DEVELOPMENTAL

BIOLOGY

Identification

148, 355-364

(19%)

and Characterization of a Ventricular-Specific Avian Myosin Heavy Chain, VMHCI : Expression in Differentiating Cardiac and Skeletal Muscle JOSEPHG.BISAHAANDDAVIDBADER

DeparPment

of Cell

Biology

and Anatomy,

Cornell Accepted

University August

Medical

College,

New

York,

New

York

10021

12, 1991

To investigate the initial diffcrentiative processes of avian cardiac and skeletal myogcnesis, we have isolated and characterized a molecular marker of the cardiac myocyte cell lineage, ventricular myosin heavy chain 1 (VMHCl). Our goal in this initial study was to use a gene-specific probe to analyze the expression pattern of VMHCl RNA during development. DNA sequence analysis confirmed that VMHCl represented a novel member of the MHC gene family. PCR analysis using gene-specific primers determined that the VMHCl RNA is first expressed in the stage 7 cardiac primordia, much earlier than the appearance of a tubular beating heart. RNA blot analyses determined that the VMHCl message was present in the embryonic and adult ventricles but not in the embryonic or adult atria or skeletal muscle tissues of either the fast or slow type after definitive muscle structures were formed. Still, PCR and in situ hybridization analyses of the initial phases of cardiac and skeletal myogenic differentiation determined that VMHCl was expressed in both progenitor populations at the initiation of myogenesis regardless of the source of myoblast or site of initial differentiation. The transient expression in skeletal muscle precursors coincided with the onset of differentiation in these cells. These data suggest that the differentiative programs of cardiac and skeletal myocytes overlap during their initial phases, then quickly become distinct.. The VMHCl gene should provide a model for identification of transcription factors involved in cardiac myocyte differentiation. o 1991 Academic press. hc.

myocyte transition and use this gene to isolate putative cardiac-specific regulatory factors. In the avian system, most of the available data concerning cardiac myogenesis have come from biochemical and immunochemical studies of muscle-specific protein expression (Masaki and Yoshizaki, 19’74; Sartore et al., 1978; Obinata et al., 1984; Gonzalez-Sanchez and Bader, 1984,199O; Toyota and Shimada, 1981; Sweeney et aZ., 1984, 1987, 1989; Evans et ah, 1988). In addition, molecular biological techniques have been used to study the expression of certain cloned muscle-specific genes including actin; troponins I, T, and C; and myosin heavy chain (MHC) (Hayward and Schwartz, 1982; Hastings et ah, 1991; Bucher et al, 1988; Swiderski and Solursh, 1990; Kropp et al., 1987). However, these and other studies do not address the events which occur during the transition from committed cardiac progenitors to differentiated myocytes. To examine these primary events we sought to isolate a cloned marker of the cardiac myocyte cell lineage which was expressed at the initiation of the cardiac myogenic program. Myosin is a major protein component of the contractile apparatus. Each hexameric myosin molecule is comprised of two heavy and four light chains. Because of the many physiologically important properties of MHC, the expression of a specific MHC isoform is potentially significant to the contractile properties of a particular cell

INTRODUCTION

Early in avian development two separate groups of myogenic progenitor cells become committed to the cardiogenic and skeletal myogenic cell lineages. Cells of the lateral plate mesoderm are committed to the cardiogenie cell lineage subsequent to gastrulation (GonzalezSanchez and Bader, 1990), approximately stage 4 (Hamburger and Hamilton, 1951), and begin to differentiate soon thereafter, with rhythmic heart beats commencing at stage 10. Skeletal myogenic progenitors are derived from somitic mesoderm and begin to differentiate at approximately stage 13 (Holtzer et al., 1957). Because of the different origins of cardiac and skeletal myocyte progenitors, the temporal and spatial differences between the initiation of cardiac and skeletal myogenic differentiation, and the inability to detect expression of previously described skeletal myogenic regulatory genes in cardiac tissue (Braun et ah, 1989; Davis et ah, 1987; Wright et aZ., 1989), it is believed that the mechanisms regulating cardiac and skeletal myogenic differentiation are distinct. Unlike skeletal myogenesis, little is known about the regulatory factors or the cellular and molecular mechanisms involved in commitment and the initial differentiative processes of cardiac myocytes. One method of investigating cardiac myocyte differentiation is to isolate a gene expressed at the myoblast355

0012-1606/91 Copyright All rights

$3.00

0 1991 by Academic Press, Inc. of reproduction in any form reaervcd.

