myocardialization of the cardiac outflow tract

Developmental Biology 212, 477–490 (1999)Article ID dbio.1999.9366, available online at http://www.idealibrary.com on

Myocardialization of the Cardiac Outflow Tract

Maurice J. B. van den Hoff,* Antoon F. M. Moorman,* Jan M. Ruijter,*Wout H. Lamers,* Rossi W. Bennington,† Roger R. Markwald,†and Andy Wessels†,1

*Department of Anatomy and Embryology, Academic Medical Center, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands; and †Department of Cell Biology and Anatomy, MedicalUniversity of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by acomplex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of theatrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components ofthe developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of theOFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscularstructure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbedthis process “myocardialization.” Our immunohistochemical analysis of staged chicken hearts demonstrates thatmyocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared toother events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basicallycompleted around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulationof myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated wasdeveloped. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) wholeOFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants fromstage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could beinduced to myocardialize and was therefore “myocardialization-competent” at all stages tested (H/H16–30), (iv) myocar-dialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at noneof the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricularmyocardium did not produce factors capable of supporting myocardialization. © 1999 Academic Press

Key Words: cardiac development; chick embryos; endocardial cushion; myocardialization; neural crest.

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INTRODUCTION

Congenital malformations of the cardiovascular systemare observed in at least 1% of newborn babies in the UnitedStates and are held responsible for an estimated 20% ofspontaneous abortions and 10% of all stillbirths. The ma-jority of these cardiac malformations involve abnormaldevelopment of the septal structures (Hoffman, 1995a, b;Hoffman and Christianson, 1978). Particularly prone toerrors are the formation and alignment of the septal com-

1 To whom correspondence and reprint requests should be ad-dressed at Medical University of South Carolina, Department ofCell Biology and Anatomy, 173 Ashley Avenue, Suite 601, P.O. Box

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250508, Charleston, SC 29425, USA. Fax: (843) 792-0664. E-mail:[email protected].

0012-1606/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

onents derived from the endocardial cushions, includinghe outflow septum separating the right from the leftentricular outlet. Improper development of the outfloweptum can lead to malformations that range from annfundibular (perimembranous) ventricular–septal defect toouble outlet right ventricle.The remodeling process responsible for the transforma-

ion of the tubular heart into a fully septated four-hambered heart, supporting two separate, yet interdepen-ent, blood circulations, involves the interaction of manyell types on a cellular and molecular level (Mjaatvedt et al.,999). The number of genetically modified animal modelsith cardiac malformations increases almost daily. In order

o be able to understand how perturbations in single genese.g., knockouts, transgenes) can lead to congenital malfor-

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478 van den Hoff et al.

mations reminiscent of those observed in the human popu-lation, it is crucial to obtain insight into the molecular andcellular processes that reign over normal development.

The primary heart tube consists of two concentric epi-thelial cell layers, being the endocardium and myocardium,that differentiate from the cardiogenic fields in the splanch-nic mesoderm (see for a review De Jong et al., 1997). Thendocardium and myocardium are separated by extracellu-ar matrix known as cardiac jelly produced mainly by the

yocardium (Markwald et al., 1975, 1977, 1981). By Ham-urger and Hamilton stage 14 (H/H14)2 in the chicken, the

primary straight heart tube loops to the right and differentsegments of the heart can be distinguished, i.e., the inflowtract, atrioventricular canal, ventricle, and outflow tract(OFT) (De la Cruz et al., 1989). With development, thesingle-circuited cardiac tube transforms into the double-circuited four-chambered heart by a complex process ofremodeling and septation. This requires the proper forma-tion and alignment of the interatrial and interventricularsepta and the formation of functional one-way valves, themesenchymal components of these structures being derivedmainly from the endocardial ridges (De Jong et al., 1997;Wessels et al., 1996). The fusion of the endocardial ridges inthe proximal portion of the OFT in the embryonic heartresults, initially, in the formation of a mesenchymal outletseptum. In the adult heart, however, the part of the outletseptum that is incorporated into the ventricular componentis muscular. Hence, the mesenchyme of the proximal outletseptum has to be replaced by cardiomyocytes. In earlierstudies we have already presented evidence that this occursby growth of existing myocardium into the endocardialcushion-derived tissues. We have dubbed this process“myocardialization” (Lamers et al., 1995; Ya et al., 1998;Franco et al., 1999). In the mammalian heart, the only siteof significant myocardialization is in the endocardial cush-ion tissue of the outflow tract. This raises the question ofhow myocardialization is regulated.

Although the phenomenon of myocardialization was re-ferred to in previous studies (Okamoto et al., 1981; De laCruz et al., 1989; Franco et al., 1999; Lamers et al., 1995; Yaet al., 1998), it has not yet been described in sufficient detailto display potential relations to other developmental pro-cesses occurring in the heart at the same time. Therefore, inthis study we present a comprehensive description of myo-cardialization of the developing outflow tract in thechicken based on immunohistochemical analysis.

In order to be able to test hypotheses regarding regulationof myocardialization, an in vitro assay that allows the studyof different aspects of myocardialization was developed.Using this three-dimensional collagen gel culture assay, wehave obtained the first set of results that might lead to theelucidation of the mechanisms involved in regulation of

2 Abbreviations used: BSA, bovine serum albumin; DMSO, di-methyl sulfoxide; dOFT, distal outflow tract; H/H, Hamburger and
Hamilton stages; OFT, outflow tract; pOFT, proximal outflowtract; PBS, phosphate-buffered saline.
Copyright © 1999 by Academic Press. All right

myocardialization. Specifically, we show that whole-OFTexplants from stage H/H21 onward spontaneously myocar-dialize collagen gels in vitro. In addition, we demonstratethat factors produced and secreted by the nonmyocardialdistal component (or truncus) of the OFT can stimulatemyocardialization of the proximal portion of the OFT, butthat these factors are not able to induce myocardializationof explants isolated from ventricular myocardium.

