DEVELOPMENTAL DYNAMICS 208:491–504 (1997) Expression of Fast Myosin Heavy Chain Transcripts in Developing and Dystrophic Chicken Skeletal Muscle WILLIAM E. TIDYMAN, LAURIE A. MOORE, AND EVERETT BANDMAN* Department of Food Science and Technology, University of California, Davis, California ABSTRACT The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation. Dev. Dyn. 208:491–504, 1997. r 1997 Wiley-Liss, Inc. Key words: myosin; myosin heavy chain; MyHC genes; muscle; muscle development; muscular dystrophy; gene expression INTRODUCTION Myosin is the principal component of the contractile apparatus in muscle. Myosin diversity is primarily r 1997 WILEY-LISS, INC. produced by the differential expression of multiple isoforms of the myosin heavy chain (MyHC) subunit. In all vertebrates which have been examined, MyHC is encoded by a large, highly conserved multigene family whose expression is regulated in a developmental and tissue-specific fashion (Nguyen et al., 1982; Wydro et al., 1983; Buckingham, 1985). The number of sarcomeric MyHC genes ranges from 7 to 12 in rats and humans, respectively (Leinwand et al., 1983; Wydro et al., 1983), to more than 30 in the chicken (Robbins et al., 1986). Initially, using peptide mapping and immunological techniques, an embryonic, neonatal, and adult MyHC isoform were identified in mammals (Whalen et al., 1981) and the chicken (Bader et al., 1982; Bandman et al., 1982; Winkelmann et al., 1983). The expression of these MyHC isoforms was extensively characterized in developing, regenerating, and dystrophic muscle fibers of the chicken using isoform specific MyHC monoclonal antibodies (Cerny and Bandman, 1987; Bandman and Bennett, 1988; Bandman et al., 1989). However, subsequent molecular genetic studies revealed that three embryonic MyHC isoforms are expressed in the developing pectoralis major (PM) muscle in ovo (Umeda et al., 1983; Robbins et al., 1986; Lagrutta et al., 1989). These embryonic MyHC isoforms have been termed Cemb1, Cemb2, and Cemb3 by Moore et al. (1992). The Cemb1 MyHC isoform corresponds to that identified by Umeda et al. (1983) with cDNA clone p251 and by Robbins et al. (1986) with the corresponding genomic clone, N118. Cemb2 corresponds to that identified with cDNA clone p110 (Umeda et al., 1983) and Cemb3 to clone pCM4.1 described by Lagrutta et al. (1989) who characterized it as a ‘‘late embryonic’’ MyHC isoform. In addition, it has recently been shown that the initial stages of skeletal muscle myogenesis is characterized by the expression of a ventricular MyHC isoform, termed Cvent here (Sweeny et al., 1989; Bisha and Bader, 1991). Thus, at least six MyHC isoforms are expressed in the PM muscle during development (Hofmann et al., 1988; Rushbrook et al., 1991); however, the complexity of their expression pattern remains unclear. Contract Grant sponsor: NIH; Contract Grant number: AG08573; Contract Grant sponsor: USDA; Contract Grant number: 94-372051034. *Correspondence to: Dr. Everett Bandman, Department of Food Science & Technology, University of California, Davis, CA 95616. Received September 16 1996; accepted December 23 1996. 492 TIDYMAN ET AL. Although the embryonic, neonatal, and adult MyHCs were initially named based on their temporal appearance in the PM muscle during development, subsequent studies using biochemical and immunological methods revealed that embryonic and neonatal MyHC isoforms were also expressed in several other adult fast muscles (Crow and Stockdale, 1986; Bandman and Bennett, 1988). However, little information exists on the extent of embryonic MyHC gene expression in adult chicken fast muscles. Thus, one of the objectives of our study was to examine the diversity of MyHC gene expression at a mRNA level in different adult fast muscles of the chicken. Avian muscular dystrophy has been shown to affect MyHC expression in adult fast muscles in the chicken. Although the precise myogenic defect is not known, it is not caused by a defect in the dystrophin gene which is responsible for Duchenne muscular dystrophy in humans (Monaco et al., 1986; Hoffman et al., 1988). Avian muscular dystrophy appears to prevent the transition to the adult phenotype in fast muscle (Gordon and Vrbrova, 1975; Wilson et al., 1979). It inhibits the expression of the adult MyHC isoform, resulting in the continued expression of the neonatal MyHC isoform in adult fast muscle (Bandman, 1985; Bandman and Bennett, 1988). The extent to which the expression of other fast MyHC isoforms are affected is unknown, since it has not been examined at the transcriptional level. The goal of this study was to examine the complexity and temporal profile of MyHC gene expression during development using a set of transcript-specific oligonucleotide probes. Our results demonstrate the sequential expression of the five fast MyHC genes in the PM and gastrocnemius muscles during development. We have shown that the pattern of MyHC gene expression was similar in these two muscles during embryonic development, but diverged significantly following hatching. In addition, we compared MyHC expression pattern observed during development in vivo with that in vitro in a primary muscle cell culture. Our results showed that the expression of the Cemb3 MyHC gene, which was predominant during late embryonic development in vivo, was not expressed in vitro. In adult fast muscle other than the PM, two distinct patterns of MyHC gene expression were identified. One, present in fast muscles other than the leg, consisted of the expression of the Cadult and Cemb3 MyHC genes. The other pattern, present in fast leg muscles, consisted of the expression of the Cneo and Cemb3 MyHC genes. These data suggest Cemb3 is a major adult MyHC isoform. We also showed that MyHC genes expressed in adult dystrophic muscles corresponded to those which were expressed in the same normal muscle during the neonatal developmental period, providing further evidence that avian muscular dystrophy is a disorder of muscle maturation. Fig. 1. Nucleotide sequence of each 38-UTR oligonucleotide probe and the fast MyHC ‘‘generic’’ oligonucleotide probe, Myo-4. Fig. 2. Isoform specificities of the 38UTR oligonucleotide probes. Identical DNA dot blots were prepared on which 10 ng DNA from each of the fast MyHC isoform plasmids, N118 (Cemb1, Molina et al., 1987), D1 (Cemb2), D7 (Cemb3), E1 (Cneo), and AN4 (Cadult) and for the ventricular MyHC isoform plasmid (CV) were spotted. DNA from the Bluescript plasmid (BS), without MyHC insert DNA, was used as a negative control. The blots were hybridized, each with one of the 38UTR oligonucleotide probes, or with the fast MyHC probe, Myo-4. Each blot was washed at 5°C below the Tm50 of the respective probe. RESULTS Isoform Specificities of the Oligonucleotide Probes The isoform specificities of the oligonucleotide probes were determined by DNA dot-blot hybridizations to cDNA plasmids coding for the fast MyHC isoforms (Fig. 2). Each of the 38UTR oligonucleotide probes hybridized specifically with the respective plasmid to which the probe’s sequence was complementary and did not crosshybridize with any of the other plasmids. The ‘‘generic’’ fast MyHC probe, Myo-4, hybridized with equal efficiency to each of the fast MyHC-encoding plasmids. In addition, the oligonucleotide probes were MyHC TRANSCRIPTS IN CHICKEN SKELETAL MUSCLE 493 extensively examined by northern hybridizations to establish their