DEVELOPMENTALBIOLOGY VOLUME148,1991

356

type. Multiple MHC isoforms have been detected in all vertebrates studied, with different isoforms being expressed in both tissue- and stage-specific manners (Emerson and Bernstein, 1987; Epstein and Fischman, 1991). In addition, because of the obvious phenotypic diversity of cardiac and skeletal myocytes, it is conceivable that MHC gene expression reflects the initial divergence of the two myogenic phenotypes. Still, little or no molecular data on MHC gene expression during the earliest stages of myogenic differentiation have been reported. Therefore, we reasoned that an embryonic cardiac MHC clone may represent a useful marker of the cardiac myocyte lineage. Our goal in the present study was to examine gene expression during early cardiac and skeletal muscle development. To this end, we have cloned an avian MHC cDNA (ventricular myosin heavy chain 1, VMHCl). This 3-kb cDNA represents the 3’-most half of the corresponding mRNA and is sufficient for use as a probe in studying gene expression during early development. Our data have determined that the VMHCl RNA is expressed in cardiogenic mesoderm at stage ‘7 prior to the formation of the tubular heart and approximately 10 hr before the first signs of rhythmic heart beating (stage 10). Thus cardiomyogenic differentiation occurs at an earlier stage than previously suggested (DeHaan, 1965). VMHCl expression was detected in the ventricles throughout development but was undetectable in any skeletal muscles during later developmental stages (stage 40 through adulthood), which is consistent with cardiac-specific gene expression. Examination of the earliest stages of cardiac and skeletal myogenic differentiation demonstrated that VMHCl was transiently expressed during the initial phases of atria1 and skeletal myogenesis. Indeed, we found that VMHCl was expressed in all myogenic populations during the primary phases of myogenesis regardless of myoblast origin or site of differentiation, but its expression is later restricted to ventricular myocytes.

(Auffray and Rougeon, 1980) or (2) the guanidinium thiocyanate-phenol method described by Chomczynski and Sacchi (1987). The second method was used preferentially for small amounts of tissue (i.e., early stage embryonic tissue). Comparable yields were obtained using both methods. Poly(A)+ RNA was enriched using oligo(dT) cellulose chromatography (Maniatis et ab, 1982). cDNA

Library

Construction

and Screening

Double-stranded cDNA was synthesized from 1 pg of poly(A)+ RNA isolated from Embryonic Day 13 (E13) chick ventricles using the Boehringer-Mannheim cDNA synthesis system. A cDNA library was constructed in the X gtl0 vector using the Amersham cDNA cloning system according to the method of Hyunh et al. (1985). This library contained approximately 4 X lo6 recombinants. The amplified cDNA library was screened at 10,000 plaques per 150-mm-diameter culture dish using nitrocellulose filters (Shleicher & Schuell Inc.). A 2.2-kb neonatal pectoralis myosin heavy chain cDNA (HC-1) (a gift of D. A. Fischman) was radiolabeled using the random priming method of Feinberg and Vogelstein (1983) and used to probe the El3 ventricular cDNA library. Membranes were prehybridized in a solution containing 50% deionized formamide, 5~ SSPE [1X SSPE is 0.18 M NaCl, 1 mM EDTA, 10 mM sodium phosphate (pH 7.5)], 1X Denhardt’s solution, 0.1% SDS, 100 pg/ml denatured salmon testis DNA, and 10% dextran sulfate for 1 hr at 42°C and then hybridized overnight after the addition of radiolabeled probe. The membranes were washed twice in 2~ SSPE and 0.1% SDS at room temperature for 10 min each and then once in 1X SSPE and 0.1% SDS at 37°C for 30 min and then in the same solution at 42°C for 30 min. Positive plaques were purified by secondary screening under similar conditions and recombinant clones were identified following restriction enzyme digestion.

MATERIALS AND METHODS Animals

and Tissues

White Leghorn chickens and fertilized eggs were purchased from commercial sources. Eggs were maintained under high humidity in a 37°C incubator. Embryos were staged according to Hamburger and Hamilton (1951). Cardiogenic regions of staged embryos were based on the fate maps of Rawles (1943). All tissues were dissected immediately postmortem and stored frozen at -70°C until use. RNA

Isolatimz

Total RNA was prepared from frozen tissue by either of two methods: (1) LiCl-urea-ethanol precipitation

DNA

Sequencing

Our goal in this initial study was to obtain a probe which could be used to analyze the expression of VMHCl during development. cDNA inserts were cloned into the EcoRI site of the sequencing vector M13mp18 (Boehringer-Mannheim). Sequence analysis was performed using the dideoxy-chain termination method of Sanger et al. (1977) with a modified T7 DNA polymerase (Sequenase 2.0; United States Biochemical Corp.). RNA

Blot Hybridization

The integrity of all RNA preparations were monitored by ethidium bromide staining of RNA gels. RNAs