MATERIALS AND METHODS

Chicken Embryos

Fertilized chicken eggs were obtained from local hatcheries(Drost BV, Nieuw Loosdrecht, The Netherlands and PeeDee Hatch-ery, Hartsville, SC), incubated at 37°C in a moist atmosphere, andautomatically turned every hour. After the appropriate incubationtimes, embryos were isolated and staged according to Hamburgerand Hamilton (1951).

Immunohistochemistry

Embryos were fixed in ice-cold modified Amsterdam’s fixative(40% methanol:40% acetone:20% water) for 4 h, dehydrated in agraded alcohol series, and embedded in Paraplast. Serial 7-mmections were prepared and mounted onto polylysine-coated slides.fter deparaffinization and hydration in a graded alcohol series,ndogenous peroxidase activity was blocked using 3% H2O2 in

phosphate-buffered saline (PBS; 150 mmol/L NaCl and 10 mmol/Lsodium phosphate, pH 7.4). Following a pretreatment for 30 min inTENG-T (10 mmol/L Tris, 5 mmol/L EDTA, 150 mmol/L NaCl,0.25% (w/v) gelatin and 0.05% (v/v) Tween-20, pH 8.0), to reducenonspecific binding, the sections were incubated overnight withMF20, a mouse monoclonal antibody specific to myosin heavychain (Hybridomabank, Iowa City, IA). Antibody binding wasvisualized using the indirect unconjugated peroxidase–antiperoxidase technique as described previously (Wessels et al.,1990).

Hearts, to be prepared for whole-mount staining, were fixed inDMSO and methanol (1:4) overnight, cleared in 3% H2O2 inmethanol, and hydrated in a graded alcohol series. After beingblocked in PBS–BSA (PBS containing 1% (w/v) BSA; Sigma) supple-mented with 5% (v/v) rabbit serum for 1 h, the hearts wereincubated with MF20 overnight. Following extensive washing inPBS–BSA, the hearts were incubated in FITC-labeled rabbit anti-mouse serum (Nordic) for at least 3 h. Finally, after a series ofwashes, the hearts were mounted in PBS–BSA, 50% (v/v) glycerol,and 50 mg/ml sodium azide. The specimens were analyzed byonfocal laser scanning microscopy (Bio-Rad MRC1024).

In Vitro Myocardialization Assay

Preparation of collagen gels. Collagen gels were prepared es-sentially according to procedures previously described (Runyan andMarkwald, 1983). In short, the day prior to the use of the collagengels, rat tail collagen type I (Collaborative Research, Inc.) wasdiluted to a final concentration of 1.5 mg/ml in M199 (103; LifeTechnologies) culture medium and distilled water on ice. Polymer-
ization of the collagen was initiated by adding NaOH to a finalconcentration of 15 mmol/L. Directly after addition of NaOH,
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479Myocardialization of the Outflow Tract

aliquots of approximately 500 ml of collagen solution were pipettedinto the wells of 4- or 24-well Nunc plates. Subsequently, the gelswere placed in a 37°C tissue-culture incubator at 5% CO2 andllowed to polymerize. After 30 min, 500 ml M199 (Life Technolo-

gies) culture medium containing penicillin/streptomycin (pen/strep; Life Technologies) was added to the gels. The medium waschanged once after another 30 min and eventually replaced withcomplete medium consisting of M199 culture medium with pen/strep, supplemented with 1% chicken serum (Life Technologies), 5mg/ml insulin, 5 mg/ml transferrin, and 5 ng/ml selenium (ITS;Collaborative Research Inc.). The gels were then conditionedovernight in a 5% CO2 incubator at 37°C.

Isolation of explants. Embryonic chicken hearts were isolatedunder sterile conditions in filter-sterilized Earl’s balanced saltsolution (Life Technologies). The cardiac regions of interest wereisolated, cut open longitudinally, and positioned on top of thedrained collagen gel with the endocardial surface facing the gel.Prior to the addition of complete medium M199, the explants wereallowed to attach to the gel for 4 h. After a culture period of 1 week(37°C, 5% CO2), the gels containing the explant were rinsed with

BS and fixed by incubation for 30 min in 70% ethanol and 1 h in00% ethanol at room temperature. Next, the gels were hydrated in

graded ethanol series and incubated with MF20 diluted inBS–BSA supplemented with 5% (v/v) rabbit serum overnight.fter extensive washing in PBS–BSA, the gels were incubated inITC- or DTAF-labeled rabbit anti-mouse serum (Nordic or JacksonmmunoResearch, respectively) for at least 3 h. After extensiveashing, the gels were mounted in a solution of PBS–BSA/glycerol

50% v/v) containing 50 mg/ml sodium azide and analyzed byconfocal microscopy (Bio-Rad MRC1024).

Using conditioned medium. The experimental setup of theseexperiments was similar to that described above, except for the factthat the cardiac explants were cultured in medium that wasconditioned for 1 week with the respective cardiac explants. Priorto their use, the conditioned media were filtered through a 0.2-mm

lter (Schleicher and Schuell PF030/3 filter) and diluted with 3olumes of fresh complete medium.