isoform specificities (data not shown) and to demonstrate that each probe hybridized exclusively with MyHC mRNA (Fig. 3). Each probe was hybridized to total RNA isolated from skeletal muscle presumed to contain the MyHC mRNA species to which the probe was complementary, based on earlier studies using MyHC isoform-specific monoclonal antibodies (Cerny and Bandman, 1987). The hybridization of each probe resulted in a single 6-kb band, consistent with the size of MyHC mRNA. Expression of the Fast MyHC Genes During Development The temporal expression of the fast MyHC isoform genes was examined in the PM muscle during development by RNA dot-blot hybridizations using the 38UTR, isoform-specific, oligonucleotide probes. Total RNA was isolated from the PM at days 10, 12, 14, 16, 18, and 20 of embryonic development and at progressive ages, through adult, during post-hatch development. The resulting autoradiogram of the RNA dot-blot hybridizations is shown in Figure 4A. The relative percentage of each of the fast MyHC mRNAs was estimated by radiographic quantitation of the RNA dot blots (Fig. 4B). During development in ovo in the PM muscle, three embryonic MyHC genes, Cemb1, Cemb2, and Cemb3, were sequentially expressed. Although there was a sequential pattern to their expression, all three of the embryonic MyHC mRNAs could be detected at each time point in ovo. At embryonic day (ED) 10, Cemb1 MyHC mRNA was present in the greatest relative abundance, accounting for approximately 60% of the total MyHC mRNA transcripts. The relative abundance of Cemb1 mRNA progressively decreased between ED 10 and ED 16; however, Cemb1 mRNA could still be detected at ED 16 at very low levels. The relative abundance of Cemb2 mRNA increased from ED 10 to ED 14. Cemb2 mRNA was the most abundant at ED 14, accounting for approximately 40% of the MyHC mRNA transcripts. Following ED 14, Cemb2 mRNA levels decreased in parallel with the decline in Cemb1 mRNA levels. As the expression of the Cemb1 and Cemb2 MyHC genes decreased after ED 14, there was a dramatic increase in the expression of the Cemb3 MyHC gene. Maximum expression was at ED 18, with Cemb3 mRNA accounting for more than 80% of the MyHC transcripts. Following ED 18 Cemb3 expression rapidly declined; however, it was still significantly expressed at hatching. Just prior to hatching, beginning at ED 18, a second period of expression of the Cemb1 and Cemb2 MyHC genes occurred. In addition, the rate of decline in Cemb3 gene expression decreased just before hatching, resulting in all three of the embryonic MyHC genes being coexpressed at hatching. Following hatching, the amounts of Cemb2 and Cemb3 mRNAs decreased to barely detectable levels by 5 days post-hatch. Cemb1 mRNA was present longer after hatching, being undetectable after 10 days post- Fig. 3. The specificity of oligonucleotide probes on northern hybridizations. Each probe was hybridized to 15 µg total RNA isolated from muscle containing the MyHC mRNA species to which the probe’s sequence was complementary. The Cvent (CV)-specific probe was hybridized to RNA isolated from 5-day post-hatch ventricular muscle. Cemb1, Cemb2, and Cemb3 specific probes were hybridized to RNA isolated from 15-day embryonic PM muscle. The Cneo specific probe was hybridized to RNA isolated from 2-week-old PM muscle. The Cadult specific probe (CA) and the fast MyHC probe Myo-4 were hybridized to RNA isolated from adult PM muscle. The lane labeled (M) contained RNA molecular weight marker. Each blot was washed at 5°C below the Tm50 of the respective probe. hatch. After 14 days post-hatch no embryonic mRNAs could usually be detected. Cneo mRNA was first detected at very low levels at ED 20. The relative abundance of Cneo mRNA progressively increased following hatching, such that by 10 days post-hatch, it accounted for nearly 100% of the MyHC mRNA transcripts. Cadult mRNA was first detected at 10 days post-hatch. Following 10 days post-hatch, the relative abundance of Cneo mRNA began to gradually decrease concomitantly with an increase in the relative abundance of the Cadult mRNA. The relative abundance of the Cadult mRNA increased during post-hatch development, until by 70 days posthatch only Cadult mRNA could be detected in the PM muscle. The temporal expression of the fast MyHC genes was also examined in the gastrocnemius muscle during development (Fig. 5A,B). The pattern of expression of the fast MyHC genes in the gastrocnemius muscle was very similar to that in the PM muscle during embryonic development. As in the PM muscle, there was a sequential pattern of expression of the three embryonic MyHC genes. At ED 10, Cemb1 MyHC mRNA was initially the Fig. 4. RNA dot blot analysis of MyHC gene expression in the PM muscle during development. A: Total RNA was isolated from PM muscle at the indicated ages, from 10 days embryonic through adult (240 days). Blots were prepared, each containing identical samples of 2.5 µg total RNA isolated from the PM muscle at the indicated age. Each blot was hybridized with one of the MyHC transcript-specific, 38UTR probes, or with the fast MyHC probe, Myo-4. Each blot was washed at 5°C below the Tm50 of the respective probe. Total RNA isolated from ventricular muscle (V) from a 5-day-old chick was used as a positive control for the Cvent-specific probe. Total RNA isolated from brain (B) was used as a negative control. B: The relative percentage of each of the fast MyHC transcripts in the PM muscle during development was determined from the RNA dot-blot hybridizations shown in A. The hybridizations were quantified as described in Experimental Procedures. The normalized hybridization signals from the five fast MyHC 38UTR probes were summed, and the relative percentage was determined for each of the probes. 494 TIDYMAN ET AL. MyHC TRANSCRIPTS IN CHICKEN SKELETAL MUSCLE most abundant. From ED 10 to ED 14 there was a progressive decrease in relative Cemb1 mRNA levels. As the percentage of Cemb1 mRNA levels was decreasing, the relative abundance of Cemb2 mRNA increased between ED 10 and ED 14. However, unlike Cemb2 MyHC gene expression in the PM muscle, it continued to be abundantly expressed throughout the remainder of embryonic development. Similar to what was observed in the PM muscle, the Cemb3 MyHC gene was the predominant MyHC gene expressed from ED 14 until just prior to hatching. Relative Cemb3 mRNA levels were maximum at ED 16, accounting for approximately 60% of the MyHC mRNA transcripts. In addition, as in the PM muscle, there was an increase in the expression of the Cemb1 and Cemb2 MyHC genes just prior to hatching, however, the relative abundance of Cemb2 mRNA was much higher in the gastrocnemius muscle, accounting for approximately 50% of the MyHC mRNA transcripts at ED 20. Following hatching, the expression of MyHC genes in the gastrocnemius muscle was very different from that observed in the PM muscle. The relative abundance of Cemb2 mRNA was higher not only at hatching, but remained higher for a prolonged period in the gastrocnemius muscle. Cemb2 MyHC transcripts were still detectable at low levels in the adult gastrocnemius. In contrast to what occurred in the PM muscle, the Cemb3 MyHC gene was re-expressed in the gastrocnemius muscle following hatching. Between 5 and 14 days post-hatch, Cemb3 mRNA levels increased, accounting for approximately 25% of MyHC mRNA transcripts at 14 days post-hatch. In addition, Cemb3 