THE J O U R N A L OF BIOLOGICAL CHEMISTRY IC! 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 266. No. 33, Issue of November 25, pp. 22419-22426.1991 Printed in U. S . A.

Myosin Heavy Chain Gene Expression in Mouse Embryoid Bodies AN IN VITRO DEVELOPMENTAL STUDY*

(Received for publication, July 3, 1991)

Alejandro Sanchez$, W. Keith Jones$, James Gulick$, Thomas DoetschmanQ, and Jeffrey Robbins$T From the Departments of $Pharmacology and Cell Biophysics and §Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575

Embryoid bodies (EBs) are obtained when mouse pluripotential embryonic stem cells are grown in the absence of an embryonic fibroblast feeder layer. Seven- to 9-day-old EBs undergo rhythmical, sponta- neous contractions and express the appropriate tissue- and developmental stage-specific cardiac and skeletal myosin heavy chain (MHC) genes (18). To study the expression patterns of these MHC genes in vitro we isolated and partially sequenced the cDNAs expressed in EBs such that specific oligonucleotides suitable for polymerase chain reaction analyses and appropriate riboprobes for in situ hybridizations could be made. The data show the &cardiac gene is expressed first during EB development (days 3 and 4), and a-cardiac gene expression begins at -day 8. A similar pattern of expression is also detected during mouse embryogene- sis in utero. Only those EBs that expressed both the a- and &cardiac transcripts contracted. In situ hybridi- zation of EBs using riboprobes shows that the spatial distribution of the cardiac MHC transcripts differs. No expression of the genes was detected in day 8 or older nonbeating EBs. These data suggest that developing EBs closely mimic the pre- and early postsomitic pat- terns of in vivo expression of the cardiac MHC genes and thus provide a useful system in which to study early aspects of mammalian cardiogenesis.

During murine development, cardiogenesis takes place be- fore other organogenic events. Morphological studies of mouse embryonic development (1, 2) have shown that the cardiac compartment arises in a manner similar to that of other vertebrates (3). Generally, vertebrate cardiogenesis occurs early during gestation and involves a paired area of the lateral mesoderm (4) near the cephalic end of the embryo. The differentiation of this region gives rise to two heart tubes (3) usually referred to as the heart primordia or heart anlage.

In the mouse the cellular strands (2) of lateral mesoderm involved in the generation of the heart rudiment are detected first at 7.5 days postcoitum ventral to the foregut furrow, which itself arises as an entodermal indentation as early as

* This work was supported by National Institutes of Health Grants HD13225, HL41496, and HL22619 and by a grant from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this DaDer has been submitted to the GenBankTM)EMBL Data Bank with accession number(s) M74751, M74752, and M74753.

(I Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 513-558-2318; Fax: 613-558-1169.

day 6 postcoitum (1, 2). By day 8 postcoitum, thickening of the mesodermal cells results in the formation of the epimy- ocardium (5). Unattached mesenchymal cells located between the thickened mesoderm and the endodermal floor begin to differentiate into two bilaterally opposed endocardial tubes (1). These bilateral primordia come together at the midline axis of the embryo, first by the migration and fusion of the epimyocardial crescents and soon after by fusion of the en- docardial tubes (5, 6) . The result is a single cardiac “tube within a tube” of epimyocardium and endocardium separated by an extracellular matrix known as the cardiac jelly. At this stage, irregular but spontaneous contractions of the tube are observed, and by day 9 postcoitum blood flow through the heart tube occurs.

Several studies dealing with chick embryological develop- ment indicate that the expression of muscle-specific genes does not necessarily coincide with the morphologically visible, early stages of cardiogenesis. 5-Bromodeoxyuridine is known to induce the loss of phenotypic traits in differentiating cells (7,8). In myoblasts this nucleotide analog suppresses myotube development, presumably by inhibiting the synthesis of the transcripts which encode muscle specific proteins (9, 10). 5- Bromodeoxyuridine treatment of isolated, precardiac meso- dermal cells did not affect the synthesis of cardiac muscle proteins or the beating of these cells when allowed to differ- entiate i n vitro (11). Furthermore, polyclonal antibodies against myosin (12) and troponin (13) demonstrated the pres- ence of these proteins in stage 3 chick embryos (14). This corresponds to approximately day 7 postcoitum in the mouse, a stage at which only the most primitive heart rudiment is present. Immunofluorescence surveys of nonstriated chick embryonic cardiomyocytes (15) have shown that these cells contain the proteins that will eventually comprise both thin and thick sarcomeric filaments. All of these observations taken together indicate that muscle-specific genes may be activated prior to the overt differentiation of mesodermal cells into the heart primordia, i.e. before the appearance of striated muscle.