Scoring system. The extent of myocardialization was scored onn arbitrary scale, with scores ranging from 0 to 4, using theollowing criteria. A score of “0” was assigned to explants of whichhe border was smooth and no myocardial projections could bebserved. When the edge of the explant was rough, containing onlyfew small myocardial protrusions (“spikes”) on the surface of theollagen matrix, the explants were given a score of “1”. When theyocardial protrusions consisted of isolated groups of ramifyingyocardial cells, the explants received the score of “2”. A score of

3” was assigned to explants in which the ramifying myocardialrotrusion had locally formed a myocardial 3-D network into theollagen matrix. Finally, a score of “4” was given to explants whichhowed extensive myocardial networks localized on top and intohe collagen gel around the entire explant.

Statistics

Since the scoring system for the extent of myocardializationresults in values on an ordinal scale and more than two groups hadto be compared simultaneously, the Kruskal–Wallis nonparametricANOVA had to be used for statistical analysis. With this test, firstthe null hypothesis (H0), “all groups have equal means,” is testedagainst the alternative hypothesis (H1), “at least one is different.”
Only when this overall H0 is rejected may a multiple comparisonof groups be performed to identify which one (ones) is (are)

different. As this latter extension of the Kruskal–Wallis test is notavailable in most of the commercially available statistical analysispackages, a customized computer program was developed (avail-able via e-mail from [email protected]; subject: KruskalWallis). For both the overall test and the multiple comparison ofgroups a significance level of 0.05 was used. Only differences thatwere statistically significant are mentioned as such under Results.

RESULTS

In Vivo MyocardializationTo determine the developmental window of myocardial-

ization in the OFT in vivo, sections of staged chick embryosere immunohistochemically stained for myosin heavy

hain delineating myocardial cells from nonmyocardialells. Initially, at H/H16 the OFT consists of two epithelialell layers, a myosin heavy chain-positive myocardial outerayer and an endocardial inner layer (Figs. 1A and 1B). Athis stage, mesenchymal cells have not yet populated theardiac jelly, the extracellular matrix-rich space separatinghe myocardium and the endocardium (Fig. 1B). By H/H21,esenchymal cells have started to populate the cardiac

elly at the most cranial side of the OFT (Fig. 1C) and theransformation of endocardium into mesenchyme is appar-nt. The OFT ridges are completely filled with mesenchy-al cells at H/H24 (Fig. 1D). At H/H28 (Fig. 1E) theyocardial cells flanking the endocardial ridges of the OFT

tart to form elongated cell processes into the adjoiningesenchyme at the most distal myocardial border of theFT (Fig. 1F). Subsequently, from stage H/H30 onward

Figs. 1G and 1H), overt ingrowth of cardiomyocytes fromhe surrounding myocardium into the ridges is seen. Asevelopment progresses, the myosin-positive cardiomyo-ytes become arranged in a network that is intermingledith mesenchyme of the ridges. During the subsequentevelopmental period the cardiomyocytes invade from theanking myocardium into the mesenchyme. The leadingdges of the invading cardiomyocytes reach each other athe sides that flank the lumen of the ventricles and therterial pole, leaving an area to be myocardialized in be-ween (Fig. 1H). The zones of myocardialization continue torow toward each other and fuse (Figs. 1I and 1J). Myocar-ialization is completed at H/H38. The initial mesenchy-al outlet septum, separating the left and right ventricular

utlets, is at that stage completely muscularized (nothown).

Development of the In Vitro MyocardializationAssay

To address the question of how myocardialization isregulated at the molecular level, we developed an in vitroculture system in which the process of myocardializationcan be mimicked (Fig. 2). This culture system was based onthe collagen lattice system developed for the study of
endocardial-to-mesenchymal transformation in the atrio-ventricular canal and outflow tract of the developing heart

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(Runyan and Markwald, 1983; Ramsdell and Markwald,1997). Since, in vivo, overt protrusion of cardiomyocytesnto the endocardial ridges is observed at H/H30 (Figs.G–1I), explants from the OFT at this developmental stageere explanted on the collagen gels and cultured for severalays in the presence of complete medium M199 (see Mate-ials and Methods). Inspection of these cultures usingoffman modulation contrast microscopy and Varel mi-

roscopy showed the abundance of endocardial cells on theurface and mesenchymal cells within the collagen gelFigs. 3A and 3C). In addition, the images also suggestedrotrusions of the myocardial portion of the explant extend-ng over the surface and into the collagen gel forminghree-dimensional networks (Figs. 3A and 3C). This micro-copical approach, however, did not allow unambiguous

FIG. 1. Myocardialization of the outlet septum in the developinimmunohistochemically stained sections of staged chick embryosmonoclonal antibody recognizing myosin heavy chain (MF20). It isOFT do not contain any cardiomyoctes (A–F), whereas at later stageprocess of myocardialization. The boxed areas in A, E, G, and I arestage; CS, conal septum; endo, endocardium; ECT, endocardial cusoutflow tract; RA, right atrium; RV, right ventricle.

dentification of cardiomyocytes. Hence, to distinguish theardiomyocytes from endocardial and mesenchymal cells in

