4749Development 124, 4749-4758 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV2214

Vitamin A deficiency and mutations of RXR α, RXRβ and RAR α lead to early

differentiation of embryonic ventricular cardiomyocytes

Philippe Kastner, Nadia Messaddeq, Manuel Mark, Olivia Wendling, Jesus M. Grondona 1, Simon Ward 2,Norbert Ghyselinck and Pierre Chambon*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 ILLKIRCHCedex, Strasbourg, France*Author for correspondence (e-mail: igbmc@igbmc.u-strasbg.fr)1Present address: Laboratorio de Fisiologia Animal, Departamento de Biologia Animal, Facultad de Ciencias, Universidad de Malaga, 29071 Malaga, Spain2Present address: Department of Human Anatomy, South Parks Road, Oxford, OX1 3QX, UK

Knock-out of the mouse RXRα gene was previously shownto result in a hypoplastic heart ventricular wall, histologi-cally detectable in 12.5 dpc fetuses. We show here that aprecocious differentiation can be detected as early as 8.5dpc in ventricular cardiomyocytes of RXRα−/− mutants.This precocious differentiation, which is characterized bythe presence of striated myofibrils, sarcoplasmic reticulumand intercalated disks, is found after 9.5 dpc in about 50%of RXRα−/− subepicardial myocytes. In contrast, wild-typesubepicardial myocytes remain morphologically undiffer-entiated up to at least 16.5 dpc. A similar precocious differ-entiation was observed in 9.5 dpc subepicardial myocytesof several RXRβ−/− and RARα−/− mutants, as well as invitamin A-deficient embryos. The proportion of differenti-ated subepicardial myocytes almost reached 100% in

RXRα /RXRβ double null mutants, indicating a partialfunctional redundancy between RXRα and RXRβ. Thisdifferentiation defect was always paralleled by a decreasein the mitotic index. In addition, subepicardial myocytes ofRXRα−/−, RXRα−/−/RXRβ−/− or vitamin A deficient, but notof RXRβ−/− and RARα−/− embryos, were often flattenedand more loosely connected to one another than those ofWT embryos. Thus, retinoids are required at early stagesof cardiac development to prevent differentiation, supportcell proliferation and control the shape of ventricularmyocytes, and both RXRs and RARs participate in themediation of these functions.

Key words: retinoic acid receptors, subepicardial myocyte,differentiation

SUMMARY

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INTRODUCTION

Retinoic acid (RA), the major active metabolite of vitamin Aexerts its functions through two families of nuclear receptoRetinoic acid receptors (RARα, β and γ) are activated by allactive forms of retinoic acid, whereas retinoid X recepto(RXRα, β and γ) are activated specifically by 9-cis RA. RXRsform heterodimers with RARs, which are likely to be the actufunctional units transducing the retinoid signal at the moleculevel. In addition to their role in the action of RARs, RXRmay also function as homodimers, as well as within herodimeric associations with a number of nuclear receptonotably thyroid hormone receptors, vitamin D3 receptoPPARs and several orphan receptors (for reviews, see Mgelsdorf and Evans, 1995; Chambon, 1996).

The respective roles of each RAR and RXR in the tranduction of the developmental functions of retinoids has beaddressed by engineering null mutations in each of threceptor genes (see Kastner et al., 1995 for a review). Stuof RAR and RXR single null mutants, RAR double mutanand RXR/RAR compound mutants have led to important coclusions. Firstly, as all the defects of the fetal vitamin A deciency (VAD) syndrome were recapitulated in RAR single

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double mutants, RARs are clearly required for the mediatioof the known vitamin A functions during developmen(Mendelsohn et al., 1994; Lohnes et al., 1994; Luo et al., 199Ghyselinck et al., 1997). Secondly, RXRα null mutantsexhibited some cardiac and ocular defects belonging to tfetal VAD syndrome (Sucov et al., 1994; Kastner et al., 1994and furthermore a strong synergy was observed between effects of the RXRα and RAR mutations, as RXRα/RARcompound mutants reproduced most of the defects occurrin RAR double mutants and VAD fetuses (Kastner et al., 1991997). Therefore, RXRs are also involved in the mediation the retinoid signal during development, most probably as heerodimeric partners for RARs.