mRNA continued to be present throughout post-hatch development. However, the expression of the Cemb1 MyHC gene was similar to what was observed in the PM muscle during post hatch-development. Cemb1 mRNA levels progressively decreased after hatching and were not detected after 14 days post-hatch. The expression of the Cneo MyHC gene was also quite different in the gastrocnemius muscle during post-hatch development from that observed in the PM muscle. Cneo mRNA was first detected at ED 18 in the gastrocnemius muscle. As with the PM, the relative abundance of Cneo mRNA increased following hatching; however, it continued to increase in the gastrocnemius throughout post-hatch development and accounted for approximately 80% of the MyHC transcripts in the adult gastrocnemius. In addition, in contrast to the PM muscle, little if any Cadult MyHC mRNA was detected in the gastrocnemius at any time point during development. Thus, the Cneo MyHC gene was the predominant MyHC gene expressed in the adult gastrocnemius muscle. MyHC Gene Expression in Cell Culture In order to compare the MyHC gene expression pattern observed during development in vivo to that which occurs in vitro, a primary muscle culture was established from myoblasts obtained from ED 12 pecto- 495 ralis muscle. Total RNA was isolated from culture dishes, following 1, 2, 5, 7, and 8 days of incubation. MyHC mRNA was analyzed by RNA dot-blot hybridizations using the fast MyHC 38UTR probes and a 38UTR probe specific to ventricular MyHC mRNA (Fig. 6). In addition to analysis of MyHC mRNA, the accumulation of MyHC isoforms at the protein level was analyzed by immunohistochemistry using isoform-specific monoclonal antibodies (data not shown). Myoblasts began to fuse between 1 and 2 days in culture. Fusion was complete by 3 days in culture, forming well-defined myotubes. By 5 days in culture, the developing myotubes showed cross-striations and exhibited spontaneous contractile activity. Cemb1 and Cvent mRNA transcripts were detected in myoblasts already at 1 day of incubation and in myotubes throughout their development in culture consistent with previous studies using monoclonal antibodies (Hartley et al., 1991). Cemb1 was the predominant embryonic MyHC isoform expressed in cell culture. Cemb1 mRNA was consistently detected in abundance at each time point in culture; however, Cvent mRNA levels began to decline after 5 days in culture. Cemb2 mRNA was also detected at each time point in culture—however, at much lower levels than Cemb1. Maximum Cemb2 expression was between 3 and 5 days in culture. In contrast, only trace amounts of Cemb3 mRNA could be detected at any time point in culture. Low levels of Cneo MyHC mRNA was first detected at 3 days in culture. This coincided with the first observations of occasional spontaneous contractions. The abundance of Cneo mRNA increased by 5 days in culture when spontaneous contractions were clearly apparent. Cneo mRNA levels appeared to increase throughout myotube maturation. MyHC Gene Expression in Adult Muscles Expression of the fast MyHC genes was also examined in a variety of adult muscles. Total RNA was isolated from several fast muscles, including the PM, biceps brachii, triceps, biceps femoris, quadriceps, and lateral gastrocnemius, and from several slow muscles, including the ALD, medial adductor and soleus. The autoradiogram of the RNA dot-blot hybridizations is shown in Figure 7. The relative percentage of each of the MyHC mRNA transcripts are presented in Table 1. In addition, northern hybridizations were performed on the same RNA samples using the fast MyHC transcriptspecific 38UTR probes in order to confirm the results obtained from the RNA dot-blot hybridizations. The results obtained from the northern hybridization were consistent in all respects with the RNA dot-blot hybridizations (data not shown). In the adult PM muscle only the Cadult MyHC gene was expressed. This expression pattern was unique among the fast adult muscles, since all the other fast muscles expressed one or more of the MyHC genes expressed earlier during development. With the exception of the PM muscle, there were two general patterns Fig. 5. RNA dot-blot analysis of MyHC gene expression in the gastrocnemius muscle during development. A: Total RNA was isolated from the gastrocnemius muscle at the indicated ages, from embryonic day 10 through adult (240 days). Blots were prepared and hybridized as described in the legend to Figure 4. B: The relative percentage of each of the fast MyHC transcripts in the gastrocnemius muscle during development was determined from the RNA dot-blot hybridizations shown in A. Hybridizations were quantified as described in Experimental Procedures. The normalized hybridization signals from the five fast MyHC 38UTR probes were summed, and the relative percentage was determined for each of the probes. 496 TIDYMAN ET AL. MyHC TRANSCRIPTS IN CHICKEN SKELETAL MUSCLE 497 Of the slow muscles examined, only the ALD showed no fast MyHC gene expression. The medial adductor and soleus, both slow muscles of the leg, showed signicant expression of the fast Cemb3 MyHC gene. The sum of the hybridization signals from the isoformspecific 38UTR probes was compared to the hybridization signal from the Myo-4 probe. Since Myo-4 was complementary to a conserved region in the sequences of the five fast MyHC isoforms, the Myo-4 hybridization signal was taken as an estimate of the total fast MyHC mRNA content. In the PM muscle, the hybridization signal from the Cadult 38UTR probe approximated the Myo-4 hybridization signal. However, in all the other fast adult muscles, the sum of the normalized 38UTR hybridization signals was less than the Myo-4 hybridization signal, which raises the possibility that there were additional as yet unidentified MyHC isoforms detected by Myo-4 present in these muscles. MyHC Gene Expression in Normal and Dystrophic Adult Muscles Fig. 6. RNA dot-blot analysis of MyHC gene expression in muscle cell culture. A muscle cell culture was established from myoblasts isolated from 12-day embryonic pectoralis muscle. Total RNA was isolated from muscle cell culture at 1, 2, 3, 5, 7, and 8 days of incubation. Blots were prepared, each containing identical samples of 5 µg total RNA isolated at the indicated time point. Each blot was hybridized with one of the 38UTR probes, specific to either Cvent Cemb1, Cemb2, Cemb3, or Cneo MyHC transcripts, or with the fast MyHC probe, Myo-4. The blots were washed at 5°C below the Tm50 for the respective probe. Total RNA isolated from ventricular muscle (V) from a 5-day-old chicken was used as a positive control for the Cvent-specific probe. Total RNA from 14 day embryonic pectoralis muscle (Emb. PM) was used as a positive control for the embryonic-specific probes. Total RNA isolated from a fibroblast culture (F) was used as a negative control. of MyHC gene expression in the other adult fast muscles examined. The first pattern consisted of the coexpression of the Cadult and Cemb3 MyHC genes. This pattern of expression was observed in the biceps brachii, triceps, and PLD muscles. In the PLD and triceps, the Cadult MyHC mRNA was expressed in the greatest relative abundance. However, in the biceps brachii, Cemb3 MyHC mRNA was the most abundant. The second pattern consisted of the coexpression of the Cemb3 and Cneo MyHC genes. This pattern of expression was observed in the fast muscles