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ultures, we adapted an immunohistochemical stainingrotocol to immunohistochemically stain the explants.sing confocal laser scanning microscopy it proved possible

i) to distinguish the different cell types within the collagenel (Fig. 3B), (ii) to determine that protrusions with ayocardial appearance as judged with Hoffman modulation

ontrast microscopy are indeed myocardial (Figs. 3C andD), (iii) to visualize the three-dimensional organization ofhe explant (Figs. 3E and 3F), and (iv) to judge and score thextent of growth of the myocardial component of thexplant over the surface of and into the collagen matrix.Cultures with whole outflow tract explants isolated from/H30 embryos showed in the in vitro myocardialization

ssay pronounced myocardial protrusions after 2 days. Itas determined that 1 week was the optimal time to

cken heart. Nomarski (A–F) and bright-field (G–J) micrographs ofhe level of the outflow tract. All sections were incubated with awn that up to stage 28 H/H the endocardial cushion tissues in theJ) the endocardial ridges become increasingly muscularized by the

rged respectively in B, F, H, and J. H/H, Hamburger and Hamiltontissue; LA, left atrium; LV, left ventricle; myo, myocardium; OFT,

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ulture the explants before assessing the extent of myocar-ialization; myocardialization-positive cultures normally


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have formed extensive networks of myocardial projectionsat this time, while the collagen gels are still in good shapeand sturdy enough to be processed for immunohistochemi-cal staining. Beyond 8 days of culturing, the collagen gelstend to collapse. In addition, testing collagen concentra-tions, it was found that gels containing 1.0 mg/ml collagenwere too fragile to withstand the different steps of theimmunohistochemical staining procedure, whereas gelscontaining 2.0 mg/ml collagen were found not to supportmyocardialization to the extent observed in 1.5 mg/mlcollagen gels. Finally, it was established that addition ofchicken serum to the culture medium (1% v/v) was essen-

FIG. 2. The in vitro myocardialization assay. This cartoon illuisolation of the embryonic heart (a), the outflow tract is dissectexperiment, either the whole outflow tract, consisting of the proximin this cartoon, the proximal portion alone is cut open longitudinadown (d and e). The extent of myocardialization of the collagen gemonoclonal antibody recognizing myosin heavy chain (MF20) and(see Materials and Methods). h shows a typical example of an explillustrates a typical example of an explant with extensive myocayellow, endocardial cushion tissue; orange, endocardium.

tial for successfully culturing of the explants (data notshown).


Developmental Window of In VitroMyocardialization

Chicken embryos at stages H/H16–30 were isolated, andfrom the individual specimens the entire OFTs, from thetrabeculated ventricular segment up to the bifurcation intothe pharyngeal arches in the aortic sac, were dissected. Toexpose the endocardial surface of the OFT, the explantswere cut open longitudinally (see Fig. 2). The explants werepositioned on the collagen gels with the endocardial surfaceface down. After 1 week of culture at 37°C, the explantswere immunohistochemically stained for the presence of

es the procedure for the in vitro myocardialization assay. Aftertween the right ventricle and the aortic sac. Depending on theFT (purple) and distal OFT (light blue) or, for instance as depictedd placed on the collagen gel with the endocardial surface (orange)

nd g) is determined after immunohistochemical processing with aning laser confocal imaging and using an arbitrary scoring systemn which no myocardialization was observed (score “0”), whereas ization (score “4”). Purple, proximal OFT; light blue, distal OFT;

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myosin heavy chain with the monoclonal antibody MF20and the extent of myocardialization was scored according to


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the scoring system outlined under Materials and Methods.The results are summarized in Fig. 4. In most cultures ofOFTs ranging from H/H16 to H/H20 the border of theexplant was smooth and no myocardial projections were

FIG. 3. Outflow tract explants myocardialize collagen gels. Someand C show two characteristic examples of explants with protrusio(photographed using Hoffman modulation contrast microscopy). Tois essential. D shows, after immunohistochemical processing for tmonoclonal antibody MF20, the myocardial nature of the outgrowantibody against smooth muscle actin. This antibody recognizesthree-dimensional organization of an area of myocardialization in athrough a pair of red/green stereoglasses, clearly demonstrates the

observed. In some cases a few myocardial protrusions wereobserved on the surface of the collagen gel. Extensive

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yocardial networks were, however, never observed. Whenntire OFTs (see Fig. 5) from embryos ranging from H/H21p to H/H31 were cultured, the myocardial protrusionsere found to have formed extensive networks into the

e features of the in vivo myocardialization assay are illustrated. Arrows) extending from the core of the explant over the collagen gelblish the nature of the protrusions the use of specific cell markerspression of the myocardial marker myosin heavy chain with the

xed in C. B shows, in a similar culture, a control staining with anof the mesenchymal cells in the collagen gel. E and F show the

ure of H/H25 OFT. F is a red/green stereoimage which, when seennature of the myocardializing network within the collagen gel.

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ollagen gel. About half of the cultures of OFT explantssolated from embryos ranging from H/H21 to H/H23


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showed extensive myocardialization all around the entireexplant (score 4). At older stages, however, the explants hadonly formed myocardial networks in restricted areas (score3). Whole-mount immunostaining of hearts with myosinheavy chain antibodies showed that the myocardial compo-nent of the OFT extends up to the bifurcation into thepharyngeal arch arteries at H/H21, whereas at H/H26 themyocardial component extends only halfway into the OFT,i.e., the proximal portion (Fig. 5). Hence, it is likely that therestricted in vitro myocardialization observed in entire OFTexplants beyond HH23 is related to the regression of themyocardial component and the development of the non-myocardial, mesenchymal, distal portion of the OFT.