All RXRα −/− mutants die in utero and, beside ocular defectdisplay a hypoplastic ventricular wall (Sucov et al., 1994Kastner et al., 1994; Dyson et al., 1995). At the histologiclevel, the severity of this cardiac defect is variable, which moprobably accounts for the large time window during whicdeath of RXRα−/− mutants occurs (11.5-17.5 days post-coitum(dpc); Kastner et al., 1994, and our unpublished results). Tnormal embryonic myocardium is subdivided into a periphercompact zone and an inner trabecular zone. As one moves frthe periphery towards the lumen of the ventricle, the rate of c

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division decreases, whilst morphological features indicativecardiomyocyte differentiation become more promine(Rumyantsev, 1977). The rapid expansion of the compact lapermits an harmonious adjustment of the cardiac function tobody size increase. We have previously shown that, at 14.5 the RXRα−/− compact zone myocytes display aberrant diffeentiation features, in that some of these cells contained worganised striated myofibrils resembling those normally fouin trabecular myocytes (Kastner et al., 1994). These obsetions suggested that the mutant myocytes of the compact zfailed to remain in their ‘normal’, relatively undifferentiatedstate. To gain further insight into the timing of appearancethis precocious differentiation and to investigate the possibithat the variable severity of the RXRα−/− cardiac phenotypecould be due to a partial functional compensation by RXRβ, wehave now examined the ventricular myocytes at earlier staof development, both in RXRα single, RXRβ single orRXRα/RXRβ double mutants. Furthermore, in order tcorrelate this differentiation defect to a possible function vitamin A, we have also examined ventricular myocytes froVAD embryos and RAR mutant embryos.

MATERIALS AND METHODS

Mice and embryosAll single mutant mice lines employed in this study have bedescribed previously (Kastner et al., 1994, 1996; Lohnes et al., 19Lufkin et al., 1993; Ghyselinck et al., 1997). RXRα/RXRβ doublenull embryos were obtained from the mating of RXRα+/−/RXRβ+/−

males with RXRα+/−/RXRβ+/− or RXRα+/−/RXRβ−/− females. Geno-typings were usually performed on DNA extracted from yolk sac Southern blotting, as described previously. The RXRα, RXRβ andRARα genotypes could also be determined by PCR (primers and Pconditions available upon request).

Histology, electron microscopy and quantification ofdifferentiated cellsEmbryos and fetuses were fixed in 2.5% gluteraldehyde in 0.1sodium cacodylate buffer (pH 7.2) for 24 hours at 4°C, washed inM cacodylate buffer for 30 minutes and post-fixed in 1% osmiutetroxide in 0.1 M cacodylate buffer for 1 hour at 4°C. Followinstepwise dehydration with increasing concentrations of ethanol embedding in Epon 812, semi-thin sections (7.5 µm thick) werestained with toluidine blue for light microscopy. Ultrathin sectionwere contrasted with uranyl acetate and lead citrate and observeda Phillips 208 electron microscope. For all observations, care taken to consider sections that correspond to comparable positwithin the heart. Estimations of the percentage of differentiated cwere performed by scoring at least 100 cells. Scorings of differgrids from the same embryo showed that the variations in the centages of differentiated cells were within 10%.

Mitotic indexMitotic indexes were estimated on semi-thin sections by scoring number of mitotic events (metaphase and anaphase) in subepicacells. For each embryo, a minimum of 200 cells were analysed,counting 2-3 sections, each containing about 80-100 subepicarcells (to avoid counting the same cell twice, only one out of four cosecutive sections was examined). Note that, for a given embryo, twas very little variation between the percentages of mitotic cecounted from each individual section (±1%). The error margin of themitotic indexes obtained for each mutant is thus likely to be within 1

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In situ hybridisationThe MLC2a probe was a 285 bp fragment recloned by RT-PCspanning nucleotides 128-413 (Kubalak et al., 1994). The MLCprobe was a gift from K. Chien. Probe labeling, in situ hybridizatioand emulsion autoradiography were performed as described (Rubet al., 1990).

RESULTS

Differentiation features in wild-type, RXR α+/− andRXRα−/− ventricular myocytes from 8.5 dpc to 16.5dpcThe differentiation state of cardiomyocytes was assessed atultrastructural level by the presence of structures involvedmuscle contraction. Individual actin and myosin myofilamenfirst associate into bundles of myofilaments (F, Fig. 1c), whithen form sarcomeres, the smallest contractile unit of cardmyofibrils. Morphologically, a sarcomere is delimited by twsuccessive Z-lines (Z, see Fig. 1b,d,e). At cellular junctionmyofibrils from two adjacent myocytes are connected by intecalated disks (ID, Fig. 1e). The sarcoplasmic reticulum (SFig. 1e), a kind of smooth endoplasmic reticulum associawith myofibrils, is involved in the propagation of myofibrilcontraction by releasing Ca2+ ions (Krstic, 1984, and refstherein). These ultrastructural features were used to scoredifferentiation status of cardiac myocytes in wild type (WTand RXR mutant embryos.