of the leg, including the quadricepts, biceps femoris, and lateral gastrocnemius. In these muscles, the Cneo MyHC mRNA was expressed in the greatest relative abundance. In addition, no Cadult MyHC mRNA was detected in either the biceps femoris or quadriceps muscles. A very small amount of Cadult mRNA was detected in the lateral gastrocnemius on the dot-blot hybridizations; however, the amount was close to background levels, and none was detected on the northern hybridization. In addition to the expression of the Cemb3 and Cneo MyHC genes in the fast leg muscles, the Cemb2 MyHC gene was also expressed at a very low level. The expression of the fast MyHC genes was compared in muscles from normal (line 03) and dystrophic (line 433) adult chickens. Total RNA was isolated from the PM, biceps brachii, PLD, ALD, and lateral gastrocnemius muscles from normal and dystrophic 8-monthold chickens. The autoradiogram of the RNA dot-blot hybridizations is shown in Figure 8, and the relative percentages of each of the MyHC mRNAs are presented in Table 2. The pattern of expression of the fast MyHC genes differed in fast dystrophic adult muscle from that of normal muscle. Each dystrophic fast muscle showed reduced expression of MyHC genes expressed in the normal adult muscle and a specific increase in the expression of MyHC genes expressed in that muscle during the neonatal stage of development (Bandman and Bennett, 1988). The normal adult PM muscle expressed only the Cadult MyHC gene. However, in the dystrophic PM muscle, Cadult MyHC gene expression decreased and the expression of the Cneo MyHC gene increased. In addition, there was no expression of any of the embryonic MyHC genes in dystrophic PM muscle. The adult biceps brachii and PLD muscles in the normal chicken coexpressed the Cemb3 and Cadult MyHC genes. In these muscles in the dystrophic chicken, there was a reduction in the expression of both the Cadult and Cemb3 MyHC genes and an increase in the expression of the Cneo MyHC gene. As with the PM muscle, there was no expression of either Cemb1 or Cemb2 MyHC genes in either the normal or dystrophic biceps brachii or PLD muscles. In the gastronemius muscle of the normal chicken, the Cneo MyHC gene was the most abundantly expressed. Cemb2 and Cemb3 MyHC genes were also expressed but at much lower levels. In the dystrophic gastrocnemius, in contrast to the other fast muscles, the expression of the Cneo MyHC gene decreased and 498 TIDYMAN ET AL. Fig. 7. RNA dot-blot analysis of fast MyHC gene expression in adult muscles. Blots were prepared, each with identical samples of 2.5 µg total RNA isolated from the indicated muscles. Each blot was hybridized with one of the fast MyHC transcript-specific 38UTR probes, or the fast MyHC probe, Myo-4. Each blot was washed at 5°C below the Tm50 for the respective probe. Total RNA isolated from 14-day embryonic PM muscle was used as a positive control for the embryonic-specific probes. Total RNA isolated from brain was used as a negative control. TABLE 1. The Relative Percentages of Fast MyHC Transcripts in Adult Musclesa Cemb1 Cemb2 Cemb3 Cneo Cadult % Myo4 PM 0 0 0 0 100 103 Biceps 0 0 62.7 0 37.3 69.6 Triceps 0 0 26 4.5 69.5 65.5 PLD 0 0 38 0 62 74 ALD 0 0 0 0 0 0 Gast. 0 6.4 13.6 77.6 2.4 72.1 Soleus 0 0 100 0 0 103 Quad 0 4.3 39.4 61.3 0 62.5 BF 0 0 34.8 65.2 0 54.6 MA 0 0 100 0 0 106 aThe RNA dot-blot hybridizations shown in Figure 7 were quantified as described in Experimental Procedures. The normalized hybridization signals from the fast MyHC 38UTR probes were summed, and the relative percentage of each probe was calculated. The sum of the 38UTR probe’s hybridization signals was also expressed as a percentage of the normalized Myo-4 hybridization signal. the expression of the Cemb2 and Cemb3 MyHC genes were increased, reflecting the MyHC gene expression pattern seen in the normal neonatal gastrocnemius muscle. DISCUSSION We used isoform-specific oligonucleotides to the five fast MyHC genes to examine the diversity of myosin expression in fast muscles of normal and dystrophic chickens since prior studies employing biochemical and/or immunological approaches were unable to fully examine the complexity of MyHC expression. Our study confirms that the regulation of MyHC expression in the chicken is unique compared to that observed in mammals in that transcripts of embryonic and neonatal MyHC genes represent the predominant species in many normal adult muscles, thus providing further evidence that the chicken MyHC multigene family has evolved distinct regulatory mechanisms. MyHC Gene Expression in the PM and Gastrocnemius Muscles During Development The pattern of MyHC gene expression in the PM and gastrocnemius muscle was very similar during embry- MyHC TRANSCRIPTS IN CHICKEN SKELETAL MUSCLE Fig. 8. RNA dot-blot analysis of fast MyHC gene expression in adult muscles, from (N) normal (line 03) and (D) dystrophic (line 433) chickens. Identical blots were prepared, each with replicate samples of 2.5 µg total RNA isolated from the indicated muscles. Each blot was hybridized with one of the fast MyHC transcript-specific 38UTR probes, or with the fast MyHC probe, Myo-4. The blots were washed at 5°C below the Tm50 for the respective probe. Total RNA from brain (B) was used as a negative control. onic development. The three embryonic MyHC genes were expressed in a sequential, overlapping pattern in both muscles in ovo. The Cemb1 MyHC gene was the predominant isoform expressed before ED 12. This was followed by the expression of Cemb2 which showed maximum relative expression at ED 14 and finally by the Cemb3 gene which was maximally expressed between ED 16 and hatching. Our findings are consistent with previous transcriptional studies indicating the time points at which these MyHC genes were expressed (Umeda et al., 1983; Gulick et al., 1985, 1987; Kropp et al., 1987; Lagrutta et al., 1989). It is likely that the ‘‘early’’ and ‘‘late’’ embryonic MyHC isoforms, identified with monoclonal antibodies by others (Lowey et al., 1986; VanHorn and Crow, 1989) correspond, respectively, to Cemb1 and Cemb3 MyHC genes based upon their temporal pattern of expression. The absence of any Cvent mRNA in our study is not surprising, since previous results have shown that its expression is limited to before ED 8 (Sweeny et al., 1989; Bisha and Bader, 1991). There was an increase in the expression of the Cemb1 and Cemb2 MyHC genes, and a decrease in the rate of decline in the expression of the Cemb3 gene in the PM just prior to hatching which resulted in the coexpression of all three embryonic MyHC genes in the newborn chick. A similar expression pattern occurred in the gastrocnemius muscle. This second period of expression of the embryonic MyHC genes coincides with the temporal appearance of satellite cells during muscle development (Hartley et al., 1992; Feldman and Stockdale, 1992). The incorporation of satellite cells are thought to 499 be involved in the rapid phase of muscle hypertrophy in the chick. Our observations suggest that the second period of embryonic MyHC gene expression may result from the incorporation of satellite cells into existing fibers during muscle growth, since the fusion of satellite cells is known to result in the re-expression of embryonic MyHC isoforms in regenerating muscle (Cerny and Bandman, 1987; Saad et al., 1987) and stretch hypertrophied muscle fibers (Barnett et al., 