In order to determine whether the formation of myocar-dial projections on top of and into the collagen gel isintrinsic to the myocardial component of the developingOFT, we prepared explants from the ventricles of heartsranging from H/H16 up to H/H38 (Figs. 5 and 6). In general,the ventricular explants showed very little in vitro myocar-ialization. In two cases (both at H/H25–26) a myocardialetwork was observed in the collagen matrix and in fourases some myocardial protrusions had formed on theurface of the matrix. All the high scores were found in therst group of ventricular explants tested. In this first seriesf experiments the ventricular explants used were ratherarge and it is possible that they may have contained some

FIG. 4. In vitro myocardialization by outflow tract explants isstage dependent. The extent of in vitro myocardialization of 1.5mg/ml collagen gels by whole OFT explants between stage 16 andstage 30 H/H was assessed after 1 week in culture as describedunder Materials and Methods. On the x axis the different develop-mental stages are indicated according to Hamburger and Hamiltonand on the y axis the extent of in vitro myocardialization, expressedin arbitrary units, is shown. Each point in the graph is based on theanalysis of a separate in vitro experiment.

trioventricular endocardial cushion tissue (in the chicklso prone to myocardialization). In the following experi-


ents greater care was taken to use “pure” myocardialxplants isolated from the apex of the ventricle. In theseatter sets of experiments in vitro myocardialization wasever observed.Taken together, the data presented here show that, in theyocardialization assay as described above, whole-OFT

xplants from stage H/H21 onward can spontaneously myo-ardialize collagen gels, whereas explants taken from theyocardial component of the ventricular apex, in general,

o not show any in vitro myocardialization.

The Mesenchymal Distal Portion of the OFTSecretes a Stimulator(s) of Myocardialization

To further unravel the mechanism of myocardialization,H/H25–26 OFT explants were separated into myocardialproximal (conal) portions and mesenchymal distal (truncal)components. Culturing these distal and proximal explantsseparately revealed that the extent of myocardialization ofexplants from the myocardial proximal portion was signifi-cantly less than the extent of myocardialization of explantsof the entire OFT (Fig. 7). As expected, no in vitro myocar-dialization was observed when the, predominantly mesen-chymal, explants from the distal portion of the OFT werecultured alone. These observations suggest that the mesen-chymal distal OFT produces factors that stimulate myocar-dialization of the myocardial component of the proximalOFT. To test this hypothesis the separated proximal anddistal portions of the OFT were cocultured. In these cocul-tures the separated portions were positioned at least 5 mmapart on the collagen gels. In these coculture experiments itwas found that the extent of myocardialization of themyocardial component of the proximal portion of the OFTincreased to the level observed in explants of the entireOFT. Moreover, in these cocultures myocardialization wasnot induced in the mesenchymal distal portion of the OFT(Fig. 7A). These observations suggest either that the mes-enchymal distal portion of the OFT secretes a factor(s) intothe medium that stimulates myocardialization of the myo-cardial component of the proximal portion of the OFT orthat explants of the mesenchymal distal portion of the OFTalter the composition of the collagen gel in such a way as tofacilitate myocardialization.

Conditioned Medium Stimulates and InducesIn Vitro Myocardialization

To further substantiate the observation that in vitromyocardialization is regulated by factors secreted by themesenchymal distal portion of the OFT, conditioned me-dium was prepared from (a) the entire OFT, (b) the myocar-dial proximal portion of the OFT, (c) the mesenchymaldistal portion of the OFT, and (d) the left ventricle of chickembryos at H/H25–26. Culturing the proximal portion ofthe H/H25–26 OFT in medium conditioned by the entire
OFT (Fig. 8A), the myocardial proximal portion of the OFT(Fig. 8B), or the mesenchymal distal portion of the OFT (Fig.

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8C) showed that the extent of in vitro myocardializationcould be stimulated to the level of that observed whenexplants of the entire OFT were cultured in standard

FIG. 5. The developing outflow tract in embryonic chicken hearts30 (C), and 36 H/H (D). The cartoons are based on scanning EM anline a (junction between distal OFT (dOFT) and aortic sac) and line cOFT (wOFT). In experiments in which myocardialization of the pOthe inductive capacity of the dOFT was tested, the wOFT was separachicken hearts at stages H/H21 (E) and H/H26 (F) processed for whdirected against myosin heavy chain. The immunoreactivity was viimages is shown as a brightest point projection. The arrow in E indwith the aortic sac. The arrow in F indicates how this boundary haorto; LV, left ventricle; RV, right ventricle; V, ventricle.

medium (Fig. 7). The extent of in vitro myocardialization ofhe proximal portion of the OFT could not be rescued,

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owever, by culturing in medium conditioned by left ven-ricular myocardium (Fig. 8D). To test whether the respec-ive conditioned media could induce in vitro myocardial-

show cartoons of embryonic chick hearts at stages 16 (A), 26 (B),secting-microscope images. The area of the OFT located between

ction between RV and proximal OFT (pOFT)) is referred to as wholeas compared with that of the dOFT, and in experiments in which

nto pOFT and dOFT along the lines labeled b. E and F show isolatedount immunofluorescence using the monoclonal antibody MF20

zed using scanning laser confocal microscopy. The stack of opticals the distal boundary of the myocardial OFT close to the junctionifted as a result of differential growth of the tissues involved. Ao,

. A–Dd dis(junFT wted iole-msualiicate

zation in tissues that under standard conditions do nothow myocardialization (Fig. 7), these media were tested in


vti e extent of in vitro myocardialization, expressed in arbitrary units, iss ate in vitro experiment.

