Myosin expression and specialization among the earliest muscle fibers of the developing avian limb

DEVELOPMENTAL

113,238-254 (1986)

BIOLOGY

Myosin Expression and Specialization among the Earliest Muscle Fibers of the Developing Avian Limb MICHAEL Depurtment Received

T. CROWS AND FRANK

of Medicine, May

Stanford

University,

16, 1985; accepted

E. STOCKDALE Stanford,

in revisedjkrm

California

August

9&‘&i

15, 1985

Monoclonal antibodies specific to the lightand heavy-chain subunits of chicken skeletal muscle myosin have been used to identify fast and slow myosin-containing fibers in the thigh muscles of embryonic and adult chickens and to determine when in development diversification of muscle fiber types first occurs. Primary generation fibers which expressed different MLC and MHC types were evident within the dorsal and ventral premuscle masses and in the first muscles to form in the limb. These early embryonic muscle fiber types became distributed among and within the individual muscles of the thigh in a characteristic spatial pattern which served as a “blueprint.” for guiding future muscle development and predicting the future fiber composition of the muscle. Despite the continuous addition of muscle fibers to the limb throughout development, the pattern remained unchanged. Neither the time of appearance, initial specialization, nor characteristic distribution of these primary fiber types within the limb was altered during the early embryonic period by chronic neuromuscular paralysis induced by D-tubocurarine. In contrast, muscles at later stages of embryonic development were markedly affected by such treatments and underwent atrophy and loss of differential staining characteristics. These results demonstrate that diversification of fibers in terms of myosin content is one of the earliest events in the formation of these muscles and suggest that the development of avian muscles be divided into two phases: an embryonic phase during which fibers of differing myosin content appear independently of innervation to become distributed in a specific topographic pattern within each muscle as it forms, followed by a fetal phase during which innervation becomes essential for maintaining this pattern and modulating the myosin content of its fibers. IZZ1986 Academic Press, Inc.

Crow et ab, 1983a,b; Lowey et ah, 1983; Shafig et ab, 1984). Because of the multiplicity of isoform types and factors that can influence their expression, the cellular basis for the divergence of muscle fibers into fast and slow fiber types has remained unclear. The developmental origin of the different muscle fiber types has been pursued using a variety of cytological and biochemical techniques but differences in the interpretation of these measurements have emerged due to differences in the methods of analyses and in the species and embryonic ages studied. A number of studies which have relied on differences in the pH stability of the myofibrillar ATPase to identify different fiber types have shown that the early fibers which occupy the developing limbs do exhibit differences (Ashmore et al., 1972; Butler and Cosmos, 1981; McLennan, 1983a; Phillips and Bennett, 1984). In contrast, analyses of developing animals with antisera specific for the fast and slow myosins of the adult have shown that muscles containing fibers with differing morphologies and antibody reactivity patterns do not appear in mammals until later embryonic stages (Gauthier et al., 1978,1982; Rubinstein and Kelly, 1981). While all these data indicate that differences among fibers first appear in the embryo, it is not known if muscle fibers differing in molecular isoform composition are found among the earliest muscles of the embryo or if

INTRODUCTION

Most vertebrate skeletal muscles consist of a heterogeneous population of fibers displaying a wide range of biochemical and physiological properties. At the molecular level, differences in these properties have been closely linked to the expression of isoforms of a number of contractile and metabolic proteins (Peter et al., 1972; Whalen et al, 1981, 1982; Barnard et ah, 1982). The particular pattern of metabolic and contractile isoforms expressed by a given fiber is not invariant but subject to developmental influences and modulation caused by changing pattern of usage, innervation, and hormonal status (Streter et al., 1976; Ianuzzo et al., 197’7; Winder et al, 1980; Saltin and Gollnick, 1984). Fibers have been designated as “fast” and “slow” based upon differences in their mechanical properties, ATPase staining, and myosin isoform content (Burke and Edgerton, 1975; Guth and Samaha, 1970). Multiple “fast” and “slow” isoforms of the myosin heavy chain (MHC) and light chain (MLC) are known to exist and to be expressed in both fiberspecific and developmental stages-specific fashions (Hoh and Yeoh, 1979; Whalen et ab, 1981; Bandman et al, 1982; 1 To whom correspondence ology, University of Houston,

should be addressed: Department of BiUniversity Park, Houston, Tex. 77004.