BISAHA

AND

BADER

Carcliovv~yo~e~~ic

were separated by electrophoresis through 1% agarose gels containing 2.2 M formaldehyde and transferred to nitrocellulose filters (Schleicher & Schuell, Inc.) by capillary action. Hybridizations were performed in 50% deionized formamide, 0.1% SDS, 5~ Denhardt’s reagent, 5X SSPE, 10% dextran sulfate, and 100 pg/ml denatured salmon testis DNA at 42°C. Placmids

and Probes

Plasmid pVMHC1 was constructed by subcloning the 3-kb cDNA insert of the h gtl0 MHC clone into the EcoRI site of the transcription vector pGem-4Z (Promega). A vector containing the unique 3’sequence of this clone, pVMHClA, was constructed by digesting plasmid pVMHC1 with Csp451 (Promega) which cleaves the VMHCl cDNA at a site 251 bp from the 3’ terminus of the cDNA (see Fig. 1). The cDNA was then blunt ended with the Klenow fragment of DNA polymerase I and digested with EcoRI to liberate the 251-bp fragment. This fragment was then cloned into a SmaI/EcoRI digested pGem4Z vector. cDNA probes were prepared by the random primed labeling technique of Feinberg and Vogelstein (1983) using [a-32P]dCTP. RNA Analysis (PCRJ

Using the Polymerase

Chain Reaction

RNA concentrations used for PCR analyses were determined and standardized by slot blot hybridization with a P-a&in probe (data not shown). Single-stranded cDNA was synthesized from total RNA in a 20-~1 reaction containing 50 mMTris-Cl (pH 8.3), 6 mMMgCl,, 60 mMNaCl,20 mMDTT, 20 U RNasin (Boehringer-Mannheim), 20 fig/ml oligo(dT), 1 mM dNTP’s (1 mM each of dATP, DCTP, dGTP, dTTP), and 20 U MMLV reverse transcriptase (Boehringer-Mannheim). Each 50-~1 PCR reaction contained: 1X reaction buffer [lo mMTris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl,], 200 pm dNTP’s, 60 nm of each PCR primer (see Fig. l), 1.25 U Tag DNA polymerase (Perkin-Elmer Cetus). An initial 4-min denaturation at 94°C was followed by 25 cycles of amplification. Each cycle included a 1-min denaturation at 94°C followed by 1.5 min of primer annealing at 50°C and a 3-min elongation at 72°C. The last cycle was followed by a lo-min elongation at 72°C. PCR amplified DNA products were subjected to electrophoresis in 1% agarose gels and transferred to nylon filters (Schleicher & Schuell) as described above. Prehybridization was performed in 6~ SSPE, 10X Denhardt’s reagent, 0.5% SDS, and 100 pg/ml denatured salmon testis DNA at 42°C for 1 hr. Hybridization was performed in 50% deionized formamide, 6~ SSPE, 0.5% SDS, and 50 pg/ml denatured salmon testis DNA. Positive (El3 ventricle) and negative (El3 brain) controls were performed for

Diflermtiu

357

f ion

all experiments, although not necessarily shown on each blot. In all experiments the VMHCl probe always hybridized to a PCR product from El3 ventricular RNA and never hybridized to a product from brain. Control experiments were performed using RNA from tissue that was shown not to contain the VMHCl RNA by RNA blot analysis. No amplification product was detected in embryonic brain or adult skeletal muscle using our standard experimental conditions (data not shown). In all the experiments the sizes of the amplification products were determined by comparison to standard molecular weight markers which were not visible on the autoradiograms. In Situ Hybridixatior~

Procedures were similar to those of Cox et al. (1984) with some changes. Whole chick embryos were fixed in 4% paraformaldehyde in PBS, dehydrated with ethanol, and embedded in paraplast. Serial sections were cut at 8 pm and mounted on poly-L-lysine (Sigma) coated slides (50 @g/ml). Hybridizations were performed using a synthetic 50-bp oligonucleotide which hybridizes to the 3’ untranslated region of the VMHCl RNA (see Fig. 1). This DNA probe was radiolabeled with terminal deoxynucluotidyl transferase in the presence of 35S-labeled dATP. Fifty microliters of hybridization solution (10% deionized formamide, 1X SSPE, 1X Denhardt’s, 10% dextran sulfate, 10 mM DTT, 500 pg/ml calf thymus tRNA, and 2 X lo6 cpm radiolabeled probe) was applied to each slide. Hybridization occurred for 16 hr at 50°C in a moist chamber. Slides were washed in 2~ SSC, 10 mM DTT overnight at 50°C and then in 0.1X SSC for 15 min at room temperature. Autoradiography was performed using Kodak NTB-2 emulsion diluted 1:l with distilled water. After exposure, slides were developed for 1.5 min in a 1:2 dilution of Kodak D-19 developer, stopped in Kodak stop bath for 15 see, and fixed in Kodak rapid fix for 3 min. Sections were stained with hematoxylin and eosin and mounted under coverslips. RESULTS

