurred along the

por-tion of the condensapor-tion located between the

proxi-mal part of the aorticopulmonary septum and the

anticipated pars fibrosa of the ventricular horizontal

septum (Figs. 1, 6). In the Y24 to Y26 embryos, the

production of type II collagen had increased toward

the periphery of the condensation (Fig. 7).

Cardiac Cartilage in Young and Adult

Animals

In the young terrapin, age 3 months, there was a

well-developed cartilaginous deposit, which

ex-tended along the proximal part of the

aorticopulmo-nary septum and pars fibrosa of the horizontal

sep-tum. The cartilage appeared as an elongated bar and

was hyaline. The chondrocytes were embedded in a

type II collagen-positive cellular matrix (Fig. 8). The

deposit was surrounded by a thin perichondrium

Fig. 1. Diagram of the chelonian heart in right anteroventral view to show the location of the cardiac cartilages. The antero-ventral half of the ventricle wall was removed to show the hori-zontal septum (HS), which divides the ventricular cavity into a cavum dorsale (CD) and a cavum ventrale (CV). The proximal portions of the ventral walls of both the left aorta (LAo) and pulmonary artery (PA) were removed to show a cartilaginous deposit (arrow) located in the proximal portion of the aorticopul-monary septum and the pars fibrosa (star) of the horizontal septum. The arrowhead points to a cartilaginous focus placed in the proximal wall of the right aorta. AoR, aortic root; AV, atrio-ventricular valve leaflet; LA, left atrium; LC, left common carotid artery; LS, left subclavian artery; RA, right atrium; RAo, right aorta; RC, right common carotid artery; RS, right subclavian artery.

(Fig. 9), composed of collagen fibers that ran in a

circumferential direction and contained a single

layer of flattened cells.

In the other young animal, age 18 months, and in

all six adult terrapins, 4 –10 years old, there were

two cartilaginous deposits in the heart. One of them

was located in the pars fibrosa of the horizontal

septum and proximal portion of the

aorticopulmo-nary septum, reaching the proximal level of the

in-teraortic septum (Figs. 1, 10A). Histological sections

revealed that the cartilage was hyaline, with small,

spheroidal chondrocytes (Fig. 10B). The

perichon-drium was thin, sometimes even incomplete. At the

sites that were devoid of perichondrium the

cartilag-inous tissue merged gradually with the surrounding

connective tissue.

The other cardiac deposit extended along the

si-nus wall of the right semilunar valve of the right

aorta (Figs. 1, 10A), penetrating the fibrous cushion

that constitutes the proximal support of the

corre-sponding valve leaflet (cusp). In the 18-month

ter-rapin, this second focus consisted of a small group of

chondrocytes, which were embedded in a type II

collagen-positive cellular matrix that stained

mark-edly with hematoxylin and was devoid of

perichon-drium. In the adult animals, the focus was of hyaline

cartilaginous tissue and was surrounded by a thin

perichondrium.

Finally, it should be noted that the cartilaginous

deposits of the adult animals displayed no

hypertro-phied chondrocytes, nor mineralization of the

ma-trix.

DISCUSSION

In 1938, Matumoto stated that in reptiles the

for-mation of cardiac cartilage is initiated after birth.

He based his conclusion on the fact that he detected

no cartilaginous focus in embryonic hearts of the

gekkonid

Hemidactylus bowringii

and the viperid

Gloydius blomhofii

, referred to as

Ancistrodon

blom-hofii

. Subsequently, Van Mierop and Kutsche (1985)

mentioned the occurrence of cartilage in the base of

the heart and near the valves in an alligator embryo

about half-way through incubation (see the

discus-sion following the oral presentation of the

contribu-tion by the authors cited). To our knowledge, no

other data concerning the ontogeny of cardiac

carti-lage in reptiles have been reported.