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cultures with explants isolated from (i) the entire OFT atH/H16, (ii) the left ventricular apex of H/H26, and (iii) themesenchymal distal portion of the OFT at H/H26. It wasfound that the media conditioned by the entire OFT, theproximal portion of the OFT, and the distal portion of theOFT were able to induce myocardialization of the H/H16OFT up to the level of the entire H/H26 OFT when culturedin standard medium (Figs. 8A–8C). However, medium con-ditioned by ventricular explants did not induce in vitromyocardialization of the H/H16 OFT (Fig. 8D). The obser-vation that medium conditioned by the proximal portion ofthe OFT at H/H26 is able to stimulate in vitro myocardial-ization of the proximal portion of the OFT at H/H26 andinduce in vitro myocardialization of the entire OFT atH/H16 implies that the proximal portion of the OFT atH/H26 produces the myocardialization inducer(s) in limit-ing amounts. This might reside in the relative abundance ofneural crest-derived mesenchyme in the proximal portionof the OFT at H/H26 (Waldo et al., 1998). As expected, noneof the conditioned media was able to induce in vitromyocardialization in cultures of the mesenchymal distalportion of the H/H26 OFT and, interestingly, also not incultures of the H/H26 left ventricle.

DISCUSSION

The Formation of the Muscular Outlet Septum

Whereas in the adult heart the outlet septum, separating

FIG. 6. In vitro myocardialization of ventricular explants. The eentricular apex of embryos at stages H/H16–38 after 1 week in cuhe MF20 mouse monoclonal antibody and confocal laser scannindicated according to Hamburger and Hamilton. On the y axis thhown. Each point in the graph is based on the analysis of a separ

xtent of in vitro myocardialization by explants isolated from the leftlture was assessed by immunofluorescent staining of the explant usingng microscopy. On the x axis the different developmental stages are

the subaortic from the subpulmonic outlet, is completelymuscular, in the embryonic heart this septum initially

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FIG. 7. Myocardialization is induced by factors produced by thedOFT. The extent of in vitro myocardialization after culturingdifferent cardiac segments isolated from H/H16 or H/H26 chickenembryos for 1 week was assessed by immunofluorescent staining ofthe cardiomyocytes utilizing the MF20 mouse monoclonal directedagainst myosin heavy chain and confocal laser scanning micros-copy. On the x axis the different cardiac segments and the devel-opmental stages are indicated and on the y axis the extent of initro myocardialization is shown. It is shown that explants of thentire OFT at H/H16 show virtually no myocardialization, whereasxplants from entire OFT at H/H26 show extensive myocardializa-ion. Separating the OFT into a proximal and a distal portionesults in decreased myocardialization of either component. Myo-
ardialization of the pOFT can be stimulated by coculturing withhe dOFT; the dOFT cannot be induced to myocardialize.

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develops as a mesenchymal structure. Hence, the embry-onic mesenchyme has to be replaced by cardiomyocytes.Two different mechanisms for this replacement are pos-sible. The cardiomyocytes found in the adult muscularoutlet septum can either be derived from a mesenchymal-to-cardiomyocyte transdifferentiation or result from theingrowth of OFT myocardium into the mesenchymal outletseptum. Our morphological observations presented heredemonstrate that the latter process, dubbed myocardializa-

FIG. 8. Conditioned medium from H/H26 OFT induces myocar-dialization. The inducing capacity of conditioned medium fromdifferent regions of the embryonic chicken heart at H/H26 wastested on OFTs isolated from H/H16 embryos and on explants ofthe myocardial proximal OFT, mesenchymal distal OFT, and leftventricle at H/H26. It was found that conditioned medium fromthe whole OFT (A), from the pOFT (B), and from the dOFT (C)induced myocardialization in the H/H16 explant and stimulatedmyocardialization in the H/H26 pOFT explant, but not in explantsisolated from the ventricular apex or the dOFT. Conditionedmedium from ventricular explants never induced or stimulatedmyocardialization (D).

tion, is the one actually taking place, though we cannotexclude the possibility of some transdifferentiation at the


ame time. The immunohistochemical data demonstratehat, in the chicken, myocardialization is initialized at/H27–28 and is basically completed around stage H/H38.Compared to other cell-migratory events in the outflow

ract, myocardialization can be considered a relatively latevent. Several other cell populations migrate into the endo-ardial cushions of the outflow tract preceding the processf myocardialization. These populations include the mes-nchymal cells derived from the endocardium, the cardiaceural crest, and the epicardium (see Fig. 9). The endocar-ially derived mesenchymal cells originate from a subpopu-ation of endocardial cells lining the OFT ridges. Afterecoming hypertrophic, these cells detach from their epi-helial context, undergo an endocardial-to-mesenchymalransformation, and, finally, migrate into the cardiac jellyrom H/H17 onward (Bolender and Markwald, 1979; Mark-ald et al., 1975, 1977; Markwald and Funderburg, 1983;akajima et al., 1994; Moreno-Rodriguez et al., 1997). At

pproximately H/H22, the aorticopulmonary septum startso develop at the most cranial side of the OFT in the aorticac and subsequently expands caudally (Epstein, 1996;irby and Waldo, 1995; Waldo et al., 1998; Somida et al.,989). The cardiac neural crest-derived cells, which contrib-te mesenchyme to this aorticopulmonary septum, alsoorm two prongs of condensed mesenchyme that extend tohe future level of the semilunar valves. In the proximalFT, below the level of the future valves, these neural

rest-derived cells become dispersed in the endocardialidges and flanking myocardium (Poelmann et al., 1998;

aldo et al., 1998; Ya et al., 1998; Creazzo et al., 1998). Thehird population of mesenchymal cells, and presumably theast to arrive in the outflow tract ridges, is derived from thepicardium (Dettman et al., 1998; Viragh et al., 1993).hese epicardially derived cells (or EPDCs) are first ob-erved in the outflow tract ridges at H/H35 (Gittenberger-deroot et al., 1998). At this stage a subpopulation of OFTesenchymal cells becomes apoptotic. Although it has

een suggested that these apoptotic cells are neural cresterived (Cheng et al., 1999; Poelmann et al., 1998; Ya et al.,