In the murine system mostly postsomitic stages of cardi- ogenesis have been studied. I n situ hybridization analyses of sectioned mouse embryos revealed similar signals for both cardiac myosin heavy chain transcripts in the cardiac com- partment as early as day 8 postcoitum, approximately 12 h after somites are first detected (16). However, the expression of muscle-specific genes during the very early, presomitic developmental stages of the mammalian embryo has not been examined.

The definition of a suitable mammalian system in which early developmental patterns of cardiac-specific gene expres- sion could be easily studied and, perhaps, manipulated i n vitro would be of considerable value. When blastocyst-derived

22419

22420 Mouse Cardiac Myosin Gene Expression

mouse ES' cells are allowed to differentiate in vitro under appropriate conditions, they develop into EBs (17). EBs ex- hibit localized regions of spontaneous contractions by day 8 of in vitro differentiation. Morphologically, visceral yolk sacs as well as blood islets have been noted (17). EBs express the appropriate developmental stage-specific cardiac and skeletal MHC transcripts (18), and similar results have been obtained for a-tropomyosin (19). Lindenbaum and Grosveld (20) have shown that EBs are capable of expressing, in the correct temporal order, the full complement of mouse embryonic globin genes.

To define further the EB as a system for the study of certain aspects of cardiogenesis i n vitro, we have undertaken an analysis of muscle-specific gene expression using the po- lymerase chain reaction (PCR). The expression of the cardiac and embryonic skeletal MHC genes during the in vitro differ- entiation of EBs and during early i n vivo embryogenesis was analyzed. We report here that ES cell-derived EBs recapitu- late the pre- and early postsomitic patterns of expression of the murine a-cardiac, P-cardiac, and embryonic skeletal MHC genes.

EXPERIMENTAL PROCEDURES

ES and EB Cultures-EBs were derived from the ES cell line ES- D3, which is derived from the inner cell mass of a 129/Sv+/+ blastocyst (17). The ES cell line was maintained undifferentiated by culturing them in the presence of 14-16-day BALB/c primary embry- onic fibroblasts that had been treated with 10 pg/ml mitomycin C for 1.5 h (17, 18). EBs were obtained by treating the ES cells and embryonic fibroblast feeder layer with 0.05% trypsin type XI (Sigma) and placing them in 100-mm plastic bacterial Petri dishes (Fisher Scientific) for 1 h to allow fibroblast attachment. The unattached ES cells were then placed in sterile 100-mm plastic bacterial Petri dishes (Fisher Scientific) containing 10 ml of Dulbecco's modified Eagle's medium (GIBCO), 15% heat-inactivated fetal calf serum (Flow Lab- oratories, Inc., McLean, VA), and 0.1 mM 2-mercaptoethanol (Sigma). Thyroxine levels in the serum were 17 ng/dl, as determined by radioimmunoassay. The medium was changed every other day, and on day 4 the serum was raised to 20%. On the odd days when the medium was not changed, 5 ml of additional medium was added (17, 18).

Analyses of EBs and Mouse Embryos Using PCR-Three sets of primers specific for the a- and 8-cardiac and embryonic skeletal transcripts were designed such that they would be suitable for both PCR and cDNA synthesis (18). al, 5"CTGCTGGAGAGGT- TATTCCTCG-3'; L Y ~ , 5"GGAAGAGTGAGCGGCGCATCAAGG-3'; 81, 5"TGCAAAGGCTCCAGGTCTGAGGGC-3'; 82, 5"GCCAA- CACCAACCTGTCCAAGTTC-3'; E.Skel.,, 5"GCATGTGGAAAA- GTGATACGTGG-3'; E.Skel.2, 5"GCAAAGACCCGTGACTTCA- CCTCTAG-3'.

The oligonucleotides complementary to the cardiac and embryonic skeletal MHC 3"untranslated regions (UTRs) (a', PI, and E.Skel.l) were used to direct the synthesis of cDNA. Total EB RNA used for this reaction was obtained by boiling a single EB in 12 p1 of H 2 0 for 5 min and then dividing this volume into three 4-pl aliquots. Total RNA from strain FVB/N embryos was prepared using RNazol (Cinna Biotecx, Friendswood, TX). Oligonucleotide annealing to the RNAs was carried out by heating the samples to 90 "C in the presence of 20 pmol of the corresponding primer. The reaction was cooled slowly to 24 "C and brought to 10 pl in the presence of 1 X PCR buffer (10 mM Tris-HC1, pH 7.5; 50 mM KC1; 1.5 mM MgC1.J; 1 mM each dATP, dCTP, dGTP, dTTP; and 6 units of avian myeloblastosis virus reverse transcriptase (Kodak-International Biotechnologies, Inc., New Ha- ven, CT). No product was observed if the enzyme was omitted from the reaction (data not shown). The cDNA synthesis was allowed to proceed a t 42 "C for 45 min. After bringing the volume of the sample to 50 p1 in 1 X PCR buffer containing 20 pmol of the appropriate primers, 2.5 units of AmpliTaq (U. S. Biochemical Corp.) were added.