Our observations demonstrate that in the

chelo-nian heart chondrogenesis can start during

embry-onic life. In the Spanish terrapin, synthesis of type II

collagen, which can be considered the first

unequiv-ocal sign of cartilage formation, begins as early as

stage Y23 (38 – 47 days of incubation). This occurs in

the core of the mesenchymal cellular condensation

that extends along the aorticopulmonary septum

and incipient pars fibrosa of the ventricular

horizon-tal septum. Therefore, this mesenchymal

condensa-tion acts as a prechondrogenic condensacondensa-tion. The

Fig. 2. Transverse sections of the cardiac outflow tract of a Y19 embryo at the level of the proximal portion of the aorticop-ulmonary septum (A), and at the level of the anterior portion of the ventricular horizontal septum (B). Resorcin-fuchsin. The ar-rows point to a condensation of mesenchymal cells, which extends along the central part of the aorticopulmonary septum (A) and which penetrates the incipient pars fibrosa of the horizontal sep-tum (B). AoR, aortic root; PA, pulmonary artery; V, ventricle. Scale bars_⫽100_␮m.

differentiation of the mesenchymal cells into

chon-drocytes seems to proceed from the center of the

condensation to its periphery. The type II

collagen-positive cellular condensation remains devoid of

perichondrium prior to birth. Thereafter, it becomes

gradually converted into hyaline cartilage.

The chondrogenetic process occurring in the heart

of the Spanish terrapin is very similar to that which

takes place in the aortic and pulmonary valves of

birds. In these valves, formation of cartilaginous

tissue also begins during embryonic life (Stiefel,

1926; Matumoto, 1938; Tsusaki et al., 1956; Sumida

et al., 1989; Lo´pez et al., 2000). Chondrogenesis

starts with the formation of conspicuous

prechon-drogenic condensations, which consist of loosely

packed mesenchymal cells embedded in a type II

collagen-negative extracellular matrix (Lo´pez et al.,

2000). Then the prechondrogenic condensations

dif-ferentiate into chondrogenic (type II

collagen-positive) condensations, which become transformed

into hyaline cartilage after hatching. In mammals,

the formation of cartilage in the cardiac semilunar

valves is somewhat different. It starts within the

first month of life, and does not involve formation of

prechondrogenic condensations (Lo´pez et al., 2001).

The cartilaginous foci occurring in the aortic and

pulmonary valves of birds are believed to originate

from neural crest-derived cells (Sumida et al., 1989;

Bachnou et al., 1996) of a nonmuscular nature

(Lo´pez et al., 2000), which invade the cardiac

out-flow tract during embryonic life (Takamura, 1990;

Yablonka-Reuveni et al., 1995,1998; Bergwerff et

al., 1996,1998; Poelmann et al., 1998; Waldo et al.,

1998,1999). The cardiac chondrocytes in mammals

are also presumed to derive from neural crest cells

(Lo´pez et al., 2001) occurring in the fibrous skeleton

of the heart during both embryonic (Waldo et al.,

1999; Jiang et al., 2000) and postnatal life (Jiang et

al., 2000). The contribution of the neural crest to the

configuration of the cardiac outflow tract in reptiles

is still uncertain. Nonetheless, it seems plausible to

assume that, as in birds and mammals (Poelmann et

al., 1998; Waldo et al., 1998; Jiang et al., 2000), cells

from the cardiac neural crest may play a decisive

role in the septation of the embryonic conotruncus in

reptiles, contributing to the formation of the

aorti-copulmonary septum. Indirect evidence supporting

this suggestion is provided by the fact that the

mes-enchymal cellular condensation that extends along

the developing aorticopulmonary septum of the

Spanish terrapin is anatomically similar to that of

neural crest-derived cells occurring in birds

(Taka-mura, 1990; Yablonka-Reuveni et al., 1995, 1998;

Bergwerff et al., 1996,1998; Poelmann et al., 1998;

Waldo et al., 1998,1999) and mammals (Waldo et al.,

1999; Jiang et al., 2000). Therefore, we presume

that, as in these two groups, the precursors of the

cardiac chondroblasts in the Spanish terrapin are

neural crest-derived elements. Moreover, the SM

␣

-actin-negative condition of these neural crest

ele-ments in the embryos examined is consistent with

the assumption that they are of a nonmuscular

na-ture.