In WT embryos at 8.5 dpc (3-5 somites), the ventriculmyocardium consists of 1-2 layers of myocytes, which usuaexhibit a cuboidal shape (Figs 1a, 2a). Only a few of themyocytes contained bundles of myofilaments (F, Fig. 1c) aZ-lines were never seen. Note that the 8.5 dpc heart lacksepicardium. At all subsequent stages from 9.5 dpc to 16.5 dthe vast majority of WT myocytes located immediately beneathe epicardium (the subepicardial myocytes, SMs) remainrelatively undifferentiated; they displayed numerous shobundles of myofilaments scattered throughout their cytoplaand occasional Z-lines (Table 1), but only rarely contained scomeres, intercalated disks or sarcoplasmic reticulum. Intereingly, the proportion of sarcomere-containing SMs in Wembryos appears to vary depending on the genetic backgrobeing totally absent in WT pure 129/Sv embryos (derived froRXRα+/− intercrosses; Table 2), while present at a lofrequency in WT embryos from a mixed 129/Sv/C57Bl/genetic background (derived from RARα+/− and RARβ+/− inter-crosses; see below and Table 3). Note that there is a graincrease in the degree of myocyte differentiation as one mofrom the subepicardial cell layer to the trabecular region (F3, 16.5 dpc WT fetus). Given the constancy of this absencedifferentiation in the subepicardial cell layer in WT embryoderived from RXRα+/− intercrosses at all stages examined (sTables 1 and 2), we focused on this cell layer to assess the dientiation status of RXRα mutant ventricular myocytes.

The RXRα+/− ventricular myocardium was indistinguishablefrom its WT counterparts at the histological level (not shownHowever, the differentiation status of ventricular myocytes (8.5 dpc) or subepicardial myocytes (from 9.5 dpc onwardclearly differed in mutant and WT embryos. At 8.5 dpc, moof the RXRα+/− myocytes already contained bundles of myofiaments, and isolated Z-lines and even sarcomeres were sesome of them (Fig. 1d, Table 1). Note that at this stage the p

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c and e are enlargements of the boxed areas in a and b, respectively. F,lated disk; M, myocyte; N, nucleus; S, sarcomere; SR, sarcoplasmicification: 3800× (a,b); 24000×(c-e).

portion of RXRα+/− cardiomyocytes containing Z-lines and/osarcomeres varied greatly from embryo to embryo, rangfrom 4% to 60%. Sarcomeres, intercalated disks and scoplasmic reticulum were visible in RXRα+/− SMs from 9.5dpc onwards (Tables 1 and 2). However, more than two csecutive Z-lines were never observed in these myocytes.

At all stages examined, the RXRα−/− ventricular myocardiumappeared thinner than its WT and RXRα+/− counterparts, andoften formed a single layer of abnormal flattened and loosassociated cells [compare Fig. 2a,b (8.5 dpc) and c,e (9.5 dAt 8.5 dpc, bundles of myofilaments were identified in all vetricular myocytes. About 50% of these cells contained striamyofibrils, displaying on average 3-4 consecutive sarcome(Fig. 1b,e) as well as a sarcoplasmic reticulum, and these ferentiated myocytes were connected by intercalated disks (1e). All these features persisted in RXRα−/− SMs at subsequentdevelopmental stages (Tables 1 and2). Note that, as dying cells were seenvery infrequently in both WT andmutant myocardia at all stagesanalysed, increased cell death isunlikely to be a major cause leadingto the thinning of the ventricular wallin RXRα mutants.

Trabeculae were present in most9.5 dpc WT and mutant ventricles,but they were, on average, thinner inRXRα−/− mutants (not shown). BothWT and mutant trabecular myocytesalways displayed numerous sarco-meres and intercalated disks, andabundant sarcoplasmic reticulum(Table 1, and data not shown).

Early morphologicaldifferentiation features inRXRβ mutant ventricularmyocytesTo investigate whether the cardiacdefects reflect specific functions ofRXRα, we analysed the hearts of six9.5 dpc RXRβ−/− mutants. At the his-tological level, their myocardiaappeared normal, with globular andtightly associated SMs (Fig. 2d;compare with Fig. 2c). In contrast,electron microscopic examinationrevealed the presence of wellorganised striated myofibrils, sar-coplasmic reticulum and intercalateddisks in SMs of four out of six 9.5dpc RXRβ−/− mutants examined(Fig. 4; Table 2). The remaining two9.5 dpc RXRβ−/− mutants wereaffected to a lesser degree, as theydisplayed only isolated sarcomeres ina restricted population of SMs(embryos 13 and 16 in Table 2).Therefore, either RXRα or RXRβdeficiency can affect the differen-tiation status of subepicardial ven-

Fig. 1.Ultrastructural feat(b,e) embryos at 8.5 dpc.myofilaments; ID, intercareticulum; Z, Z-line. Magn

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tricular myocytes. However, there are some interesting diffences between the RXRα and RXRβmutant phenotypes: (1)the abnormal flattened aspect and the loose associabetween myocytes was seen only in RXRαnull mutants(compare Fig. 2d with e); thus this alteration of cell shape cbe dissociated from an abnormal presence of sarcomeres (that the RXRβmutant illustrated in Fig. 2d was as affected aRXRα null mutants with respect to the percentage of SMs cotaining sarcomeres); (2) individual RXRβ−/− embryos weremuch more variably affected than RXRα−/− embryos, withphenotypes varying from very mild to as severe as that RXRα−/− embryos.