Isolation

and Ch,aracterixa,tion

of VMHCl

To isolate molecular markers for the study of early cardiac and skeletal muscle development, a cDNA library was constructed using poly(A)+ RNA prepared from Embryonic Day 13 (E13) chick ventricles. The amplified ventricular cDNA library was screened at low stringency (see Materials and Methods) with a radiolabeled chicken myosin heavy chain (MHC) cDNA isolated from the neonatal pectoralis (HC-1). A total of 16,000 phage were screened and 14 plaques gave positive hybridization signals, from which 7 positive phage were

358

DEVELOPMENTAL BIOLOGY

VOLUME 148, 1991

A 1

GTG AAG Val Lys

AGC Ser

TAC Tyr

AAA Lys

CAC His

CM Gin

GCA Ala

GAG GAA Glu Glu

GCC Ala

17

CTG Leu

TAC Tyr

t TCG AA0 Ser Lys

TAC Tyr

AGA

Arg

AAA Lys

CAG Gln

CM Gln

CAT His

GAT Asp

34

AGO

GCA

Arg

Ala

GM Glu

ATA Ile

GCT Ala

GM Glu

TCT Ser

CM Gin

GTT MC Val Asn

AAG Lys

51

OAT Asp

ATT Ile

GGC ATG Gly Met

MLA Lys

AAG Lys

GTT CAT Val His

GM Glu

GAG GAG Glu Glu

68

205

TM GTG CCA OAT ATA AGA Stop- - COG - - T-s- C -T GG - - CTC -_--_---------------------------m------

TGA

TGA

TTC

ATA

CM

TCA

GGT

256

ATA --

ACC

AAA

AGC

AGA

TOT

ATT

CTA

AAA

CGA

GCA

CTT

ACA

AAA

307

ATA

MT

ATC

AAG

TGC AAA

CCA

52 103 154

GAT Asp GM Glu

AA0 Lys GCA Ala

CTA Leu

CM Gin

CTA Leu MT Asn

AAA Lys

CM Gin

GCC Ala

CTG GAC Leu Asp

GAT Asp

GCT GM Ala Glu

GM Glu

CTG AGO Leu Arg

AGC

Ser

AAG Lys

Arg

TCA Ser

AGG

CTT Leu

AAA

ACA

AAA

B VMHC ~MHC

KMLKvXSYKHQAEEAEAQILYLSKYRKQQHDLDDAEERHEEE :::::::::: ::::::: : .. .. .. .. .. .. .. .. .. ::: :: :: :: :::::

::::::

: : : ::

KLQLKVKAYKRQAEEAEEQATNLSKFRKVQHELDEAEERKSRDIGAKQKIHDEE

VNHC DXLQLKVKSYKHQAEEAEAQANLYLSKYRXQQHDLDDAEE~EIAESQVNK~SKSRDIGMK~HEEE :::::::::: :::::: : :: :::::::: :: .. .. .. .. .. .. .. .. .. ::: :: :: :: ::::: P MHC DXLQLKVKAYKRQAEEAEEQANTNLSKFRKVQHELDEAEE~DIAESQVNKL~KSRDIGAKGLNEE VMHC 251

DKLQLKVKSYKHQAEEAEAQANLYLSKYRKQQHDLDDAEERAEIAESQVNKLRSKSRDIGMKKVHEEE ,......... .. .. .. .. .. .. :::::: :: : . . . . . . . .. . :::: . ::: :: :: : DKLQMKVKSYKRQAEEAEELSNVNLSKFRKIQHELEEAEGRTDIAESQVNKL~KSREIG-K~ESEE

:::

::

::

: :

FIG. 1. Partial nucleotide and deduced amino acid sequence analysis of VMHCl cDNA. (A) Sequence of terminal 327 bp of the VMHCl cDNA. The predicted protein sequence is shown below the sense strand of the DNA sequence. The sequences used for synthesis of PCR primers are underlined. The 50-bp oligonucleotide in silu hybridization probe was synthesized from the sequence denoted with a dashed line. An arrow indicates the unique Cs@51 restriction site which was used to create the VMHCl3’ specific cDNA probe. (B) Comparisons of the deduced amino acid sequences of VMHCl and rat (Yand /3MHC (Mahdavi et al., 1982) as well as an embryonic skeletal MHC (Kavinsky et al, 1983). Percentage identities are 79.1, 76.5, and 69.1, respectively, in a 68 amino acid overlap.

obtained. One of these clones which contained a 3-kb insert (VMHCl) was chosen for further analysis. The identity of VMHCl was determined by DNA sequence analysis. The sequence of the terminal 327 bp of the VMHCl cDNA including a 204-bp open reading frame and a 120-bp 3’ untranslated region is shown in Fig. 1A. The deduced amino acid sequence of the VMHCl open reading frame was compared to those of other cloned MHCs (Fig. 1B). High degrees of sequence similarity (80%) were observed with the carboxy-terminal regions of the ct and 0 cardiac MHCs from rat (Mahdavi et al., 1982) and an avian embryonic skeletal muscle MHC (251) (Kavinsky et ah, 1983). Sequence comparisons at the nucleotide level also yielded high degrees of nucleotide identity (>65%) in the protein coding regions of these four clones. No similarity between the 3’ untranslated region of VMHCl and the other MHCs was observed (data not shown). We are currently constructing a full-length VMHCl cDNA for use in structural analysis of this MHC. Our primary goal in the present study was to obtain a gene-specific probe to analyze the expression of the corresponding transcript during cardiac and skeletal myogenesis. The present data demon-

strate that VMHCl represents a novel member avian myosin heavy chain gene family.