An interesting point is that, prior to the synthesis

of type II collagen in the portion of the cellular

condensation located in the pars fibrosa of the

ven-tricular horizontal septum, several cells of the

con-densation are transiently SM

␣

-actin-positive. SM

␣

-actin expression is usually associated with the

differentiation of mesenchymal cells into smooth

muscle cells. However, it is known that transient

expression of SM

␣

-actin may indicate cell migration

(Waller et al., 2000). Bearing in mind that in the

adult Spanish terrapin the pars fibrosa of the

ven-tricular horizontal septum is devoid of smooth

mus-cle cells, cell migration seems to be the only

expla-nation for the SM

␣

-actin immunoreactivity detected

at this site. This assumption, together with the

pre-sumed neural crest origin of the condensed cells in

the pars fibrosa, leads to the following suggestion: In

the Spanish terrapin, cells from the neural crest

may populate the embryonic pars fibrosa of the

hor-izontal septum, thereby contributing to its

align-ment with the aorticopulmonary septum. Further

studies using appropriate techniques to detect

neu-ral crest cells are needed to verify this hypothesis.

In the heart of the Spanish terrapin, formation of

cartilage can also be initiated after birth. This is the

case for the cartilaginous focus that forms in the

wall of the right semilunar valve of the right aorta,

entering the fibrous cushion that supports the

prox-imal attachment of the valve leaflet. We were unable

to detect any sign of chondrogenesis at these sites in

the embryos, nor in the 3-month animal. In the

6-month specimen, however, the focus placed in the

aortic sinus wall displayed a relatively advanced

stage of development. These data indicate that the

formation of the focus had begun between the 3rd

and 18th month of life.

The morphogenetic origin of this cartilaginous

focus cannot be decided from the present data.

Neural crest cells have been found to occur in all

aortic and pulmonary valvular cushions of

quail-chick chimera embryos (Takamura et al., 1990).

Whether such cells also invade the cushions of the

cardiac semilunar valves in reptiles remains an

open question. The only conclusion derived from

our observations at this point in time is that the

differentiation into chondrocytes begins much

later in the semilunar valve than in the

aortico-pulmonary septum and pars fibrosa of the

ventric-ular horizontal septum. However, the reason for

this difference is unknown.

The functional significance of the cardiac

carti-lages remains a problem. It is generally accepted

that, when present, they act as pivots resisting

mechanical tensions. The deposits form in cardiac

portions that are exposed to large stresses during

the cardiac cycle, i.e., the cardiac septa and the

cardiac

valves

in

reptiles

(Matumoto,

1938;

Kashyap, 1950; White, 1956,1959; Webb, 1979;

Young, 1994; present data) and the attachments of

the cardiac semilunar valves to their respective

sinuses and the fibrous trigones in both birds

(Stiefel, 1926; Matumoto, 1938; Tsusaki et al.,

1956; Lo´pez et al., 2000) and mammals

(Matu-moto, 1938; Hueper, 1939; Hollander, 1968;

Wex-ler, 1964; Kelsall and Visci, 1970; Sans-Coma et

al., 1994; Lo´pez et al., 2001). In the Syrian

ham-ster the location of the cardiac cartilaginous foci is

significantly associated with the morphology of

the aortic valves; the foci appear where

mechani-cal stress is particularly intense (Sans-Coma et

al., 1994). Therefore, it has been suggested that

locally strong mechanical stimulation might be a

key factor in the formation of cartilage in the

vertebrate heart (Matumoto, 1938; Hueper, 1939;

Hollander, 1968; Sans-Coma et al., 1994).

The turtle heart begins to beat between the 6th

and 7th day of incubation (see Ewert, 1985, for a

review of the literature), and the embryonic heart

rate varies according to the incubation temperature.

In

Chelydra serpentina

, the estimated mean heart

rate amounts to 51.8 beats per minute at 24°C and

72.3 beats per minute at 29°C (Birchard and Reiber,

1996). In the heart of the Spanish terrapin,

chondro-genesis can already start between embryonic days

38 and 47, that is, at about 32– 41 days after the

commencement of the first pulsations. Whether or

not the mechanical stimulation generated during

this embryonic period on the cardiac structures

con-taining the foci are strong enough to play a primary

role in the induction of the chondrogenetic process is

an open question.