The abbreviations used are: ES, embryonic stem; MHC, myosin heavy chain; EB, embryoid body; E.Ske1, embryonic skeletal; LMM, light meromysin; PCR, polymerase chain reaction; UTR, untrans- lated region; hp, base pair(s).

Under these conditions, saturation was not observed until >42 cycles were performed. Mineral oil was used to overlay the reactions and an Ericomp temperature cycler (Ericomp, San Diego, CA) was used to amplify the cDNAs. The cycling sequence consisted of an initial denaturation for 3 min at 94 "C, followed by 30 cycles of 1 min a t 94 "C, 30 s a t 65 "C, and 30 s a t 72 "C. A final 10-min extension a t 72 "C was done. Ten p1 of the resulting samples were analyzed by electrophoresis in 2% agarose gels.

Preparation of E B Sections for in Situ Analyses-After 10 days in culture, beating EBs were easily identified by brief (1-3 min) micro- scopic examination, since they exhibited consistent spontaneous con- tractions of about 30 beats/min. Nonbeaters were carefully observed for a slightly longer period of time (2-4 min) to ensure that weak or slow beaters were not included. PCR results confirmed the reproduc- ible separation of beaters and nonbeaters using this procedure (see data below). Once segregated, the EBs were washed separately several times in diethylpyrocarbonate (Sigma)-treated phosphate-buffered saline. Fixation was carried out using a chilled solution of 4% para- formaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in RNAse-free phosphate-buffered saline and allowed to proceed for 16 h on ice. The fixative was removed by several washes with chilled phosphate-buffered saline, and the EBs were cryoprotected by resus- pension in cold, RNAse-free 20% sucrose for 24-36 h on ice. After removal of the sucrose solution, the EBs were gently resuspended in Tissue-Tek OCT embedding compound (Miles, Elkhart, IN), placed on dry ice until the compound hardened, and stored a t -70 "C. Seven- to 8-pm-thick serial sections were cut using a Leitz 1720 digital cryostat and a 185-mm steel blade (Lipshaw, Detroit, MI). Four to six EB sections were mounted onto 1,3-aminopropyltriethoxysilane (Sigma)-coated slides and allowed to dry for a t least 2 h a t room temperature. The sections were fixed again in 4% paraformaldehyde, postfixed, treated with triethanolamine/acetic anhydride, washed, and dehydrated through a graded series of ethanol solutions (23). a- and @-Cardiac MHC Riboprobe Construction and Synthesis-

Double-stranded DNA fragments homologous to the 3'-UTRs of the a-cardiac and 8-cardiac MHC transcripts were amplified by PCR and cloned into the multiple cloning regions of pIBI30 and pIBI31 (Ko- dak-IBI). The a-cardiac MHC-specific region was 107 bp, spanning nucleotides 5762-5869 (Fig. 1, boxed) The 8-cardiac MHC-specific region was 67 bp, spanning nucleotides 5816-5883 (Fig. 1, boxed). To generate the riboprobes, 1 pg of plasmid DNA was linearized with either Sal1 or EcoRI. Both sense and antisense cRNA probes were then synthesized using a-35SS-labeled UTP (1,500 Ci/mmol; Du Pont- New England Nuclear) and 10-15 units of T3 RNA polymerase (Kodak-IBI) essentially as described by Melton et al. (21). Reactions were incubated for 2 h a t 40 "C. The cRNA was separated from unincorporated radioactive nucleotides by centrifugation through a 0.5-ml Sephadex G-25 (Pharmacia LKB Biotechnology Inc.) column that had been equilibrated with 25 mg of yeast tRNA in 0.5 ml of 10 mM Tris-HC1, pH 7.5; 5 mM EDTA; 0.1% sodium dodecyl sulfate; 10 mM dithiothreitol. The cRNA was isolated from the eluate using RNAzol. The volumes were adjusted to give a probe concentration of 1 X lo6 cpm/pl.

Hybridization and Washing of E B Sections-The hybridization and posthybridization procedures were as described previously (22, 23) with modifications. Briefly, sections were hybridized overnight a t 55 "C in 50% ultrapure formamide (Electron Microscopy Sciences), 4 x SSC (1 x SSC is 0.15 M NaC1, 0.015 M sodium citrate solution), 10% dextran sulfate, 5 X Denhardt's, 100 pg/ml denatured salmon sperm DNA, 100 pg/ml yeast tRNA, 10 mM dithiothreitol, and 1 X lo5 cpm/pl of the [a-35S]UTP-labeled cRNA probe. The sections were then washed twice at 65 "C for 30 min (24) followed by two 15-min washes in 2 X SSC and a final wash in 0.1 X SSC (24 "C for the a- cardiac MHC riboprobe and 50 "C for the 8-cardiac MHC riboprobe). The slides were dehydrated, dipped in Ilford K5 emulsion (Electron Microscopy Sciences), and exposed for 5-20 days in light-tight boxes with desiccant a t 4 "C. Kodak Dl9 developer was used to process the slides, and the sections were analyzed using the phase-contrast and dark-field optics of an Olympus BHTU microscope.

