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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
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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.
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