Increased proportion of differentiated subepicardialmyocytes in RXR α/RXRβ double mutants The incomplete penetrance, at the cellular level, of the pre

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Fig. 2. Semi-thin sectionsthrough the ventricular wall ofWT and RXR mutant embryosat 8.5 dpc (a,b) or 9.5 dpc (c-f).E, epicardium; EN,endocardium; M, myocyte; SM,subepicardial myocyte; T,trabeculae. Magnification: 370×.

cious differentiation phenotype seen in either RXRα−/− orRXRβ−/− myocardia could be due to a partial functional redudancy between these receptors. We therefore examiRXRα−/−/RXRβ−/− double mutants. Many of the doublemutant embryos were growth-retarded and displayed sev

Table 1. Differentiation status of subepicardial, Subepicar

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Gestation

Presence of Genotype 8.5(d) 9.5

Myofilaments RXRα+/+ −a +RXRα+/− + +RXRα−/− + +

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Intercalated disk RXRα+/+ − −RXRα+/− − +/−a

RXRα−/− + +

Sarcoplasmic reticulum RXRα+/+ − −RXRα+/− − +/−a

RXRα−/− + +

aPresent in a few cells (,10%).bIsolated Z lines were occasionally present.cNot more than 2 consecutive Z lines were seen.dNote that at 8.5 dpc, the epicardium is not formed.

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malformations at 9.5 dpc, and most of them died between 9and 10.5 dpc (our unpublished results). The myocardial wof these growth-retarded mutants was extremely thin aconsisted almost exclusively of elongated and looseconnected cells (Fig. 2f). Nearly all these cells displayed sa

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Table 2. Sacomeres and mitoses in subepicardial cells of9.5 dpc RXR mutant embryos

IndividualType of embryo Cells with Mitoticembryo number sarcomeres cells

WTb 1 0% 17%2 0% 16%3 0% 15%4 0% ND5 0% ND6 0% ND

RXRα+/− 7 27%a ND8 25%a ND9 38%a ND

RXRα−/− 10 64% 6.5%11 52% 7.5%12 48% 9.5%

RXRβ−/− 13 35%a 12%14 50% 7%15 59% 5%16 12%a 11.5%17 55% ND18 56% ND

RXRα+/−/RXRβ−/− 19 30%a ND

RXRα−/−/RXRβ+/− 20 77% ND21 59% ND22 60% ND23 60% ND24 70% ND25 58% ND

RXRα−/−/RXRβ−/− 26 92% 6%27 82% 6%28 96% 6%

aNot more than two consecutive Z lines were observed.bObtained from the same litters as the RXRα+/− and RXRα−/− embryos

(pure 129/Sv background).ND, not determined.

Table 3. Sacomeres and mitoses in vitamin A-deficient(VAD) and RAR mutant 9.5 dpc embryos

IndividualType of embryo Cells with Mitoticembryo number sarcomeres cells

Cultured WT embryosb

Control 1 16%a 10%2 20%a 11.5%3 30%a 11%

VAD 4 53% 6.5%5 65% 6%6 72% 5.5%

RARα mutantsc

RARα+/+ 7 14% 13.4%8 13% 13.8%

RARα+/− 9 12% 10.6%10 10% 8.7%

RARα−/− 11 90% 5.5%12 75% 6.5%13 62% 6.5%14 65% 5%

RARβ mutantsc

RARβ+/+ 15 16% ND16 18% ND

RARβ−/− 17 34% ND18 22% ND19 32% ND

RARγmutantsc

RARγ+/+ 20 0% ND21 9% ND

RARγ−/− 22 13% ND23 0% ND

aNot more than 2 consecutive Z lines were observed.bCD1 strain.cMixed 129/Sv/C57BL/6 genetic background.ND, not determined.