RESULTS

Sequence Analysis of the Light Meromyosin (LMM) and 3'- UTRs of the a- and p-Cardiac and Embryonic Skeletal MHCs-We wished to examine transcript accumulation of the cardiac and embryonic skeletal MHCs during develop- ment. However, only limited sequence data for the cardiac

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and embryonic skeletal murine MHC isoforms exist (25). Therefore, to facilitate the design of the appropriate tran- script-specific probes we screened a cDNA library prepared from 12-14-day mouse EBs and isolated the two cardiac and the embryonic skeletal cDNAs (18). The nucleotide sequences encompassing the LMMs of the three isoforms were then determined (Fig. 1). Previous analyses have shown that the rod of the protein is the most divergent domain between the MHC isoforms. Yet, the conservation between the mouse a- and P-cardiac isoforms is striking, with >94% homology at the nucleotide level. The 0-cardiac cDNA is 92 and 95%

homologous to the human (26) and rat (27) 0-cardiac LMM sequences, respectively, whereas the mouse a-cardiac LMM cDNA shows 90% homology with the human (28) and 95% homology with the rat a-cardiac (29) sequences. The sequenc- ing of multiple cDNA clones isolated from the library indi- cates that alternative polyadenylation sites exist in the a- cardiac transcript (Fig. 1). Alternative polyadenylation of the a-cardiac transcript has been observed previously in the rat (30), but its significance, if any, remains unknown.

As expected, a comparison of the embryonic skeletal cDNA sequence with the a- and P-cardiac MHC cDNAs showed less

22422 Mouse Card iac Myos in Gene Expression

conservation in the LMM region (-77% homology). However, when the murine embryonic skeletal MHC cDNA sequence is compared with t.he human (31) and rat (32) orthologous sequences, the homologies in the LMM region are rather high (>90 and 95% respectively). When the embryonic skeletal sequence is compared wit,h ot,her skeletal MHCs such as mouse perinatal (33), some homology in t,he LMM encoding region is also detected (-75%). However, in the mouse (18) as in other mammals (28), the 3’-UTR of the (r- and &cardiac and embryonic skeletal MHC t.ranscripts were divergent (less than 46% homology). Because of the high degree of conser- vation of the rod-encoding regions in the (r- and +cardiac MHC cDNAs, as well as the embryonic skeletal’s rod to other MHCs, it became apparent in preliminary experiments that the design of transcript-specific probes had to be restricted to the divergent 3”UTRs. Fig. 1 shows the nucleotides selected for the synthesis of PCR-competent oligonucleotides and the regions utilized for the construction of t.he cardiac riboprobes to be used for the in si tu analyses (see below).

Myosin Transcript Amplification from Individual ERs-We showed previously that the only striated MHC transcripts expressed in EBs were the cardiac and embryonic skeletal RNAs and that their co-expression could he detected in a single EB using PCR (18). To compare expression hetween different EBs a t different developmental time points, PCR amplification of the three MHC transcripts was defined fur- ther. The methodology employed to prepare the ERs for PCR analysis does not involve the separation of DNA from RNA (see “Experimental Procedures”). As EBs are composed of several thousands of cells, it was necessary to demonstrate that the amplified fragments were originating from mRNA and not from genomic DNA. PCR products resulting from amplification of the ru-cardiac (302 bp), 8-cardiac (205 bp), and embryonic skeletal (151 bp) transcripts were defined previously (18). To confirm that the primer pairs would allow us to distinguish between genomic DNA and mRNA amplifi- cations, PCR was carried out in the presence of either the RNAs or the cognate genomic sequences (Fig. 2). The frag- ments obtained for the a- and @-cardiac gene amplifications

using a genomic clone (34) as template (Fig. 2, lanes 2 and . I ) were -1,500 and -800 bp, respectively. When 1 pg of total mouse genomic DNA was amplified using the embryonic skeletal PCR oligomers (an embryonic skeletal MHC genomic clone was not available) a fragment of -600 bp was observed (Fig. 2, lane 6) . The size differences between the amplifica- tions resulting from EB mRNA and the genomic clone or DNA are presumably a result, in all three cases, of introns positioned between the 5‘ and 3’ oligonucleotides used. The size differences allowed us to discriminate easily between fragments amplified from DNA or mRNA and thus confirm that the 302 (tu)-, 205 (@)-, and 151 (embryonic skeletal)-bp fragments (Fig. 2, lanes I, 3 , and 5, respectively) are diagnostic for the ru- and +cardiac and embryonic skeletal MHC tran- scripts, respectively.