Another fact that hinders an accurate

assess-ment of the significance of the cardiac cartilage in

vertebrates is that its presence is not a

prerequi-site for normal heart performance. This conclusion

relies on the fact that both the incidence and

lo-cation of the cartilaginous foci vary widely

be-tween species and bebe-tween individuals of the same

species (Stiefel, 1926; Matumoto, 1938; Hueper,

1939; Tsusaki et al., 1956; Kashyap, 1959; Wexler,

1964; Hollander, 1968; Kelsall and Visci, 1970;

Sans-Coma et al., 1994; Young, 1994). Several

species are even devoid of cardiac cartilages

(Ma-tumoto, 1938). In this regard, it should be

empha-sized that the presumed precursors of

chondro-cytes, i.e., nonmuscular neural crest-derived cells,

are present in the cardiac outflow tract of all

am-Fig. 9. Oblique sagittal section of the cartilage located in the aorticopulmonary septum of the Spanish terrapin age 3 months. Hematoxylin-eosin. A thin perichondrium (arrows) surrounds partially the cartilage. Scale bar⫽50␮m.

Fig. 3. Transverse section of the cardiac outflow tract of a Y22 embryo at the level of the proximal portion of the aorticopulmonary septum. Smooth muscle␣-actin (SM␣-actin) immunostaining. The medial tissue of both the aortic root (AoR) and pulmonary artery (PA) displays an organization of SM_␣-actin-positive lamellar cells and SM_␣-actin-negative interlamellar cells. An SM_␣-actin-negative cellular condensation (arrow) can be recognized in the central part of the aorticopulmonary septum. Scale bar⫽200␮m.

Fig. 4. Transverse section of the cardiac outflow tract of a Y20 embryo at the level of the anterior portion of the ventricular horizontal septum. SM␣-actin immunostaining. The arrowheads point to a small spot of immunoreactivity located in the central core of the cellular condensation situated in the anticipated pars fibrosa of the septum. Scale bar_⫽25_␮m.

Fig. 5. Transverse section of the cardiac outflow tract of a Y22 embryo at the level of the anterior portion of the ventricular horizontal septum. SM␣-actin immunostaining. A considerable amount of SM␣-actin-positive cells (arrow) are present at the caudal end of the cellular condensation occurring in the anticipated pars fibrosa of the ventricular horizontal septum. AoR, aortic root; PA, pulmonary artery. Scale bar⫽200␮m.

Fig. 6. Longitudinal section of the cardiac outflow tract of a Y23 embryo at the level of the anterior portion of the ventricular horizontal septum. Type II collagen immunostaining counterstained with hematoxylin. The cellular condensation located in the anticipated pars fibrosa of the horizontal septum is type II collagen-positive. Scale bar_⫽25_␮m.

Fig. 7. Longitudinal section of the cardiac outflow tract of a Y25 embryo. Type II collagen immunostaining counterstained with hematoxylin. The arrow points to cartilaginous tissue located in the aorticopulmonary septum. AoR, aortic root; LAo, left aorta; PA, pulmonary artery. Scale bar_⫽200_␮m.

Fig. 8. Oblique sagittal section of the heart from a Spanish terrapin age 3 months. Type II collagen immunostaining counterstained with hematoxylin. A cartilaginous deposit (arrow) is located in the aorticopulmonary septum. AoR, aortic root; LAo, left aorta; PA, pulmonary artery; RAo, right aorta; V, ventricle. Scale bar⫽500␮m.

niotes. However, the reason why some of these

cells differentiate into chondrocytes, whereas

oth-ers do not, remains an open question. It is well

known that the morphogenesis of skeletal

carti-lage is a complex process that involves multiple

factors such as the expression of specific genes,

synthesis of gene products, and cell interactions

(Hall and Newman, 1991; Adolphe, 1992; Hall and

Miyake, 1992,1995; De Crombrugghe et al., 1996;

Lefebvre et al., 1998). For the present, however,

the role of these factors in the ontogeny of

spon-taneous extraskeletal cartilages, like those

occur-ring in vertebrate hearts, remains unknown.

So far, the presence of cartilages in the cardiac

outflow tract of vertebrates can only be regarded as

an outcome of the chondrogenetic potential of this

cardiac region (see also Young, 1994; Lo´pez et al.,

2000), yet the expression of this potential

presum-ably relies on the action of various causative factors,

which appear to diverge between species, resulting

in a wide individual variation.

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