of the

VMHCl Is Expressed in Cardiogenic Mesoderm before Heart Formatiun

One of our first goals was to determine when the VMHCl RNA was first expressed in the developing avian embryo. Therefore tissue was isolated from pregastrulated embryos (stages 2 and 3) and from gastrulated cardiogenic regions (stages 4-8) and subjected to polymerase chain reaction (PCR) analysis. Total RNA was isolated and cDNA was synthesized and subjected to amplification using synthetic gene-specific primers corresponding to the 3’ region of VMHCl (see Fig. 1). DNA sequence and genomic DNA blot analyses (data not shown) demonstrated that the 3’ untranslated region of VMHCl was unique; hence, a PCR primer corresponding to this region would ensure amplification of only VMHCl sequences and not those of other similar MHCs. PCR analysis was employed because of the paucity of tissue obtainable at these early developmental stages. The abundance and integrity of each RNA prepa-

BISAHA

AND

BADER

Cardiomyogenic

VMHC I -

FIG. 2. PCR analysis of VMHCl expression in the differentiating heart. Total RNA was isolated from stage 2-8 precardiac mesoderm and subjected to reverse transcription followed by amplification using gene-specific primers (see Fig. 1). Amplification products were analyzed on 1% agarose gels, transferred to nylon membranes, and probed with the 3’-specific VMHCl cDNA. An amplification product of 208 bp can be seen at stages 7 and 8. The minor band present in the stage 8 lane is a nonspecific band that does not appear in other similar experiments (see Figs. 5 and 6).

ration was determined by hybridization with an actin probe prior to PCR analysis. Amplification products of staged tissues were analyzed by DNA blot hybridization using a 3’-specific VMHCl cDNA probe (see Materials and Methods). As seen in Fig. 2, the VMHCl RNA was first detectable in stage 7 cardiogenic mesoderm. At stage 7 the tubular heart has not yet formed, and dehnitive heart beats, once thought to be the hallmark of cardiac myogenic differentiation, are not detected until stage 10 or approximately 10 hr later. Thus, the present data demonstrate the presence of the VMHCl transcript before the definitive heart is formed.

359

Differentiation

sorbance at 260 nm and ethidium bromide staining of the gel (Fig. 3), were used for RNA hybridization studies. Hybridization of the 3’-specific VMHCl cDNA probe to an RNA present in the ventricles from as early as E5 was observed (Fig. 3). This RNA was not detected in the atrium after E5 (stage 27). The weak hybridization signal seen in the El5 ventricles is most likely due to experimental variation and not to a decrease in messenger RNA levels. The ventricular-specific nature of this RNA with a has been confirmed using in situ hybridization gene-specific probe (data not shown). The VMHCl probe did not hybridize to RNA from embryonic brain or skeletal muscle tissues (see below). These data indicated that VMHCl expression was ventricular-specific as soon as the atria and ventricles are morphologically distinguishable (E5) through adulthood. Biochemical, immunochemical, and molecular studies have shown that several cardiac MHC genes are expressed in developing and adult skeletal muscles (Kropp et al., 1987; Narusawa et al., 1987; Sweeney et al., 1984; Masaki and Yoshizaki, 1974). In the rat, the ventricular or p MHC is identical to the major slow skeletal MHC isoform (Narusawa et al., 1987), and in the chicken (Can-

E5 VA

E9 El5 V AV AVNAVA

A

28s 18s

VMHCl Expression Is Ventricular-SpeciIc after the Formation of Definitive Heart Chambers Previous studies have shown MHC isoform expression in the avian heart to be complex and regulated in chamber- and developmental stage-specific manners (Sweeney et al., 1987; Evans et al., 1988; Gonzalez-Sanchez and Bader, 1984; 1985). To determine the expression pattern of VMHCl in the definitive chambers of the heart, RNA blot analysis was performed. Total RNA was obtained from chick atria and ventricles of various developmental ages from E5, when the atria and ventricles are first morphologically distinguishable, to the adult. Equal amounts of RNA, as approximated by ab-

FIG. 3. Ventricular-specific expression of VMHCl RNA during early cardiac development. Total atria1 (A) or ventricular (V) RNA (5 pg) from Embryonic Day 5,9, and 15 (E5, E9, E15), 2-day-posthatch neonatal (N), and adult (A) tissues were electrophoretically separated, transferred to nitrocellulose, and probed with the 3’-specific VMHCl cDNA. Below the autoradiogram is a photograph of the ethidium bromide stained gel showing equivalent amounts of RNA in each lane.