OOlZ-1606/86 $3.00 Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved

238

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the motor nervous system plays a significant role in establishing such differences. As an approach to understanding the developmental origin of different fiber types, we have used protein analyses and monoclonal antibodies to specific subunits of myosin to identify, quantitate, and determine the time course of myosin isoform transitions in avian muscle development (Stockdale et al., 1981; Crow and Stockdale, 1983; Crow et al., 1983a,b). The results obtained from this approach confirmed and extended the observations of others that there are specific periods of transition in myosin isoform expression common to both developing fast and slow muscles and that both fast and slow myosin isoforms are expressed in embryonic muscle regardless of the type of muscle to which they give rise. These studies, however, did not provide information on the cellular distribution of these isoforms and on the chronology of appearance of distinct fiber types. Because monoclonal antibodies specific to the fast and slow isoforms of both the MHC and MLC subunits are particularly effective probes for defining muscle fiber types, we have used such antibodies to determine the appearance of fast and slow muscle fiber types during development. We find that differences in both MLC and MHC expression exist among the first fibers to populate the developing hindlimb. The embryonic fiber types defined by these differences are distributed within each muscle in a unique and characteristic arrangement that served as a “blueprint” for guiding future fiber development and predicting future muscle function. The establishment of the different fiber types and their “blueprints” occurs in the absence of functional neuromuscular contacts.

MATERIALS

AND

METHODS

Chick embryos. White Leghorn eggs were obtained from a local hatchery (Donsing Farms, San Rafael, Calif.). The eggs were incubated at 38°C in a humidified, forced-draught incubator and staged according to the morphological guidelines of Hamburger and Hamilton (1951). With the exception of the curare-treated embryos, the designation “days in ova” refers to the corresponding morphological stage, regardless of the actual time of incubation. Extraction of myosin. Muscles were dissected, minced into small pieces, and homogenized in a Waring blender for 20 set at 4°C in lo-15 vol of low-salt buffer (LSB) containing 20 mM KCl, 2 mM sodium phosphate buffer (pH 6.8), and 1 mM EGTA. The precipitate was washed three times in LSB and then extracted in 3 vol (based on the original weight of tissue) of myosin extracting solution containing 0.6 M KCl, 10 mM potassium phosphate buffer (pH 6.8), 1 mMNaqPe07, 1 mMdithiothreito1

of Muscle

Fibw

Typm

239

(DTT), 5 mM MgClz, and 1 mM EGTA. The supernatant from this extraction was saved and combined with that from a second extraction of the pellet and dialyzed overnight against a X LSB. The resulting crude myosin precipitate was collected by centrifugation and dissolved in 80 mMNa4P207, 2 mMMgC12, and 2 mMEGTA (pH 9.5). Undissolved material was removed by centrifugation and an equal volume of glycerol added to the supernatant. All preparations were stored at -20°C. PuriIcation of myosin. Myosin was purified using a modification (Pollard, 1982) of the method of Keilley and Bradley (1956). Briefly, isolated muscles containing a mixture of fast and slow muscle fiber types were homogenized in a meat grinder and then extracted in 0.6 M KCl, 10 mM potassium phosphate (pH 6.8), 1 mM Na4P207, 1 mM DTT, 5 mM MgClz, 1 mM EGTA in at a tissue:buffer ratio of 1:3. The extract was clarified by centrifugation and dialyzed against 5 mMsodium-phosphate buffer (pH 6.8) for 4 hr. The precipitated myosin was dissolved in i vol (based on the original weight of muscle) of 2 M KCl, 0.5 mM DTT, 25 mM EGTA in 0.2 M potassium phosphate buffer (pH 6.8), and dialyzed overnight at 4°C against 0.6 M KCl, 10 mM EDTA, 1 mMDTT in 25 mMpotassium phosphate buffer (pH 6.8). An equal volume of cold distilled water was then added to the contents of the dialysis sack which was then stirred for 30 min at 4°C. The resulting precipitate of actomyosin was collected by centrifugation and discarded. The myosin remaining in solution was reprecipitated and collected by centrifugation. Preparation of myosin subfragments. Light meromyosin (LMM), rod, and subfragment 1 (Sl) were prepared as described by Weeds and Taylor (1975) from myosin purified from muscles composed of a mixture of fast and slow fiber types. For LMM subfragments, the myosin was diluted to a concentration of 15-20 mg/ml and dialyzed overnight against two changes of 0.5 M KCl, 0.05 M potassium phosphate buffer (pH 6.5). This dialysate was then warmed to room temperature and reacted with cu-chymotrypsin (type CDS; Worthington Biochemicals, Freehold, N. J.) at 50 pg/ml for 7.5 min. This reaction was terminated by the addition of phenylmethylsulfonylfluoride (PMSF) to a final concentration of 1 mM and the reaction products were dialyzed overnight at 4°C against 20 mMNaC1, 1 mMPMSF, in 5 mM sodium-phosphate buffer (pH 7.0). LMM was recovered by low-salt precipitation. For the production of rod and Sl, purified myosin was diluted to 12.5 mg/ml and dialyzed at 4°C overnight against 0.12 M NaCl, 5 mM ethylenediamine EDTA in 20 mM sodium-phosphate buffer (pH 7.0) to induce the formation of “mini-filaments.” The dialysate was then warmed to room temperature and reacted with a-chymotrypsin as above. The reaction was terminated after