1980; Essig et al., 1991). This provides further evidence that satellite cells play a role in neonatal muscle growth. Following hatching the expression of MyHC genes diverged in the PM and gastrocnemius and began to reflect the MyHC content of the adult muscles. Based upon our present observations one can correlate specific MyHC genes with the MyHC isoforms described in previous studies using monoclonal antibodies (Bandman, 1985; Crow and Stockdale, 1986; Bandman and Bennett, 1988) and cDNA probes (Gulick et al., 1987; Kropp et al., 1987), thus providing a molecular explanation for the diversity of MyHC expression in chicken skeletal muscle. As a proportion of total MyHC mRNA, Cemb1 was predominant before ED 12, while Cemb3 was predominant at ED 18. The appearance of Cemb1 corresponds with an early phase of myogenesis and primary muscle fiber development. Fast embryonic MyHCs are known to be expressed prior to ED 4 (Sweeny et al., 1989; Bourke et al., 1991), but no transcriptional studies have been done to confirm the presence of Cemb1 mRNA prior to ED 8. Since development and growth of primary fibers proceeds normally in the absence of innervation (McLennan, 1983) and Cemb1 MyHC gene expression is the predominant embryonic isoform present in muscle cell cultures (Subramaniam et al., 1990), its expression is not dependent on innervation. On the other hand, Cemb3 was predominantly expressed only after functional innervation had occurred (Gordon and Vbrova, 1975; McLennan, 1983). Furthermore, embryos paralyzed by chronic treatment with d-tubocurarine failed to express this isoform (VanHorn and Crow, 1989), suggesting that the expression of the Cemb3 MyHC gene may be dependent on functional innervation. Our observation that muscle cell cultures failed to express this isoform further supports this conclusion. MyHC Gene Expression in Culture Cemb1 was the predominant embryonic MyHC gene expressed at each time point in culture in agreement with previous studies using monoclonal antibodies (Cerny and Bandman, 1986; VanHorn and Crow, 1989), as well as with studies in which an oligonucleotide probe was used to demonstrate the presence of the N118 transcript in culture (Subramaniam et al., 1990). We demonstrate for the first time that Cemb2 mRNA is also expressed in culture. Similar to what was observed in vivo, Cemb2 was detected at lower levels than Cemb1. However, in contrast to what was observed in vivo, little Cemb3 mRNA was detected in vitro. The 500 TIDYMAN ET AL. TABLE 2. The Relative Percentages of Fast MyHC Transcripts in Normal and Dystrophic Adult Musclesa PM Probe Cemb1 Cemb2 Cemb3 Cneo Cadult % Myo4 Norm. 0 0 0 0 100 98.5 Dys. 0 0 0 36.9 62.9 96 Biceps Norm. 0 0 65.4 0 34.6 57.8 Dys. 0 0 14.6 52.6 32.8 45.2 ALD Norm. Dys. 0 0 0 0 0 0 0 0 0 0 0 0 PLD Norm. 0 0 41.0 0 58.9 69.9 Dys. 0 0 31.6 34.0 34.3 58.1 Gastrocnemius Norm. Dys. 0 0 7.8 13.0 5.3 54.1 86.7 32.9 0 0 68.0 96.6 aThe relative percentage of each of the fast MyHC mRNA transcripts in adult muscles from normal (line 03) and dystrophic (line 433) chickens. The RNA dot-blot hybridizations shown in Figure 9 were quantified as described in Experimental Procedures. The normalized hybridization signals from the fast MyHC 38UTR probes were summed, and the relative percentage for each probe was calculated. The sum of the 38UTR probe’s hybridization signals was also expressed as a percentage of the normalized Myo-4 hybridization signal. absence of Cemb3 expression in culture may reflect the aneural cell culture environment, consistent with the studies showing that expression of a ‘‘late’’ embryonic MyHC isoform was blocked in curarized chick embryos (VanHorn and Crow, 1989). This suggests that Cemb3 expression may be dependent on functional nerve– muscle interactions. This interpretation is also supported by electrical stimulation studies which showed that a ‘‘late embryonic’’ MyHC protein was detected by SDS-polyacrylamide gel electrophoresis (PAGE) following electrical stimulation of myogenic cultures (Dusterhoft and Pette 1990). Previously we demonstrated using monoclonal antibodies that contractile activity is required for the expression of the Cneo MyHC isoform in cell culture (Cerny and Bandman, 1986). In the present study, the expression of Cneo mRNA also corresponded to the appearance of spontaneous contractile activity. Apparently spontaneous contractions are not sufficient to induce Cemb3 MyHC gene expression. Thus the differences in expression between Cemb3 and Cneo in culture suggest that distinct regulatory mechanisms are involved in determining transcriptional activity of these genes. MyHC Gene Expression in Adult Muscles MyHC composition of the adult PM is unique in that it was the only muscle in which the Cadult MyHC gene was exclusively expressed. In other fast muscles two distinct patterns of MyHC gene expression were observed. One pattern, found in the biceps, triceps, and PLD, consisted of the coexpression of the Cadult and Cemb3 MyHC genes. A second pattern, present in the fast leg muscles, consisted of the coexpression of the Cneo and Cemb3 MyHC genes. Previous studies had shown that diversity existed in MyHC expression in adult fast muscles other than the PM (Crow and Stockdale, 1986; Bandman and Bennett, 1988). Our results are the first to show that the Cemb3 MyHC gene is abundantly expressed in all adult muscles with the exception of the PM and ALD. In all of the adult muscles examined, except the PM and ALD, the sum of the normalized hybridization signals from the 38UTR probes was much lower than the hybridization signal from the fast MyHC probe, Myo-4. Since Myo-4 is complementary to a conserved region among the five fast MyHC gene sequences, this may indicate the presence of additional unidentified fast MyHC transcripts in these muscles. The presence of an additional adult fast MyHC isoform is consistent with studies of seven fast MyHC genomic clones by Robbins et al. (1986). One of the clones identified a transcript that was expressed in adult gastrocnemiums muscle but was not expressed in the PM at any stage of development. Additional evidence for a second adult fast MyHC isoform expressed in leg muscles has also been reported (Umeda et al., 1983). MyHC Gene Expression in Avian Muscular Dystrophy MyHC genes expressed in a given adult dystrophic muscle correspond to those expressed in the normal muscle during neonatal development. In addition, MyHC genes which were expressed in normal adult muscle were expressed at lower levels in dystrophic muscle. No fast MyHC genes were expressed in the dystrophic ALD muscle, consistent with previous findings that slow-tonic muscle is unaffected in avian muscular dystrophy (Ashmore and Doerr, 1971). It has been reported that the expression of immature MyHC isoforms in Duchenne’s muscular dystrophy is the result of muscle fiber regeneration (Webster et al., 1988). In contrast, in avian muscular dystrophy, no evidence of muscle fiber regeneration has been observed (Yorita et al., 1980). Muscle regeneration is characterized by the recapituation of MyHC expression during normal development (Cerny and Bandman, 1987; Saad et al., 1987). The absence of Cemb1 and Cemb2 transcripts in dystrophic muscle further suggests that avian muscular dystrophy does not induce muscle regeneration, since in dystrophic muscle there was not a general re-expression of immature MyHCs, but a specific expression of those