comeres, intercalated disks and sarcoplasmic reticulum (Ta2, and data not shown). Even though the thinness of the vtricular wall and the absence of trabeculae seen in these domutants may be related to their developmental retardation,elongated shapes and differentiated state of their myocytesno equivalent in WT hearts at earlier developmental sta(compare Fig. 2f with Fig. 1a). Some RXRα−/−/RXRβ−/−

mutants were developmentally less affected, of similar sizetheir littermates, and survived until 10.5 dpc. Morphologicalthe myocardia of the double mutants belonging to this seccategory were similar to those of RXRα−/− mutants (data notshown). Moreover, the percentage of SMs exhibiting differetiated features in these less affected mutants (examined at dpc) was also similar to that of RXRα−/− mutants (not shown).In any event, the severe abnormal cardiac phenotype seeseveral RXRα−/−/RXRβ−/− double mutants clearly underscorethe essential role of RXRs in preventing an early differentiatiof ventricular myocytes, and also indicates that RXRα andRXRβ are partially redundant. It is also noteworthy thwhenever a SM was differentiated, its degree of differentiat(as judged from myofibrillar organisation) was similar in singRXRα−/−, RXRβ−/− and RXRα−/−/RXRβ−/− mutants and never

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At 9.5 dpc, RXRα−/−/RXRβ+/− embryos were externallyindistinguishable from their RXRα−/− or WT littermates. Theirventricles were histologically indistinguishable from those oRXRα−/− mutants and the proportion of SMs exhibiting dif-ferentiated features was similar to that of RXRα−/− mutants(Table 2). Interestingly, one 9.5 dpc RXRα+/−/RXRβ−/− mutantdisplayed features that were essentially similar to those seenRXRα+/− or mildly affected RXRβ−/− embryos, in that isolatedsarcomeres were occasionally detected in approximately 30of the cells (Table 2). Thus, the combination of RXRα het-erozygocity and RXRβ homozygocity does not result in anaggravation of the cardiac phenotype associated with these twgenotypes (note that RXRα+/−/RXRβ−/− mutants are viable).

Reduced proliferation of subepicardial cardiacmyocytes in RXR mutants Cell proliferation in 9.5 dpc SMs was evaluated by countingcells in metaphase and anaphase (Table 2). In WT myocarda high proportion (about 16%) of SMs were engaged intmitosis. The fraction of cells in mitosis was markedly reducein RXRα−/−/RXRβ−/− mutants, as well as in RXRα−/− mutants.Interestingly, in the case of RXRβ−/− mutants, there was an

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. Differentiation gradient of the myocytes through the myocardial wallT fetus at 16.5 dpc. Brackets indicate sarcomeres, and the large arrow

s towards the lumen of the ventricle. C, capillary, E, epicardium, N,us, SM, subepicardial myocyte. Magnification: 5300×.

apparent correlation between a reduced mitotic index andpresence of organised myofibrils (Table 2; compare embr13 and 16 with embryos 14 and 15). These observatisuggest that a decrease in the rate of cell proliferation is aciated with the precocious differentiation of myocardial cein RXRα and β mutants.

Correct ventricular specification in RXRmutantsMorphological features of differentiation are seen inthe cells localised at the posterior part of the hearttube, the presumptive atrium, in 5- to 7-somitesembryos (our unpublished result). Thus, the questionarises as to whether the precocious differentiation ofRXR mutant ventricular cardiomyocytes could reflectan aberrant, atrial-like specification. In WT embryos,the myosin light chain 2v (MLC2v) exhibits a ven-tricular specificity from very early embryonic stages(e.g. 9.5 dpc, Kubalak et al., 1994). This gene wascorrectly expressed in the ventricles of 2RXRα−/−/RXRβ−/− double mutants (compare Fig. 5dand f; data not shown), demonstrating that ventricu-lar specification occurs correctly in these mutants. Wealso examined the expression of the MLC2a gene at14.5 dpc in RXRα−/− mutants. This gene, which isfirst expressed in both atria and ventricles, wasreported by Kubalak et al. (1994) to be specificallydown-regulated in ventricles by 11.5 dpc. We found,however, that MLC2a expression, even though higherin the atria, still persisted in WT ventricles at 14.5dpc. Moreover, we observed a consistently higherexpression in the left atrium when compared to theright atrium (Fig. 5a). The differential expression ofthis gene can therefore be used to assess ventricular,as well as left versus right atrium, specifications. Nodifference in the pattern of expression of this genecould be observed between RXRα−/− mutant and WThearts at 14.5 dpc (Fig. 5a,b), thus suggesting that theventricular and atrial identities have been correctlyspecified in these mutants.