360

DEVELOPMENTAL

Pet EN

A

E

Leg N

A

BIOLOGY

Ald NAV

28s 18s

FIG. 4. Tissue-specific expression of VMHCl RNA in developing and adult skeletal muscle. Total RNA (5 pg) isolated from Embryonic Day 14 (E), 2-day-posthatch neonatal (N), and adult (A) pectoralis (Pet), leg (a mixture of soleus and gastrocnemius) and anterior latissimus dorsi (Ald) were analyzed on formaldehyde gels, transferred to nitrocellulose, and probed with the 3’-specific VMHCl cDNA. The last lane contains El5 ventricular RNA (V). An ethidium bromide stained gel similar to the one in Fig. 3 showed equivalent amounts of RNA present in every lane (data not shown).

tini et al., 1980; Masaki and Yoshizaki, 1974) immunological data have suggested that a ventricular or ventricular-like MHC is present in developing skeletal muscles. In order to examine VMHCl expression in developing skeletal muscles after the initial transition of myoblasts to the differentiated state, RNA blot analysis was performed using embryonic and adult fast and slow skeletal muscles. Total cellular RNA obtained from embryonic, neonatal, and adult pectoralis (skeletal fast twitch); gastrocnemius and soleus (skeletal slow twitch); and anterior latissimus dorsi (skeletal slow tonic) muscles were electrophoretically separated, transferred to nitrocellulose, and probed under stringent conditions using a radiolabeled fragment of VMHCl which contained the 3’ gene-specific sequences. As shown in Fig. 4, VMHCl RNA was present at high levels in the embryonic ventricles but was not detected in developing embryonic or adult skeletal muscles even after prolonged exposure times. In addition, PCR analysis of RNA from adult muscle other than ventricular myocardium, as described below, did not detect the VMHCl message (data not shown). VMHCl was therefore a candidate for a cardiac-specific avian gene. VMHCl Is Transiently Expressed at the Initiation Skeletal Myogenesis

of

While VMHCl expression was confined to the ventricle at a point when definitive embryonic muscle structures were observed, its expression during the initial phases of cardiac and skeletal myocyte differentiation remained unclear. To examine VMHCl expression at

VOLUME

148,1991

these early stages of differentiation, presumptive cardiac and somitic mesodermal tissues were isolated from stages 818, and 25 chick embryos and from developing limb and truncal musculature at stages 29 (E6) and 35 (E9). An amplification product of the predicted size (208 bp) which hybridized to the VMHCl cDNA probe was obtained using cardiac RNA from all three stages (Fig. 5). While no amplification product was observed using RNA from stage 8 somites, analysis of stage 18 somitic RNA demonstrated the presence of the VMHCl transcript. A VMHCl amplification product was not detectable in stage 25 somites even though MHC was abundantly expressed at this developmental stage (Gonzalez-Sanchez and Bader, unpublished data). The VMHCl RNA was present in the stage 25 atria coinciding with the initial differentiation of this tissue. The VMHCl message was also detected in the limbs and skeletal muscles at E6 (stage 29) and E9 (stage 35), respectively, which corresponds to differentiation of secondary myoblasts in these tissues (Fig. 6). To confirm the results described above, as well as to determine the cellular localization of the VMHCl RNA, in situ hybridization analysis of stage 18 chick embryos was performed. A synthetic 50-bp antisense oligonucleotide which hybridizes to the 3’ untranslated region of the VMHCl RNA (see Fig. 1) was used to identify VMHCl transcripts. The presence of the VMHCl RNA in the somites was determined in transverse sections (Fig. 7). As is shown in Fig. 5, somitic myotome contained VMHCl RNA at stage 18. No other tissues, excluding the heart, elicited positive hybridization sigSt

a

18

25

VHSHSAVS

VMHCl-

FIG. 5. PCR analysis of VMHCl RNA expression in differentiating cardiac and skeletal myocytes. Total RNA was isolated from stages 8, 18, and 25 heart (H) and somitic mesoderm (S) and subjected to reverse transcription followed by amplification using gene-specific primers. Amplification products were separated on agarose gels, transferred to nylon membranes, and probed with the 3’-specific VMHCl cDNA. Amplification products from Embryonic Day 15 ventricle RNA (V) were used in the first lane. Stage 25 hearts were separated into atria (A) and ventricles (V) and are designated as such.