transcripts present during normal neonatal development. Although the specific mechanism by which MyHC genes normally expressed during neonatal development are induced in dystrophic muscle is not known, avian muscular dystrophy may prove to be a useful model in elucidating the MyHC TRANSCRIPTS IN CHICKEN SKELETAL MUSCLE regulatory mechanisms which govern MyHC gene expression during development. Functional Role of Developmental MyHC Isoforms At the present time, the role of developmentally distinct MyHC isoforms is not known; however, evidence suggests that they may have functionally distinct properties during fibrillogenesis. In developing muscle fibers, the neonatal MyHC isoform was not randomly distributed in native thick filaments (Taylor and Bandman, 1989). In addition, individual MyHC isoforms interact only as homodimers during the thick filament formation in the chicken (Kerwin and Bandman, 1991). Although there is an overall high degree of sequence homology among the chicken fast MyHC isoforms, we have previously identified specific regions of diversity in the amino acid rod sequence which may be responsible for these observed unique properties during fibrillogenesis (Moore et al., 1992). The ‘‘hinge’’ region of the rod was also found to be very divergent, suggesting possible differences in the contractile properties among the chicken fast MyHC isoforms, although no differences in the myosin ATPase activities have been observed (Lowey et al., 1986). Sequence variation in the ‘‘hinge’’ region in Drosophila, produced by alternate exon splicing, results in myosin isoforms with vastly different contractile properties (Collier et al., 1990). Regulation of the Chicken MyHC Gene Family In this study we show that there are different patterns of MyHC expression seen in different chicken muscle groups. We also show that avian muscular dystrophy is associated with alterations in the pattern of MyHC gene expression. While neural influences and/or patterns of activity have been implicated as factors coordinating the regulation of MyHC gene transcription, the mechanisms by which MyHC gene families are differentially regulated following muscle differentiation are unknown. While the muscle regulatory factor (MRF) family has been shown to activate genes involved in muscle differentiation (see reviews by Olson, 1990; Weintraub, 1993; Olson and Klein, 1994) and are differentially expressed during embryonic development (Buckingham, 1992), the role of MRFs in the regulation of MyHC genes during muscle maturation is unclear (Ontell et al., 1995; Smith et al., 1993). The cooperative role of other transcription factor families, such as the MEF2 family, may also be essential in the developmental regulation of MyHC genes (see Olson et al., 1995, for review). In addition, recent evidence has shown that the mouse adult IIB and the chicken Cemb1 MyHC promoters share a conserved MEF2 binding element which may activate gene expression via an E-Box–independent mechanism (Takeda et al., 1995). However, since the regulatory regions of the other fast MyHC genes have not been analyzed for functional elements, it is unknown whether there are unique cis-regulatory sequences that may contribute to the 501 diversity of gene expression observed in this study. Nevertheless, it is clear that patterns of MyHC gene activation in the chicken are not analogous to those observed in mammals and that future studies aimed at elucidating the molecular mechanisms of differential gene activation in the chicken are likely to uncover distinct pathways from those found in mammalian systems. EXPERIMENTAL PROCEDURES Animals The animals used in this study were White Leghorn chickens (Gallus domesticus) from the inbred line 03 and its genetically related dystrophic line, 433 (Asmundson and Julian, 1956; Wilson, et al., 1979). Chickens and fertile eggs were obtained from the Department of Avian Sciences, University of California, Davis. All chickens and embryos were sacrificed by decapitation. RNA Isolation Total RNA was isolated from the indicated tissues using the guanidinium isothiocyanate method followed by CsCl ultracentrifugation as described by Chirgwin et al. (1979). Total RNA concentration was determined spectrophotometrically, and the integrity of the RNA was examined on ethidium bromide-stained agarose gels (Sambrook et al., 1989). Total RNA was isolated from the pectoralis major (PM) and lateral gastrocnemius muscles, pooled from five to ten embryos of line 03, at 10, 12, 14, 16, 18, and 20 days of embryonic development. The developmental stages of the embryos were determined according to the criteria of Hamburger and Hamilton (1951) and corresponded to stages 36, 38, 40, 42, 44, and 45, respectively. Total RNA was also prepared from the PM and lateral gastrocnemius muscles, pooled from two to three line 03 birds, at hatching and at 2, 5, 7, 10, 14, 35, and 240 days after hatch. In addition, total RNA was isolated from a variety of adult skeletal muscles, including the biceps brachii, triceps, posterior latissimus dorsi (PLD), anterior latissimus dorsi (ALD), quadriceps, biceps femoris, medial adductor, and soleus. From the dystrophic 433 line, total RNA was isolated from adult PM, biceps brachii, PLD, ALD, and lateral gastrocnemius muscle. MyHC Transcript-Specific Oligonucleotide Probes Based on sequence analysis of the fast MyHC genes, regions of diversity within the 38 untranslated region (38UTR) of each gene were selected as targets for transcript-specific oligonucleotide probes. The MyHC gene sequences were obtained from the previously published complete gene sequence of Cemb1 (Molina et al., 1987) and the sequences of cDNA clones coding for the rod portion of the Cemb2, Cemb3, Cneo, and Cadult isoforms (Moore et al., 1992). These MyHC cDNA sequences are listed in the EMBL/GeneBank library under the accession numbers M74085 (Cemb2), M74086 (Cemb3), M74087 (Cneo), and M74084 (Cadult). In 502 TIDYMAN ET AL. addition, a region of complete sequence identity among all five of the fast MyHC genes, in exon 40, was selected as a target for a ‘‘generic’’ fast MyHC oligonucleotide probe for use as a positive control. The nucleotide sequences of the oligonucleotide probes are shown in Figure 1. The Cvent oligo was based upon the published sequence for VMHC1 (S64689) (Bisha and Bader, 1991). Oligonucleotide Probe Labeling Oligonucleotide probes were labeled by the addition of a homopolymeric tail of [a32P]dATP (6,000 Ci/mmol, Amersham) to the 38 end using terminal deoxynucleotide transferase (Collins and Hunsaker, 1985). In a total volume of 40 µl, 2 pmoles of oligonucleotide were incubated with 100 µCi [a32-P]dATP in 100 mM sodium cacodylate (pH 7.2), 2 mM CaCl2, 0.2 mM 2-b-mercaptoethanol, and 40 units of enzyme. The reaction was stopped by the addition of 40 µl STE buffer (100 mM NaCl, 20 mM Tris-HCl, 10 mM EDTA, pH 7.5) and chilling on ice. Probes were purified using (NUCTRAP) push columns from Stratagene (La Jolla, CA) according to the manufacturer’s directions. This labeling method produced probes with specific activities of 2–4 3 109 cpm/µg. RNA Dot-Blot and Northern Hybridizations RNA electrophoresis and northern transfer were performed according to the methods described by Fourney et al. (1988). Samples consisted of 10–15 µg denatured total RNA. Electrophoresis was carried out on 1.2% agarose, 0.66 M formaldehyde gels. Following electrophoresis, RNA