1998; Takeda et al., 1996; Satow et al., 1981), quail–chickchimeras show conclusively that the majority of neuralcrest-derived cells in this region do not die but are incorpo-rated in the septal structure (Waldo et al., 1998). Concomi-tantly, macrophages are observed, their role likely being toremove apoptotic debris (Ya et al., 1998; Sorokin et al.,1994; Sz. Viragh, personal communication). This spatio-temporal sequence of events strongly suggests a relationbetween the molecular regulation of the process of myocar-dialization and the development of the other cell popula-tions in the ridges of the outflow tract.

Development of an In Vitro MyocardializationAssay

To unravel the molecular mechanisms that are respon-
sible for the induction and regulation of myocardializa-tion an in vitro culture system in which myocardializa-

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ing s


tion could be mimicked and manipulated was developed.The assay was based on the three-dimensional culturesystem developed by Markwald and co-workers (Mark-wald et al., 1990; Ramsdell and Markwald, 1997; Runyanand Markwald, 1983) to study the process of endocardial-to-mesenchymal transformation in the atrioventricularcanal and outflow tract. Because, in vivo, myocardializ-ng cardiomyocytes form a network in the mesenchymal-zed outlet septum, a prerequisite for a good in vitro

yocardialization assay was the formation of a networkf myocardial projections into the collagen matrix. Ex-eriments with OFT explants isolated from stages ofctive myocardialization not only showed that endocar-ial and mesenchymal cells grew out of the explant butlso showed protrusions of cardiomyocytes on and intohe collagen gel. As it is difficult to evaluate the extent of

myocardial network in a collagen gel by Varel oroffmann modulation contrast microscopy, the cardio-yocytes were immunofluorescently stained for the ex-

FIG. 9. Cell populations in the developing outflow tract. This carand cellular events involved in the development of the outflow traccartoon are still contentious and are the subject of numerous ongo

ression of myosin heavy chain and visualized usingonfocal laser scanning microscopy.


In Vitro Myocardialization Is Stage Dependent

Using this assay the developmental window of in vitromyocardialization of the OFT and ventricular myocardiumwas determined. Culturing whole-OFT explants ranging indevelopmental stage from H/H21 to H/H31 (Fig. 4) showedthat before H/H21 no in vitro myocardialization was ob-served, whereas the explants of stage H/H21 and olderformed extensive networks of myocardial protrusions intothe collagen. Ventricular myocardium showed hardly anymyocardialization of the collagen gel irrespective of thedevelopmental stage tested. These observations indicate (i)that OFT myocardium at stage H/H21 and older is “myo-cardialization-competent;” (ii) that, if myocardializationrequires external factors for induction of myocardialization,these factors are present (or produced) in cultures of stageH/H21 and older; (iii) that in OFT explants of stage H/H20and younger the myocardium is not myocardialization-competent and/or the factors supporting myocardializationare lacking; and (iv) that ventricular myocardium is not

summarizes the current knowledge on the subpopulations of cellsis important to note that some of the mechanisms depicted in thistudies.

toont. It

myocardialization-competent and/or does not produce fac-tors supporting myocardialization. The following experi-


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ments were performed to address these issues. When OFTexplants of H/H16 and ventricular explants of H/H26 heartswere cultured in conditioned medium prepared from aH/H26 OFT, which shows extensive myocardialization inour in vitro assay, myocardialization was observed in theH/H16 OFT explant, but not in the H/H26 ventricularexplant. When similar explants were cultured in the pres-ence of conditioned medium prepared from H/H26 ventric-ular explants, which do not show in vitro myocardializa-ion, it was observed that neither the H/H16 OFT nor the/H26 ventricular explants showed myocardialization.ased on these results we conclude that (i) the cardiomyo-ytes of the OFT are myocardialization-competent at alltages tested; (ii) OFTs, but not the ventricles, produce andecrete a factor(s) that can induce and support myocardial-zation; and (iii) ventricular cardiomyocytes are never

yocardialization-competent. We have previously shownhat the myocardium of the OFT is characterized by theersistence of a set of phenotypic and functional character-stics of the so-called “primary myocardium.” Among otherhings this primary myocardium displays a slow propaga-ion of the impulse and a matching peristaltic form ofontraction. Many so-called atrial- or ventricular-specificenes are coexpressed in the primary myocardium. Theworking myocardium” displays a fast propagation of thempulse and a matching synchronous contract and a uniquetrial or ventricular molecular phenotype (De Jong et al.,992; Moorman and Lamers, 1994, 1999a; Moorman et al.,

1998; Franco et al., 1998). We have also found evidence thatthe myocardium of the inflow tract, another remnant of theprimary heart tube, is myocardialization-competent (Moor-man et al., 1999b). Hence, the data presented in this paperndicate that myocardialization-competency is another fea-ure distinguishing the primary myocardium from theorking myocardium, the latter being differentiated from

he primary heart tube to form the working myocardium ofhe atria and ventricles (Moorman et al., 1999a).