To determine the relative efficiencies of the cardiac tran- script amplification reactions, we amplified an RNA sample in which the relative amounts of the (r- and [j-cardiac MHC transcripts were known. A nucleic acid blot of poly(A)+ RNA derived from day 14 EBs demonstrated that the (1- and /f- cardiac MHC transcript, levels were roughly equivalent in this RNA population (Fig. 3a). This RNA was subsequently di- luted and analyzed by PCR with the appropriate isoform- specific oligonucleotides (Fig. 3h). The data indicate that the amplification efficiencies for both transcripts are similar. Therefore, a failure to detect. either the (1- or /j-cardiac tran- script in a particular sample is more likely because of its absence or low abundance rather than because of large intrin- sic differences in the amplification efficiencies.

a- and @-Cardiac MHC Transcripts Arr Iliffprpntially E x - pressed during ER Ileuelopment-To delineate the in Litre expression patterns of the cardiac and embryonic skeletal MHC genes, ES cells were removed from the fibroblast feeder

a P M 1 2 3 M 1 2 3

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FIG. 2. Myosin transcript amplification from ERs and ge- nomic DNA. A single RH was used for each o f the three isomvosin- specific oligomer sets (see “Experimental I’rocedures“). 1,ancs 1. : I , and 5 show the resulting PCK prodrlrts for the tu-cardiac (302 hp), [j- cardiac (205 hp), and emhryonic skeletal (151 hp) transcript-specific amplifications. respectively. Lnncs 2 and 4 show the resulting I’CH products when a cosmid clone (MHC Cos- I ) containing the complete cr-cardiac MHC gene and the 3’ end o f the +cardiac gene (44) was amplified. I,nnc 6 shows the I’CR fragment(s) ohtained when 1 pg of total mouse genomic DNA WAS used. M is Hnclll-digested @X174 D N A used as a marker.

FIG. 3. Amplification efficiencies of the a- and B-cardiac MHC PCRs. I’ond n shows the blot analvsis o f differentiated E13 RNA. Eight pg o f poly(A)’ RNA was applied. and 2-fnld serial dilutions were made lor the subsequent dots. [1-”I’IATI’ ( K O f M ) ( ’ i / mmol; Du l’ont-New England Nurlear~-end-laheled ( I - and ,j-rardiar- specific oligonucleotides were used to quantitate relative trrlnsrript levels as descrihed previouslv (18). Ilrnsitometric rlnnlysis o f the autoradiogram showed an c x - to +cartliar transcript ratio o f 1:O.H. In pond h two aliquots of the same RNA sample used nhove were diluted t o 1 pg (Inn(. I ), 1 ng (Inn<, 2) , and 0.5 ng (Innr. :{) and su1)rnitted t o 30 cycles of amplification as tlescrihed under “Kxperinwntnl I’rore- dures.” M is Ho~III-digested rbS174 IINA.

Mouse Cardiac Myosin Gene Expression 22.123

layer and allowed to differentiate in oitro for 1-15 days (see “Experimental Procedures”). Typically, by day 8 of develop- ment in suspension culture, foci exhihiting spontaneous con- tractions were first detected, and by day 12 about 40% of the EBs were beating. A total of 10-12 ERs/developmental time point were collected from three different initial platings, and each EB to be analyzed by PCR was photomicrographed (Fig. 4). Although we noted a significant increase in size and appearance as the EBs differentiat,ed into cystic bodies (panels I-!?), we were unable to predict, on the basis of morphology, whether an EB would become a “heater” or “nonbeater.” Because of this, the contractile hehavior of each was noted for subsequent correlation wit,h MHC transcript levels.

To determine the expression patterns of the a- and (3- cardiac and embryonic skeletal MHC genes during the in vitro developmental process, individual EBs were submitted to PCR analysis as descrihed (see “Experimental Procedures”). Representative, single ER PCR reactions for each time point are shown (Fig. 5 ) . We demonstrated previously that undif- ferentiated ES cells do not express the cardiac or embryonic skeletal MHC transcripts (18), and these data are consistent (Fig. 5, day 1 ). Somewhat surprisingly, we noted detectahle levels of the $-cardiac MHC transcript on day 3 in approxi-

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DAY 10 DAY 15 FIG. 4. Photomicrographic record of ER development in

vitro. ERs were allowed to differentiate and were maintained in oitro as descrihed under “Experiment,al Procedures.” EHs collected for I’CH analysis were photographed individually using a phase-contrast microscope with t.ype 52 (ASA 400) Polaroid film. The numbrr on each ponrl corresponds to the day of in vitro development. Days 1-8 show ERs enlarged 50 x. EHs from days 9, 10. and 15 are magnilied 25 X. Days 8 and If EHs exhihited spontaneous Contractions. Days 1 0