Vitamin A deficiency and RAR α null mutationlead to an early differentiation ofsubepicardial myocytesFetuses from vitamin A deficient (VAD) rat damsexhibit a ‘spongy’ ventricular myocardium similar tothat of RXRa−/− mutants (Wilson and Warkany, 1949).To determine whether the correspondence between thetwo phenotypes also holds for the early differentiationand proliferation defects, vitamin A-deficient embryoswere generated by injection of antisense oligonu-cleotide for retinol binding protein into the yolk sac at7.5 dpc, followed by a 48-hour in vitro culture (Baviket al., 1996; control embryos were injected with thecomplementary sense oligonucleotide). Remarkably,the SMs of VAD embryos displayed the same advanceddifferentiation features of those of RXR mutantembryos (Fig. 6d and Table 3). In addition, the VADSMs were often elongated and loosely connected to oneanother, again resembling those of RXRα−/− andRXRα−/−/RXRβ−/− mutants (Fig. 6b). The mitotic

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index of the VAD subepicardial cells (6%) was also lower thain control embryos (11%; see Table 3). These observatiotherefore strongly support the idea that RXRs are involved the transduction of an early vitamin A function necessary tprevent a precocious differentiation of ventricular myocytes, t

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Fig. 4. Ultrastructural aspect of 9.5dpc WT and RXRβ−/− subepicardialmyocytes. Note the presence of welldeveloped myofibrils in the RXRβmutant and their absence in the WTmyocyte. Open arrows point awayfrom the myocardium. ID, intercalateddisk; N, nucleus; S, sarcomere.Magnification: 5700×.

maintain a high rate of cell proliferation and to control myocyshape and cohesiveness.

RXRs can mediate a vitamin A function either by exertina 9-cisRA-dependent transactivation, or by providing a heerodimeric partnership to a RAR (or both). As we had preously observed a spongy myocardium in some RARα/RARγdouble null mutants (Mendelsohn et al., 1994), RARs as was RXRs could be involved in mediating the function vitamin A in controlling the development of ventriculamyocytes. We thus examined SMs of 9.5 dpc RARα−/−,RARβ−/− and RARγ−/− mutants, as well as control littermates

Remarkably, all four RARα−/− mutants examined exhibited ahigh proportion of SMs containing sarcomeres (Table 3; nthat almost all the SMs of mutant no. 11 were differentiated).addition, the percentage of mitotic SMs was clearly reducedall RARα mutants when compared to their heterozygote or Wlittermates. However, abnormalities of cell shape and cohesness were not detected in these RARαmutants (data not shown).

About one third of RARβ null mutant SMs also exhibiteddifferentiated features, but this proportion was only twofohigher than in their WT littermates (Table 3). The ultrastruture of RARγ−/− ventricular myocytes was normal (Table 3Therefore, among RARs, RARα appears to play a major rolein the control of the differentiation and proliferation of ventricular myocytes.

DISCUSSION

Early function of retinoids in the control ofventricular myocyte differentiation and proliferationVitamin A deficiency (Wilson and Warkany, 1949), inactivation of both RARα and RARγ(Mendelsohn et al., 1994) andinactivation of RXRα (Sucov et al., 1994; Kastner et al.1994), were all previously shown to result in a hypoplasventricular wall. However, these studies were performedlate stages of heart development, and did not reveal the orof this defect. The present study demonstrates that ventrlar myocytes of RXR mutants (RXRα−/−, RXRβ−/− andRXRα−/−/RXRβ−/−), RAR mutants (RARα−/− and RARβ−/−)and VAD embryos all exhibit a premature differentiation o

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their ventricular myocytes, which can be visualized as eaas 8.5 dpc, as well as a reduced rate of proliferation. addition, RXRα−/−, RXRα−/−/RXRβ−/− and VAD ventricularmyocytes appeared elongated instead of globular and loosassociated. Vitamin A is therefore required from early stagof cardiogenesis to prevent differentiation, maintain a hirate of cell proliferation, and control cell shape and cohsiveness of ventricular myocytes. Furthermore, both RXand RARs are involved in the mediation of these retinofunctions, most probably acting as heterodimers (Kastneral., 1997).

Many cellular events are affected in the mutants (sarcomeintercalated disk or sarcoplasmic reticulum assembly, ccycle control, control of cell shape and control of cell-ceadhesion). As already pointed out, the precocious differetiation was always associated with a decreased mitotic indsuggesting that a common mechanism might regulate bdifferentiation and proliferation. On the other hand, abnormdifferentiation and proliferation were not always accompaniby cell shape abnormalities, as this latter defect was seen oin mutants lacking RXRα (or both RXRαand RXRβ) and notin RXRβ or RARα single mutants, even though these mutanwere often as affected as RXRα mutants in terms of prolifer-ation and differentiation. Thus, the retinoid control of ventriular myocyte differentiation and proliferation on the one hanand of cell shape and/or cell-cell association on the other omight occur via distinct mechanisms.