The Mesenchymal Distal Portion of the OutflowTract Plays a Role in Regulationof Myocardialization

Next, a series of experiments was performed to establishwhether the factors that induce and support myocardializa-tion are produced and secreted by the nonmyocardial or themyocardial component of the OFT. The H/H26 OFT wasdivided into its distal portion, being the nonmyocardialcomponent of the OFT that reaches up to the bifurcation into the pharyngeal arches, and its proximal portion, beingthe portion of the OFT immediately downstream of theright ventricle, which is covered by myocardium. Thefollowing observations suggest that the nonmyocardialcomponent of the OFT is the source of the factors thatinduce and/or support myocardialization: (i) The extent ofmyocardialization was significantly less when only the
proximal portion of the H/H26 OFT was cultured instead ofthe entire H/H26 OFT. (ii) The extent of myocardialization

f the proximal portion of the OFT was similar to the extentf the entire H/H26 OFT when the proximal portion wasocultured with the distal portion. (iii) The extent ofyocardialization of the H/H26 myocardial proximal por-

ion of the OFT could be stimulated to the extent of thentire H/H26 OFT when cultured in medium conditionedith the H/H26 mesenchymal distal portion of the OFT.

iv) Myocardialization of the H/H16 OFT could be inducedith medium conditioned with the H/H26 mesenchymalistal portion of the OFT. Furthermore, the observationshat myocardialization was never induced in the nonmyo-ardial distal OFT in coculture experiments or when cul-ured in conditioned media support our conclusion based onhe morphological analysis that myocardialization is notue to mesenchymal-to-cardiomyocyte transdifferentiationn the ridges.

Neural Crest Cells May Play an Important Rolein the Regulation of Myocardialization

The nonmyocardial component of the OFT comprisesmesenchymal cells that are derived from the endocardium,the neural crest, and the epicardium. The EPDCs are thelast to arrive in the OFT ridges, arriving roughly 3 days afterthe onset of myocardialization. It is therefore unlikely thatthey play a role in the induction of myocardialization. Itcannot, however, be ruled out that they play some role insupporting myocardialization as, in the outflow tract, theyare typically located within the myocardializing region(Gittenberger-de Groot et al., 1998). Neural crest cells startto migrate into the outflow tract around stage H/H21 (Kirbyand Waldo, 1995). As stated above, it has been suggestedthat, after having reached the proximal portion of the OFT,some of the neural crest-derived mesenchymal cells mayundergo apoptosis just prior to myocardialization (Satow etal., 1981; Kirby and Waldo, 1995; Takeda et al., 1996;Poelmann et al., 1998; Waldo et al., 1998; Ya et al., 1998;

reazzo et al., 1998; Cheng et al., 1998), but the studies byirby and colleagues indicate that the majority of neuralrest-derived cells do not die but instead contribute to theeveloping outlet septum (Waldo et al., 1998). It remains toe established whether the neural crest-derived cells arenvolved in the induction and/or regulation of apoptosis inhis region. The foci of apoptotic cells appear to locallyecruit macrophages to the region (Sorokin et al., 1994; Yat al., 1998). The correlation in time and space betweenhese processes and the onset of myocardialization suggestsrole for the neural crest cells in the regulation of myocar-ialization. This hypothesis is supported by our in vitro

data showing a significant increase in myocardialization ofthe cultured OFTs at H/H21 and beyond, correlating withthe onset of invasion of the neural crest cells into the OFTat this stage (Kirby and Waldo, 1995). The mechanism viawhich myocardialization is induced remains to be eluci-dated. Although we clearly show in this paper that diffus-
able signals can induce in vitro myocardialization and arecurrently, because of the cardiac phenotype of the TGF-b2

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knockout mouse (Sanford et al., 1997), focusing in onTGF-b as a possible candidate for the regulation of myocar-dialization in vivo, we also have strong evidence that neuralcrest-derived mesenchyme is involved in regulation ofmyocardialization by physical cell–cell interactions (Walleret al., 1999). It also cannot be ruled out that other cellpopulations contribute to the regulation of myocardializa-tion. A role for the endocardium-derived mesenchyme, forinstance, is not unlikely, as in the developing chicken notonly the outflow septum, but also other mesenchymalstructures (viz. the right atrioventricular valve and themesenchymal atrioventricular septum), in which no, oronly few, neural crest cells can be found, undergoes mus-cularization. Whether the mechanism of the process ofmuscularization in these areas is identical to that of myo-cardialization of the outlet septum in the OFT has still to beestablished.

ACKNOWLEDGMENTS

This work was financially supported by the following grants:The Netherlands Heart Foundation Grant M96.002 (M.J.B.v.d.H.,A.F.M.M.), NATO Science Fellowship S92-157 (A.W.), NIHHL33756 (A.W., R.R.M.), and NIH HL52813 (A.W., R.R.M.). Dr.M. J. B. van den Hoff visited the Department of Cell Biology andAnatomy in Charleston in the framework of this study. We aregrateful to Anita A. M. Buffing, Sabina Tesink-Taekema, Aimee L.Phelps, Kioina L. Myers, and Tanya J. Rittmann for expert technicalassistance; to Mr. Drost of Drost BV (Nieuw Loosdrecht, TheNetherlands) for expert advice on setting up the chicken eggincubation facility in Amsterdam; to Prof. Dr. Sz. Viragh (Budapest,Hungary) for morphological advice; and to Dr. F. de Jong (Amster-dam, The Netherlands) for stimulating discussion.

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Received for publication March 10, 1999
Revised June 4, 1999
Accepted June 4, 1999


myocardialization of the cardiac outflow tract

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