DAY- 1 2 3 4 5 6 7

FIG. 5. I ’ C H analysis o f K I 3 development in oitro. ‘I’tw v x - pected positions 01 the ( r - antl ,!.cardiac antl rn l l~rym~c skrletal l’(*l< products ( a , If, and h’. respectivelv) are shuwn o n the /#,/I. l ~ n c M . HnrIII-digested (/,X174 DNA. h n c s ( 1 , if, and S. I Y ‘ R products resulting from amplification using the (r-cardiac. Af-cartlinc. antl ern- hryonic skeletal oligonucleotide primers, respect i\fely. \Yhenevc.r the expression of the three M H C isoforms differed hetween single F:Hs from a particular time point, characteristic 1V.R results nf t w h are shown (dny J , ij- and non-rf-expressing EHs: dn\s H- I f ) alnd /A, twnters (+) and nonheaters I - ) .

mately 50% of the ERs analyzed (Fig. 5; see Fig. 8) . All of the ERs examined on day 4 expressed the &cardiac MHC tran- script, and we were unable to detect expression of either the tu-cardiac or embryonic skeletal MHC genes at th i s time (Fig. 5, days 3 and 4 ) . No contractions were observed during days 3 and 4 in any of the ERs, and previous detailed microscopic analyses revealed no structures characteristic of muscle (17).

The early expression of the /+transcript is transient, and from days 5-7 of development, 24/30 of the ERR studied did not. show detectahle levels of any of the MHC transcripts. Rv day 8, when a small percentage (1-5X) of the ERs begins to contract, the expression of hoth the 0- and +cardiac tran- scripts can be detected in beating ERs hut not in nonheating EBs (Fig. 5 ) . I t is interesting that we never detected in any of the ERs, at any stage of development, the expression of only the n-cardiac transcript. Detection of a-cardiac mRNA was restricted solely to those ERs expressing the (j-cardiac tran- script. We ohserved spontaneous contractions only in those EBs that expressed both cardiac MHC genes. The expression of the embryonic skeletal gene was not apparent during most of the in vitro developmental period. Occasionally it was detected in 15 day or older nonheating ERR (Fig. 5).

MHC Transcripts Arc Kxprmsed during thc Early Stagm of in Utero Embryogenesis-The precocious expression of the Lj-

cardiac transcript during EH development was striking. Therefore, it was of interest to estahlish if ER development in vitro was aberrant or mimicked transcriptional events in utero. Thus, PCR analyses were extended to the early mouse emhryo. Because of the difficulty involved in dissecting very early emhryos (days 5 and 7.5 postcoitum) it was possihle that trace uterine contaminants were present in the emhryonic RNA prepared. To eliminate the possihilitv that any PCR products detected arose from uterine-derived material, the ( 1 -

and &cardiac and embryonic skeletal primer sets were tested against total RNA derived from the uterus of a nonpregnant

and 15 depict hoth “heaters” (+) and “nonheaters” (-). animal. Extensive PCR amplification failed to ditect the

22424 Mouse Cardiac Myosin Gene Expression

Day p. c.+ 3 5 7.5 9.5 nn nn a p S a p S M a P S a P S

a - P - S“

m FIG. 6. PCR analysis of early mouse embryos. Uteri contain-

ing preimplantation (3-day postcoifurn ( p . c . ) ) and postimplantation embryos (,5, 7.5, and 9..5 dqvs postcoifurn) were isolated and 1 pg of total RNA was analyzed by PCR (see “Experimental Procedures”) using the n-cardiac (lanr ( x ) , /j-cardiac (lane 8 ) . and emhryonic skeletal (lane S) primers. The expected positions of the PCR products are shown on the lrff ( n , [j, and S , respectively). M, HarIII-digested +X174 DNA.

presence of the MHC isoform transcripts although the non- muscle a-tropomyosin transcript could be easily amplified using the same RNA sample.2 Thus, any MHC-PCR products detected were not the result of uterine wall contamination.

Both pre- and postimplantation embryos were analyzed. The expression pattern for the cardiac MHC genes (Fig. 6) was similar to that observed in the EB in vitro system (Fig. 5). At the preimplantation stage of the embryo (blastocyst) none of the three MHC isoforms was detected (Fig. 6, day 3 postcoitum). After implantation the @-cardiac MHC transcript was detected (Fig. 6, day 5 postcoitum); somites are first observed a t -day 7.5 postcoitum. In the embryo we were able to detect the a-cardiac MHC mRNA a t a developmental stage (Fig. 6, day 7.5 postcoitum) a t which the cardiac tube is beginning to form (5). Once the cardiac tube has begun to contract (day 9.5 postcoitum), the apparent levels of the PCR products obtained from the amplification of the a- and p- cardiac MHC transcripts appear to be similar (Fig. 6, day 9.5 postcoitum). It has been shown previously by RNA dot blot analysis that the a- and and P-cardiac MHC transcript levels are roughly equivalent in the day 10 postcoitum mouse heart (35). The PCR data obtained demonstrate that the early expression of the @-cardiac MHC occurs both in vitro and in vivo.