was transferred to Hybond-N (Amersham, Arlington Heights, IL) nylon membrane and bound by UV-crosslinking at 120 mJ (Church and Gilbert, 1984). Sets of six identical RNA dot blots were prepared, one for each of the probes used. Fifteen micrograms total RNA was denatured in a total volume of 300 µl containing 50% formamide, 2 M formaldehyde and 13 MOPS buffer (0.4 M morpholinopropanesulfonic acid, 100 mM sodium acetate, 10 mM EDTA) by heating at 65°C for 10 min (Sambrook et al., 1989). Following denaturation, an equal volume of ice-cold 203 standard saline citrate (SSC) was added, and the sample was chilled on ice. Identical blots were prepared by vacuum blotting equal volume aliquots, each containing 2.5 µg total RNA, onto individual strips of Hybond-N nylon membrane using a vacuum manifold dot-blotting apparatus (Minifold II) from Schleicher and Schuell (Keene, NH). The manifold’s sample wells were rinsed with 500 µl 103 SSC. The blots were air dried, and the RNA covalently bound to the nylon membrane by UV-crosslinking at 120 mJ (Church and Gilbert, 1984). Dot-blots and northern blots were prehybridized in 63 SSC, 103 Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), and 0.5 mg/ml denatured fractionated salmon sperm DNA for 5–6 hr at 50°C, with gentle agitation. Following prehybridization, 2 pmoles of labeled probe was added to each prehybridization solu- tion and hybridized for 24 hr at 50°C, with gentle agitation. Following hybridization, the blots were washed two times in 23 SSC, 0.1% SDS at room temperature for 15 min. This was followed by two 15-min high-stringency washes in 0.53 SSC, 0.1% SDS, at 5°C below the Tm50 for each respective probe. The Tm50 of each probe was determined empirically from the thermal dissociation curve of the hybridization of each probe to its homologous mRNA (data not shown). Quantitation of RNA Dot Blots In order to determine the relative level of expression of the five fast MyHC genes, the RNA dot-blot hybridizations were radiographically quantitated, and the abundance of each gene’s transcript was calculated with respect to the others. Following autoradiography, each hybridized dot-blot sample was individualized, placed in 5 ml scintillation fluid (Beckman, ReadySafe), and the total counts were determined using a scintillation counter (Beckman). The counts obtained from each hybridized dot-blot sample were corrected for nonspecific background hybridization by subtracting the counts obtained from the hybridization of each respective probe to total RNA isolated from brain. To allow comparisons of the hybridizations utilizing the different probes, the hybridization signal counts for each RNA dot-blot sample were normalized to account for differences in the specific activities of the probes. Plasmid DNA corresponding to each of the fast MyHC isoforms was hybridized and washed in parallel with the RNA dot blots. Since each plasmid has an equal amount of target sequence for its respective 38UTR probe and the Myo-4 probe, the hybridization of each 38UTR probe was compared to that of Myo-4 on the duplicate DNA samples. The ratio of the hybridization signals from the 38UTR probe to that of Myo-4 was used as a normalization factor for the hybridization of the 38UTR probe to the RNA dot-blot samples. The normalized hybridization signals from the five 38UTR probes on a set of RNA dot blots were summed, and the relative percentage of each was calculated. In addition, the sum of the hybridization signals from the five 38UTR probes was also expressed as a percentage of the Myo-4 hybridization signal which estimated the total fast MyHC mRNA content. Muscle Cell Cultures Primary muscle cell cultures were prepared from myoblasts isolated from 12-day embryonic pectoralis muscle according to methods described by Bandman et al. (1982). Myoblasts were plated at a density of 1 3 106 cells per culture dish on 0.1% collagen-coated 60-mm culture dishes. The culture medium consisted of 88% Hams F-10, 1 mM L-glutamine, 10% horse serum, 2% embryo extract, and 1 mM CaCl2. No antibiotic or antifungal agents were used. The cell cultures were incubated at 37°C in an atmosphere containing 5% MyHC TRANSCRIPTS IN CHICKEN SKELETAL MUSCLE CO2. The cultures were grown for 1–8 days, with the culture medium replaced every 3rd day. ACKNOWLEDGMENTS The authors would like to acknowledge Maria Arrizubieta and Macdonald Wick for their helpful suggestions during the course of this study and Dr. Zipora YablonkaReuveni for thoughtful comments on the preparation of this manuscript. This work was supported by grants from NIH (AG08573) and USDA (94-37205-1034) to E.B. REFERENCES Ashmore, C.R., and Doerr, L. (1971) Postnatal development of fiber types in normal and dystrophic skeletal muscle of the chick. Exp. Neurol. 30:431–446. Asmundson, V.S., and Julian, L.M. (1956) Inherited muscle abnormality in the domestic fowl. J. Hered. 47:248–252. Bader, D., Masaki, T., and Fischman, D.A. (1982) Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J. Cell Biol. 95:763–770. Bandman, E. (1985) Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle. Science 227:780–782. Bandman, E., and Bennett, T. (1988) Diversity of myosin heavy chain expression during the development of the gastrocnemius, bicep brachii, and posterior latissimus dorsi of the normal and dystrophic chicken. Dev. Biol. 130:220–231. Bandman, E., Matsuda, R., and Strohman, R.C. (1982) Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle. Dev. Biol. 93:508–518. Bandman, E., Cerny, L.C., and Bennett, T. (1989) Myogenic and non-myogenic factors regulate the expression of myosin heavy chains in developing and regenerating skeletal muscle. In: ‘‘Cellular and Molecular Biology of Muscle Development,’’ Kedes, L. and F.E. Stockdale (eds). New York: Alan R. Liss Inc., pp 429–439. Barnett, J.G., Holly, R.G., and Ashmore, C.R. (1980) Stretch-induced growth in chicken wing muscles: biochemical and morphological characterization. Am. J. Physiol. 239:C39–C46. Bisha, J.G., and Bader, D. (1991) Identification and characterization of a ventricular-specific avian myosin heavy chain, VMHC1: expression in differentiating cardiac and skeletal muscle. Dev. Biol. 148:355–364. Bourke, D.L., Wylie, S.R., Wick, M., and Bandman, E. (1991) Differentiating skeletal muscles initially express a ventricular myosin heavy chain. Basic Appl. Myol. 1:13–21. Buckingham, M.E. (1985) Actin and myosin multigene families: their expression during the formation of skeletal muscle. Essays Biochem. 20:77–109. Buckingham, M. (1992) Trends In Genetics. 8:144–148. Cerny, L.S., and Bandman, E. (1986) Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures. J. Cell Biol. 103:2153–2161. Cerny, L.S., and Bandman, E. (1987) Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle. Dev. Biol. 119:350–362. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299. Church, G.M., and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81:1991–1995. Collier, V.L., Kornert, W.A., O’Donnel, P.T., Edwards, K.A., and Bernstein, S.I. (1990) Alternate myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed. Genes Dev. 4:885–895. Collins, M.L., and Hunsaker, W.R. (1985) Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 58-end-labeled oligo-nucleotide probes and nick-translated plasmid probes. Anal. Biochem. 