BISAHA

AND BADER

Ca,rdiomyogegenic

361

Diferwhation

nals. VMHCl RNA was more abundant in the rostra1 somites but also clearly visible in the caudal somites which agrees with the differentiation of the somitic mesoderm in a rostral-caudal direction (data not shown). Taken together, these data demonstrate that the VMHCl RNA was present during the initial differentiative periods of both cardiac (stage 7) and skeletal (stage 18) myocytes. While expression continues in the cardiac myocytes throughout development, skeletal muscle precursor cells quickly cease to express this RNA after the initial phases of differentiation. DISCUSSION

To examine the initial differentiative events of cardiac and skeletal myogenesis, we have isolated and characterized a unique marker of the avian myocyte cell lineage, VMHCl. Our initial goal was to obtain a probe to analyze VMHCl expression during cardiomyogenesis. Our data demonstrate that VMHCl is expressed in cardiogenic mesoderm before the tubular heart is formed indicating that cardiomyogenic differentiation occurs much earlier than previously suggested. In addition, our studies show that VMHCl is expressed in all cardiac and skeletal myogenic populations during the initial phases of differentiation regardless of the origin of myoblast or the site of differentiation. After this initial period of myogenesis, VMHCl expression becomes quickly restricted to the ventricular myocyte cell lineage. These studies demonstrate a unique expression pattern of VMHCl and suggest the existence of an interestingregulatory mechanism governing the expression of this gene in cardiac and skeletal muscle.

St

29 HSHLP

35

FIG. ‘7.1~~ situ hybridization analysis of VMHCl RNA expression in the stage 18 chick embryo. Transverse sections were hybridized with a 50-bp DNA oligonucleotide probe derived from the gene-specific 3 untranslated region of the VMHCl cDNA. (A) A representative section showing the heart (H), somites (S), and neural tube(N). (B) Same section as in (A), viewed with darkfield optics. Cells containing VMHCl RNA are clearly visible in the heart and the somites.

Isolation

VMHCI

-

FIG. 6. PCR analysis of VMHCl RNA expression in differentiating secondary skeletal myoblasts. Total RNA was isolated from Embryonic Day 6 (stage 29) heart (H) and skeletal muscle (S) of the limb buds and Embryonic Day 9 (stage 35) heart (H), leg(L), and pectoralis (P) muscles and analyzed as described in the legend to Fig. 5. This autoradiogram was overexposed to visualize the low level of VMHCl RNA in the differentiating myoblasts.

of a Unique

MHC cDNA

The VMHCl cDNA represents a unique member of the avian MHC gene family. Significant similarity exists between the DNA sequence of VMHCl and other cloned MHC genes. The amino acid sequence derived from the 204bp open reading frame of VMHCl shows 79.1 and 76.5% amino acid identity with the rat cardiac clones pCMHC21 (a MHC) and pCMHC5 (0 MHC) rea spectively (Mahdavi et al., 1982) (Fig. 1B). Similarly, 69.1% amino acid identity was observed when compared with the embryonic skeletal MHC clone 251 (Kavinsky et al., 1983). The nucleotide sequence of the untranslated region of VMHCl showed no similarity to the sequences of CY,p, or the embryonic skeletal MHCs 110 or 251

362

DEVELOPMENTALBIOLOGY V0~~~~148.1991

cDNAs. In addition, genomic DNA blot analysis determined that VMHCl is a single-copy gene (data not shown). We are currently obtaining a full-length VMHCl cDNA for use in analysis of gene and protein structure. We conclude that VMHCl represents a MHC gene heretofore not described. Cardiac-Speci&c Heart Forms

Gene Expression

is Detected before the

One of the goals of the present study was to determine when cardiomyogenic differentiation can first be detected. VMHCl is expressed in cardiogenic mesoderm at stage 7, before the formation of the tubular heart and approximately 10 hr before the primitive heart begins to beat (DeHaan, 1965). Thus, our present data suggest that cardiac differentiation, as exemplified by VMHCl expression, proceeds more rapidly than previously suggested (Tokuyasu and Maher, 1987). While previous studies indicated that cardiogenic mesoderm is not phenotypically stable (i.e., committed) until a time frame roughly equivalent to the first signs of VMHCl expression (Orts-Llorca, 1963), our analyses suggest that mesoderm is not only committed to the cardiogenic cell lineage but that at least some of these progenitor cells have begun to differentiate. In addition, the expression of VMHCl at stage ‘7 suggests that cardiomyogenic differentiation is not dependent on the formation of definitive heart structures. The present study, along with our previous work, indicates that the conversion of mesoderm to initially differentiated cardiac myocytes begins at stage 7 and ends at stage 15 (Gonzalez-Sanchez and Bader, 1984). These data suggest that regulatory elements which initiate cardiac-specific gene expression may be activated before the heart forms. MHC Expression