We also noted (Fig. 6, day 5 postcoitum) the presence of a DNA fragment that is the size of the @-cardiac MHC DNA amplified product (800 bp; Fig. 2, lane 4 ) . It is likely this fragment results from the amplification of a relatively large pool of unprocessed RNA rather than from DNA contami- nation of the RNA sample, since DNA contamination should have resulted in the corresponding PCR products in the a- cardiac and embryonic skeletal MHC reactions as well, and these were not observed.

In Situ Hybridization Analysis of Beating and Nonbeating EBs-To discern the spatial transcriptional patterns, slides containing adjacent sections derived from day 10 EBs were probed with both sense and antisense a- and &cardiac MHC riboprobes and examined by dark-field microscopy. No hy- bridization was detected in either beating or nonbeating EBs when the sense riboprobes were used (data not shown). All of the beating EBs examined (6/6) were found to have localized regions that hybridized to the antisense a-cardiac MHC ri-

L. Pajak, personal communication.

boprobe (Fig. 7, panels a, b, and c ) . Nonbeating EBs present on the same slide showed no detectable a-cardiac MHC- specific hybridization (Fig. 7, panel e ) . The pattern of hybrid- ization indicates that a-cardiac MHC transcription is re- stricted to a subset of the cells within the beating EB. In each case, regions of a-cardiac MHC transcription in adjacent or nearby sections were related by position, suggesting a three- dimensional organization of these cells within the EB (de- picted schematically in Fig. 7, inset) consistent with previous data which localized cardiac muscle to beating foci (17, 18).

In contrast to the localized expression of the n-cardiac MHC, sections probed with the antisense &cardiac MHC riboprobe revealed a diffuse hybridization pattern in the beat- ing EBs (Fig. 7, panel d ) . As indicated (Fig. 7, inset) the section analyzed with the @-riboprobe is sandwiched between two sections that show regionally well defined signals when hybridized to the a-cardiac probe. The P-cardiac MHC signal in this section is not confined only to those areas in which the a-cardiac transcript is detected but also to areas in which no significant hybridization to the cY-cardiac probe occurred.

The hybridization signals for the cardiac riboprobes in nonbeating EBs were not significantly above background (Fig. 7, panel e, a-cardiac; panel f, @-cardiac). These results are consistent with the observation that PCR analyses of day 8 or older nonbeating EBs failed to detect the expression of the cardiac MHC genes.

DISCUSSION

A sequence analysis of the mRNAs encoding the three MHC isoforms expressed in EBs was undertaken to provide a ra- tionale for the design of specific probes that would be able to detect low levels of the cognate transcripts. However, the extensive homologies between the two cardiac LMMs, as well as the cross-hybridization we detected between the embryonic skeletal LMM probes and other striated myosin transcripts, precluded the use of any significant portion of the rod-encod- ing region as a specific probe. Rather, we took advantage of the 3‘-UTRs which we (18) and others (16) have shown to be specific for the complementary transcripts. Rased on the 3’- UTR sequences, we designed oligonucleotides for the PCR analysis of EBs and embryos and developed specific ribo- probes suitable for the detection of the cardiac MHC tran- scripts by in situ hybridizations. In preliminary experiments with the riboprobes, in which in situ analyses were coupled with a determination of relative and absolute transcript levels using RNA blots, we estimated that by using exposures of 2- 3 weeks we could detect as few as 100-200 transcripts/nucleus (data not shown).

Although the homologies between the n- and &cardiac LMMs are striking, there are both conservative and noncon- servative differences in the amino acid composition of the rods. Considering the recent observation that a single amino acid change in a cardiac MHC is linked to a form of familial hypertrophic cardiomyopathy (36), these differences cannot be overlooked in future studies in which the structure/func- tion relationships of the cardiac isoforms are explored.

Previous analyses (18) indicated that the developing ER might be suitable for studying certain early aspects of cardi- ogenesis. In the studies presented above we have extended our observations on MHC gene expression in this system to include both the temporal and spatial expression patterns of the two cardiac transcripts. Although the photomicrographic record did not reveal any obvious morphological features characteristic of either beaters or nonbeaters (Fig. 41, their molecular characterization using PCR and in situ hybridiza- tions revealed marked differences.