151:211–224. Crow, M.T., and Stockdale, F.E. (1986) The developmental program of 503 fast myosin heavy chain expression in avian skeletal muscles. Dev. Biol. 118:333–342. Dusterhoft, S., and Pette, D. (1990) Effects of electrical induced contractile activity on cultured embryonic chick breast muscle cells. Differentiation 44:178–184. Essig, D.A., Devol, D.L., Bechtel, P.J., and Trannel, T.J. (1991) Expression of embryonic myosin heavy chain mRNA in stretched adult chicken skeletal muscle. Am. J. Physiol. 260:C1325–C1331. Feldman, J.L., and Stockdale, F.E. (1992) Temporal appearance of satellite cells during myogenesis. Dev. Biol. 153:217–226. Fourney, R.M., Miyakashi, J., Day, R.S., and Paterson, M.C. (1988) Northern blotting: efficient RNA staining and transfer. Focus 10: 5–7. Gordon, T., and Vrbrova, G. (1975) Differentiation of fast and slow chick muscles. Pflugers Arch. 360:199–218. Gulick, J., Kropp, K., and Robbins, J. (1985) The structure of two fast-white myosin heavy chain promoters. J. Biol. Chem. 269:14513– 14520. Gulick, J., Kropp, K., and Robbins, J. (1987) The developmentally regulated expression of two linked myosin heavy-chain genes. Fed. Eur. Biochem. Soc. Lett. 169:79–84. Hamburger, V., and Hamilton, H. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 99:49–92. Hartley, R.S., Bandman, E., and Yablonka-Reuveni, Z. (1991) Myoblasts from fetal and adult skeletal muscle regulate myosin expression differently. Dev. Biol. 148:249–260. Hartley, R.S., Bandman, E., and Yablonka-Reuvini, Z. (1992) Skeletal muscle satellite cells appear during late chicken embryogenesis. Dev. Biol. 153:206–216. Hoffman, E.P., Hudecki, M.S., Rosenberg, P.A., Pollina, C.M., and Kunkel, L.M. (1988) Cell and fiber-type distribution of dystrophin. Neuron 1:411–420. Hofmann, S., Dusterdoft, S., and Pette, D. (1988) Six myosin heavy chain isoforms are expressed during chick breast muscle development. FEBS Lett. 238:245–248. Kerwin, B., and Bandman, E. (1991) Assembly of avian muscle myosins: evidence that homodimers of the heavy chain subunit are the thermodynamically stable form. J. Cell Biol. 113:311–320. Kropp, K., Gulick, J., and Robbins, J. (1987) A canonical sequence organization at the 58-end of the myosin heavy chain genes. J. Biol. Chem. 262:16536–16545. Lagrutta, A.A., McCarthy, J.G., Scherczinger, C.A., and Heywood, S.M. (1989) Identification and developmental expression of a novel embryonic myosin heavy-chain gene in chicken. DNA 8:39–50. Leinwand, L.A., Saez, L., McNally, E., Nadal-Ginard, B. (1983) Isolation and characterization of human myosin heavy chain genes. Proc. Natl. Acad. Sci. U.S.A. 80:3716–3720. Lowey, D., Sartore, S., Gauthier, G.F., Waller, G.S., and Hobbs, A.W. (1986) Myosin isozyme transitions in embryonic chicken pectoralis muscle. In: ‘‘Molecular Biology of Muscle Development,’’ Emerson, C.P., Fischman, D., Nadal-Ginard, B., Siddiqui, M. (eds). New York: Alan R. Liss, Inc., pp 225–236. McLennan, I.S. (1983) Neural dependence and independence of myotube production in chicken hindlimb muscles. Dev. Biol. 98:287–294. Molina, M.I., Kropp, K.E., Gulick, J., and Robbins, J. (1987) The sequence of an embryonic myosin heavy chain gene and isolation of its corresponding cDNA. J. Biol. Chem. 262:6478–6488. Monaco, A.P., Neve, R.L., Colletti-Feener, C., Bertelson, C.J., Kurnit, D.M., and Kunkel, L.M. (1986) Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature 323:646– 650. Moore, L.A., Arrizubieta, M.J., Tidyman, W.E., Herman, L., and Bandman, E. (1992) Analysis of the chicken fast myosin heavy chain family: localization of isoform-specific antibody epitopes and regions of divergence. J. Mol. Biol. 225:1143–1151. Nguyen, H.T., Gubits, R.M., Wydro, R.M., and Nadal-Ginard, B. (1982) Sarcomeric myosin heavy chain is coded by a highly conserved multigene family. Proc. Natl. Acad. Sci. U.S.A. 79:5230–5234. Olson, E.N. (1990) MyoD family: a paradigm for development? Genes Dev. 4:1454–1461. Olson, E.N., and Klein, W.H. (1994) bHLH factors in muscle develop- 504 TIDYMAN ET AL. ment: dead lines and commitments, what to leave in and what to leave out. Genes Dev. 8:1–8. Olson, E.N., Perry, M., and Schulz, R.A. (1995) Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev. Biol. 172:2–14. Ontell, M., Ontell, M.P., and Buckingham, M. (1995) Muscle-specific gene expression during myogenesis in the mouse. Microsc. Res. Tech. 30:354–365. Robbins, J., Horan, T., Gulick, J., and Kropp, K. (1986) The chicken myosin heavy chain family. J. Biol. Chem. 261:6606–6612. Rushbrook, J.I., Weiss, C., and Yao, T. (1991) Developmental myosin heavy chain progression in avian type IIB muscle fibers. J. Muscle Res. Cell Motil. 12:281–291. Saad, A.D., Obinata, T., and Fischman, D.A. (1987) Immunochemical analysis of protein isoforms in thick myofilaments of regenerating skeletal muscle. Dev. Biol. 11:336–349. Sambrook, J., Fritsch, E., and Maniatis, T. (1989) ‘‘Molecular Cloning: A Laboratory Manual,’’ 2nd ed. Cold Spring Harbor, NY: Cold Spring Laboratory. Smith, T.H., Block, N.E., Rhodes, S.J., Konieczny, S.F., and Miller, J.B. (1993) A unique pattern of expression of the four muscle regulatory factor proteins distinguishes somitic from embryonic, fetal and newborn mouse myogenic cells. Development 117:1125–1133. Subramaniam, A., Gulick, J., and Robbins, J. (1990) Analysis of the upstream regulatory region of a chicken skeletal myosin heavy chain gene. J. Biol. Chem. 265:13986–13994. Sweeny, L.J., Kennedy, J.M., Zak, R., Kokjohn, J., and Kelley, S.W. (1989) Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis. Dev. Biol. 133:361–374. Takeda, S., North, D.L., Diagana, T., Miyagoe, Y., Lakich, M.M., and Whalen, R.G. (1995) Myogenic regulatory factors can activate TATA-containing elements via an E-Box independent mechanism. J. Biol. Chem. 270:15664–15670. Taylor, L.D., and Bandman, E. (1989) Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle. J. Cell Biol. 108:533–542. Umeda, P.K., Kavinsky, C.J., Sinha, A.M., Huey-Juang, H., and Jakovcic, S. (1983) Cloned mRNA sequences for two types of embryonic myosin heavy chains from chick skeletal muscle. II. Expression during development using S1 nuclease mapping. J. Biol. Chem. 258:5206–5214. VanHorn, R., and Crow, M.T. (1989) Fast myosin heavy chain expression during early and late embryonic stages of chicken skeletal muscle development. Dev. Biol. 134:279–288. Webster, C., Silberstein, L., Hays, A.P., and Blau, H.M. (1988) Fast muscle fibers are preferentially affected in Duchenne Muscular Dystrophy. Cell 52:503–513. Weintraub, H. (1993) The MyoD family and myogenesis: redundancy, networks and thresholds. Cell 75:1241–1244. Whalen, R.G., Sell, S.M., Butler-Browne, G.S., Schwartz, K., Bouveret, P., and Pinset-Harstrom, I. (1981) Three myosin heavy chain isozymes appear sequentially in rat muscle development. Nature 292:805–809. Wilson, B.W., Randall, W.R., Patterson, G.T., and Entrikin, R.K. (1979) Major physiological and histochemical characteristics of inherited dystrophy in the chicken. Ann. N.Y. Acad. Sci. 317:224–246. Winkelmann, D.A., Lowey, S., and Press, J.L. (1983) Monoclonal antibodies localize changes on myosin heavy chain isoenzymes during avian myogenesis. Cell 34:295–306. Wydro, R.M., Nguyen, H.T., Gubits, R.M., and Nadal-Ginard, B. (1983) Characterization of sarcomeric myosin heavy chain genes. J. Biol. Chem. 258:670–678. Yorita, T., Nakamura, H., and Nonaka, I. (1980) Satellite cells and muscle regeneration in the developing dystrophic chicken. Exp. Neurol. 70:567–575.