To what extent do these early abnormalities of RXR aRAR single mutants lead to cardiac defects at later stages? tologically, none of the RXRβ−/−, RARα−/− and RARβ−/−

mutant fetuses analysed at 14.5 dpc or 18.5 dpc exhibitespongy myocardium similar to that of RXRα−/− or VAD fetuses(our unpublished observations). Whether the reduced celluproliferation observed in these mutants at 9.5 dpc is subquently compensated for, or results in a decreased myocarmass at later stages, cannot easily be assessed from examtion of histological sections, because even normal heaexhibit variations in the apparent thickness of their compalayer, due to variations in their contraction status at the timefixation. In any event, these observations indicate that

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LC2a and MLC2v transcripts in RXR mutant hearts. (a,b) Expressionin WT and RXRα−/− hearts at 14.5 dpc. The colours indicate differentith blue, yellow and red corresponding respectively to low, intermediate

nal. Note the similarity of the expression pattern in WT and mutantpect to the low ventricular expression and the higher expression in left-f) expression of MLC2v at 9.5 dpc in WT and RXRα−/−/RXRβ−/−

field exposures; (d,f) the corresponding dark fields exposures. A, atrium;eft atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; V,n: 17× (a,b); 29× (c-f).

differentiation and proliferation defects alone (which are least as pronounced in RARα−/− as in RXRα−/− mutants) arenot sufficient to lead to a RXRα−/−-like spongy myocardium.This phenotype may thus result, at least in part, from the abnmalities in cell shape and cohesivness. However, as a sigcant fraction of RXRβ and RARαnull mutants die perinatally(Kastner et al., 1996; Lufkin et al., 1993), it will be of intereto investigate whether cardiac dysfunction may cause the deof some of these mutants.

Does the differentiation gradient across themyocardium involve a local production of RA inpericardial tissues? The observation that the inhibitory effect of RA on differentiation is manifested only in the most peripheral layers myocytes suggests that RA may be produced at the exteperiphery of the myocardium, possibly by the epicardium. This supported by the presence of transcripts for retinaldehdehydrogenase 2 (an enzyme which is possibly involved in synthesis; Zhao et al., 1996), in the pericardium and thabsence in the myocardium (Niederreither et al., 1997). addition, the strong expression of a LacZ transgene driventhe RA-responsive RARβ2 promoter in the compact layermyocytes and its weaker expression in the trabeculae is alsagreement with this idea (Zimmer et al., 1994; our unpublishresults). Together, these observations are consistent withnotion that a gradient of RA emanating from pericardial tissumight be invoved in establishing thegradient of myocyte differentiationaccross the myocardium. Note however,that the epicardium is unlikely to corre-spond to that putative RA source since(1) the epicardium is not formed at 8.5dpc (Viragh and Challice, 1981), a stagewhen RXRα−/− myocytes are already dif-ferentiated, and (2) the myocardialcompact layer is apparently normal inmice embryos lacking integrin α4, inwhich the epicardium is absent.

That mutant ventricular myocytesexpress early differentiated features maynot be too surprising, as WT myocyteslocated in the presumptive atrium andbulbus cordis are already morphologi-cally differentiated at 8.5 dpc (data notshown). Thus, the absence of differen-tiation in 8.5-dpc WT ventricularmyocytes, or in WT SMs at later devel-opmental stages, might require aretinoid-induced inhibition of themolecular machinery triggering theirdifferentiation. The RXR mutants showthe physiological importance of such aninhibitory mechanism, as a delayeddifferentiation may favor growth and thusallow the production of an adequate massof tissue. Could RA play a similar role inother systems that require a balancebetween proliferation and differen-tiation? The developing limb is a partic-ularly interesting candidate, since RA

Fig. 5. Expression of Mof MLC2a transcripts intensities of signal, wand high levels of sigfetuses, both with resversus right atrium. (cembryos. (c,e) BrightC, conotruncus; LA, lventricle. Magnificatio

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may prevent limb blastema cells from being engaged intochondrogenic program (Jiang et al., 1995; Paulsen and Solu1988). Along the same vein, a gradient of RA is also thougto regulate proliferation and differentiation in the skin (seDarmon, 1991, for a review).