Mouse Cardiac Myosin Gene Expression 22425

Flc;. 7. Localization of a- and 8- cardiac transcripts in beating and nonbeating ERs. Shown in panrb a-d are the results o f in situ hyhridization to adjacent sections derived from a single 10-day heating EH. Panel9 a, b, and c were hybridized with [n-:'"S]UTP-la- heled n-cardiac riboprobe, and localized regions of hyhridization are apparent (arrows); panel d, which was hyhridized to the 8-cardiac rihoprohe, was derived from a section located hetween those shown in panrls b and c (inset) . Panels e and fdepict hybridizations performed on 10-day nonbeating ERs with the a- and {j-cardiac riboprohes, respectively. Sec- tions a-d are magnified 12.7 X, and sec- tions e and f, 50 X. The inset is a com- puter-generated three-dimensional re- construction of the sectioned EH.

The PCR data collected during EB development (n = 126) are summarized in Fig. 8. The expression of the P-MHC transcript at days 3-4 in the developing EBs appears to mimic the in uivo expression of this transcript on day 5 postcoitum of embryonic development (Fig. 6). I t is not a t all clear whether this expression actually reflects a precardiac or pres- keletal muscle determination. I t is possible that expression of the P-cardiac MHC gene at this stage merely indicates early mesodermal induction processes taking place in EBs and embryonic ectodermal cells. Such a hypothesis could be tested formally by development of suitable PCR primer pairs for the transcripts that encode known mesoderm-inducing factors such as fibroblast growth factor (37) transforming growth factor-/3 (3R), and T-brachyury (39, 40). An experimental approach of this nature has been used recently to detect early transient expression of the amphibian MyoD genes in Xeno- pus laeuis embryos a t a developmental stage where mesoderm has yet to develop (41).

That a-cardiac MHC transcripts were not detected during this early developmental time period, either in vitro or in uivo, while co-expression with the (J-cardiac gene occurred during the later, cardiogenic stages, underscores both the differences and similarities that must. exist between the regulatory regions of the two cardiac genes. The data indicate that the ER system could be utilized in promoter analyses of the c(- and +cardiac genes and may prove useful in identifying the ri.9-acting sequences that interact with mesoderm-inducing factors.

In every case analyzed, beat.ing ERs co-expressed both cardiac MHC genes (Fig. 8). This result suggests that the expression of both isoforms may he required for beating to occur in uitro. However, the present studies cannot determine whether co-expression is essential for contraction to occur. I t is possible that other gene products involved in contrartilitv may need to accumulate during ER maturation for a beating focus to form. Although it is verv unlikelv that a properly formed cardiac tube is present in the EHs, the in situ data

22426 Mouse Cardiac Myosin Gene Expression

1 4 -

c 0 4 -

1 2 3 4 5 6 7 9 1 0 1 5

Days in Culture

FIG. 8. Summary of a- and 8-cardiac MHC gene expression during EB development. Shown is a histogram that includes the data obtained for t h e 126 EBs that were analyzed by PCR (Fig. 4). F o r all days n = 12, except for days 5-7 where n = 10. The number of EBs that express the indicat,ed transcript (a , white bars; 0, black bars) is shown.

suggest the possibility that the contractions may reflect the formation of a rudimentary heart primordium. This explana- tion appears likely given that in uiuo, spontaneous contrac- tions have been observed to occur in bilaterally paired cardiac primordia prior to fusion (42).

The spatial expression data of the cardiac MHC genes in beating EBs show the a-MHC transcript is localized to de- fined regions whereas the @-MHC transcript is detected in a larger area which includes the region of a-MHC transcription. The spatial patterns of these transcripts correlate well with what is known about the transcripts' distribution in 8-9.5- day postcoitum mouse embryos. Lyons et al. (16) have shown, using in situ analyses of parasagitally sectioned embryos, that the a-cardiac MHC transcripts are detectable only in the cardiac tube whereas the @-MHC transcripts occur not only in the cardiac tube but also in the somites (43). Thus, both our studies and those of Lyons et nl. (16) indicate that the expression of the @-cardiac gene is not restricted solely to the mesodermal cells in the cardiac compartment. This is presum- ably because the @-cardiac MHC gene also encodes the slow myosin found in many skeletal muscles.

The present study demonstrates the potential of ES cell- derived EBs as an in vitro model for the study of develop- mentally regulated cardiac MHC gene expression. Recapitu- lation of aspects of early pre- and postsomitic patterns of expression of the murine a- and @-cardiac MHC genes con- firms the utility of the system for the relatively rapid analysis of structural and regulatory cassettes. In addition, the detailed characterization of the cardiac MHC genes' temporal and spatial expression patterns provides a useful standard against which EBs derived from ES cells targeted by homologous recombination can be compared. This will allow for rapid in uitro analyses of the effects a given mutation may have on cardiac MHC gene expression. These in uitro analyses should

prove useful in predicting the consequences of murine MHC gene ablations at the whole animal level and should help in directing the appropriate developmental and histological anal- yses of the targeted animals.

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