in the Chambered Heart

Recent studies have shown that avian MHC expression is quite complex. Our previous data suggest that at least one specific MHC is expressed in the atria, ventricles, and conduction system of the developing and adult heart (Gonzalez-Sanchez and Bader, 1984,1985; Zhang et al., 1986). Subsequent studies have shown coexpression of additional MHC isoforms in the avian heart. Sweeney et al. (1984) reported that the embryonic atria and ventricles express a MHC immunologically similar to that of the adult ventricle. Atria1 expression of this MHC ceases at Embryonic Day 6 while ventricular expression continues throughout development. Evans et al. (1988) have described the expression patterns of three distinct MHC isoforms in the developing atria and ventricles using monoclonal antibody analysis. They show that multiple MHC isoforms are expressed at different developmental stages in the atria, ventricles, and

conduction system of the heart. The present data, which most closely mirror the immunochemical reactivity of antibody CCM31A of Sweeney et al. (1984), demonstrate that while VMHCl RNA is expressed during the initiation of ventricular and atria1 myogenesis, its expression quickly becomes restricted to ventricular myocytes. Indeed, except for the initiation of cardiac myogenic differentiation in the atria (Fig. 5), the expression of VMHCl is tightly controlled in the developing avian heart which is in contrast to the expression of other /3or ventricular-like MHCs in other species (Lompre et al., 1981; Narusawa et al, 1987). Several studies have suggested that a common embryonic MHC(s) may be expressed in developing muscle regardless of the embryonic origin of the myogenic progenitors and that this MHC may resemble a cardiac-like protein (Masaki and Yoshizaki, 1974; Sweeney et al., 1984,1989; Lyons et ah, 1990). Our data demonstrate that the VMHCl RNA is expressed in all myogenic populations during the initial phases of cardiac and skeletal myogenesis. In addition, it is expressed in widely variant populations of cardiac and skeletal muscle progenitor cells (i.e., atria1 and ventricular myocytes, somitic mesoderm at stage 18, limb and pectoral musculature at stages 29 and 35, and in secondary skeletal myoblasts which differentiate in vitro (Bisaha and Bader, unpublished data), but always in a transient manner except for the ventricular myocytes. It is possible that VMHCl may represent the embryonic or “primordial” MHC previously hypothesized by Sweeney et al. (1984, 1989). We have obtained the CCM31A antibody (a gift of W. Clark and R. Zak) and studies are underway to determine whether the epitope recognized by this antibody is present on the VMHCl protein. The presence of this MHC isoform during the critical early phases of myogenic differentiation in such widely variant populations of myogenie progenitors suggests an important role of VMHCl in myofibrillogenesis. VMHCl Expression during among Cardiac Genes

fiflerentiation

Is Unique

With the initiation of myogenesis, sets of muscle-specific genes are activated. In skeletal myogenesis, some of these have been termed “cardiac” genes. At present, it is not known how cardiac genes are activated during skeletal myogenesis. Several basic mechanisms can be proposed to account for this situation. First, it is possible that cardiac genes transcribed in skeletal muscle are activated by members of the MyoDl family (Davis et aZ., 1987; Braun et al., 1989; Wright et al., 1989; Rhodes and Konieczny, 1989). Alternatively, it is possible that an as of yet unidentified “cardiac” regulatory system, which is transiently active in skeletal muscle, could initiate

BISAHA AND BADER

Cardicnnyogenic

gene expression. Finally, a set of regulatory factors involved in the initial activation of muscle-specific genes may bind in a general fashion to regulatory regions of certain embryonic genes, such as VMHCl, thereby activating the myogenic program. In this case, specialized factors would sustain tissue-specific expression of cardiac and skeletal myogenic programs. Analysis of the regulation of cardiac genes such as VMHCl in developing skeletal and cardiac myocytes may elucidate the mechanisms by which the diversification of cardiogenic and skeletal myogenic cell lineages is generated. In addition, the phasic expression pattern of VMHCl which includes accumulation during the initial phases of skeletal and cardiac myogenesis and its rapid removal from skeletal myocytes is unique among cardiomyogenic genes analyzed thus far. Most, if not all, of these genes such as actin (Hayward and Schwartz, 1982), troponin C (Bucher et al, 1988), and troponin T (Swiderski and Solursh, 1990) are expressed in skeletal muscles late in embryonic life and some, like the mammalian p MHC (Narusawa et al., 1987), are expressed at high levels in the adult animal. In contrast, while VMHCl expression appears to be widespread among myogenic populations, its expression is strictly limited (except for ventricular myocytes) to the initial phases of myogenesis. Thus, the regulatory elements of the VMHCl gene appear to be under different transcriptional regulatory control from other “cardiac” genes. We would like to thank Donald A. Fischman, as well as the members of our laboratory, for critical reading of the manuscript and helpful comments. This work was supported by grants from the NIH (HL 34318) and the New York Heart Association. D.B. is an Established Investigator of the American Heart Association. prooj During the preparation of this manuscript we became aware of a report detailing the cloning of an avian ventricular MHC (Stewart et al, J. Molec. EvoL 33(4) (1991)). This clone hybridizes to an RNA expressed in the ventricles, early skeletal muscle, and regenerating slow muscle.

Note

udded

in

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