Do RXR mutant ventricular cells adopt an atrialphenotype? Since WT atrial myocytes differentiate at early developmenstages, the precocious differentiation of RXR mutant vetricular myocytes might reflect an abnormal atrial specificatioas suggested by Dyson et al. (1995); these authors reporte13.5 dpc RXRα−/− mutants, an abnormal persistence of vetricular expression of the MLC2a gene, which is highexpressed in atrial myocytes. Conversely, atrial/ventricuspecifications may occur correctly, but the ventricular-specidifferentiation program per se may be perturbed. Our dsupport this second possibility since: (1) the mutant ventriclar cells never reached the degree of myofibril organisatidisplayed by atrial cells, and are therefore qualitativedifferent from atrial cells; (2) the ventricular-specific markeMLC2v is correctly expressed in ventricular myocytes oRXRα−/−/RXRβ−/− double mutants; (3) the differentialexpression of MLC2a between the atrium and ventricle normal in our WT and RXRα mutant 14.5 dpc fetuses. Thediscrepancy between our observation and that of Dyson et(1995) could be due to the very weak ISH signals detected

4757Retinoid receptors and cardiomyocyte differentiation

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Fig. 6. Effect of vitamin A deficiency (VAD) on the myocardial wall at 9.5 dpc. Semi-thin section through the myocardial wall (a,b) andultrastructural aspect of subepicardial myocytes (c,d) of control and VAD embryos. E, epicardium; F, myofilament; MI, mitochondria; N,nucleus; S, sarcomere; SM, subepicardial myocyte; Z, Z-line. Magnification: 500× (a,b); 17000×(c-d).

their WT sample, which could have prevented the detectiona low level of ventricular expression. In fact, their RXRα−/−

MLC2a expression pattern is very similar to ours, in thatclearly exhibits a weaker ventricular than atrial expressiTaken together, our observations do not support the possibthat the RXR mutant ventricles could have adopted an atidentity, and lead to the conclusion that RXRs are acting onrealisation of a ventricular-specific differentiation program.

Specific or redundant functions for distinct retinoidreceptors in the developing myocardium? The present results indicate that at least four distinct retinreceptors (RXRα, RXRβ, RARα and RARβ) are apparentlyinvolved in controling the development of the ventriculmyocardium. This raises questions about functional specificand redundancy of these receptors.

RXRα clearly has a key role in myocardial development, sinamong the single mutants analysed, RXRα−/− mutants most con-sistently reproduced the VAD phenotype. In particular, on

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RXRα, but not RXRβ, RARα or RARβ, seems to play a majorrole in determining cell shape and cell-cell adhesion propertof cardiomyocytes. In addition, in RXRβ and RARβmutant SMs,the penetrance at the cellular level of the precocious differetiation phenotype was often weak. The crucial role of RXRα incardiac development is also underscored by the presencecardiac abnormalities in heterozygote mice: RXRα+/− embryosdisplay a milder form of the early differentiation phenotype, anadult RXRα+/− mice exhibit an abnormal ventricular morphology(Gruber et al., 1996). Whether this prominence of RXRα reflectsunique properties of this receptor, or merely results from a quatitative predominance, remains to be investigated.

The high percentage of myocytes displaying the abnormdifferentiation features in RXRα−/−/RXRβ−/− mutants demon-strates that, with respect to preventing an early differentiatioRXRβ and RXRαexert similar functions. A threshold level ofRXRs in ventricular myocytes might be required for their normdifferentiation-inhibitory function, and the increase in the proportion of affected cells in double mutants probably reflects th

4758

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P. Kastner and others

requirement. The incomplete expressivity, at the cellular level,the precocious differentiation seen in RXRα and RXRβ singlenull mutants probably indicates stochastic variations, either in levels of RXR protein present in given cells, or in the thresholevel of RXR protein required for a correct function (or both).

Knock-out experiments have shown that the function several gene products is critical for myocardial morphogeesis, including the transcription factors N-myc (Moens et a1993), TEF-1(Chen et al., 1994), Nkx2.5 (Lyons et al., 1995WT-1 (Kreidberg et al., 1993), as well as the secreted facneuregulin and its receptor erbB4 (Meyer and Birchmeie1995; Gassmann et al., 1995). It should be interesting to invtigate whether absence of these genes similarly perturbs differentiation programme of the myocytes and if the pathwaaffected in these mutants intersect with that of retinoic acid

We thank K. Chien for the gift of the MLC2v plasmid, J.-L. Vonescfor computer processing of the MLC2a in situ hybridisation data, Bronn, B. Schubaur, B. Bondeau and L. Auvray for technical assistanand the photography, illustration and secretarial staff for help in tpreparation of this manuscript. This work was supported by funds frthe Centre National de la Recherche Scientifique (CNRS), the InstNational de la Santé et de la Recherche Médicale (INSERM), Collège de France, the Centre Hospitalier Universitaire Régional, Association pour la Recherche sur la Cancer (ARC), the Human FronScience Program and Bristol-Myers Squibb. J.M.G. was a recipient ofellowship from the EEC (TMR Program) and the Fondation pour Recherche Médicale, respectively; S.W. was a recipient of a fellowsfrom the EMBO and the EEC (TMR Program), respectively.

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(Accepted 24 September 1997)