240

DEVELOPMENTAL BIOLOGY

7.5 min by the addition of PMSF to 1 mM and the products dialyzed as above and then subjected to centrifugation. The Sl present in the supernatant was purified by ammonium sulfate fractionation between 43 and 58% saturation. The rod was recovered from the dissolved precipitate by ammonium sulfate fractionation between 40 and 60% saturation. Mono&ma1 antibodies. The production and characterization of monoclonal antibodies to fast and slow MLCs has been described elsewhere (Crow et al., 198313). Two monoclonal MLC antibodies, F310 and S21, specific for the fast-alkali MLCs, LClr and LCBI, and the slow-alkali MLC, LCi,, respectively, were used. Hybridomas that secreted antibodies to the MHCs of myosin were generated and screened using procedures similar to those employed for the MLCs (Crow et al, 198313). The specificity of these antibodies was then verified by immunoblotting and immunocytochemistry. Hybridoma F59 secreted an antibody specific for fast MHCs and was generated from the polyethylene glycol (PEG 1460)-mediated fusion of the myeloma cell line P3/NSl/lAg4-1 with the splenocytes of a Balb/c mouse immunized with myosin from adult chicken pectoralis major muscle. Hybridoma S58 secreted an antibody specific for slow MHCs and was generated in a similar procedure with splenocytes from a mouse immunized with myosin from 19day embryonic hindlimb muscle. Both hybridomas were repeatedly subcloned by limiting dilution before use. Hybridoma supernatants were used as the source of antibody throughout this study. Gel electrophoresis and electrophoretic transfers (immunoblots). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (1970) using 10% or 12.5% polyacrylamide gels. The electrophoretic transfer of proteins from the gels to nitrocellulose was performed overnight at 8-9 V/cm in a buffer consisting of 25 mM Tris base, 192 mM glycine, and 20% methanol (Burnette, 1981). To facilitate transfer of MHCs, the cathodic sponge of the transfer cassette was soaked in 1% SDS. Immunodetection of the transferred proteins with monoclonal antibodies was performed as described previously (Crow et ah, 1983b) using a F(ab)e fragment of an iodinated lz51-sheep antibody to mouse immunoglobulins (Amersham Corp., Arlington Heights, Ill.). The reaction pattern of the antibody was visualized following overnight exposure of the transfer to X-ray film. Immunocytochemistry. Tissues were frozen in melting isopentane and sectioned at 10 pm. These sections were transferred to gelatin/chromalum (0.1% gelatin, 0.01% chromium potassium sulfate-coated glass slides and air dried for 1 hr at room temperature. Endogenous peroxidase was blocked by a 5-min incubation in 3% H202 dissolved in absolute methanol and the sections were

VOLUME 113.1986

incubated for an additional 30 min in 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The sections were then incubated overnight at 4°C with the hybridoma supernatants diluted in BSA-PBS. After extensive washing in PBS, primary antigen/antibody complexes were detected using either an indirect peroxidase (Crow et al., 1983b) or an avidin-biotin complex (ABC) (Hsu et ah, 1981) procedure. In the indirect procedure, sections were incubated for 4 hr at room temperature with horseradish peroxidase (HPO)-conjugated rabbit antibody to mouse immunoglobulins (DAK0 Antibodies, Accurate Scientific Corp., New York). In the ABC procedure, sections were first incubated for 1 hr at room temperature with a biotinylated horse antibody to mouse immunoglobulins followed by incubation for another hour at room temperature with a complex of avidin and biotinylated-HP0 (Vector Laboratories, Burlingame, Calif.). In both procedures, normal serum from the species in which the secondary antibody was raised was incorporated in the incubations to reduce nonspecific background staining. The sections were developed in diaminobenzidine (Graham and Karnovsky, 1966), dehydrated, cleared, and mounted. Fiber types were identified and designated on the basis of their reaction with antibodies to fast and slow myosin isoforms. In adult muscles, three fiber groups designated type F, type S, and type F/S were recognized by antibody staining to contain fast isoforms alone, slow isoforms alone, or various mixtures of fast and slow isoforms, respectively. Antibody staining of the muscle fibers present in the embryos suggested that fibers contained either fast isoforms alone or a mixture of fast and slow isoforms. To avoid confusion, the embryonic fibers have been designated with subscripts as type Femb (fast) and type F/S,,I, (fast and slow) fibers. These designations have been adopted to describe differences in antibody reactivity and are not meant to imply that fibers characterized by similar appellations are related or contain the same myosin isoforms. The fiber type composition of the different adult and embryonic muscles was determined from antibodystained serial cross sections. The distribution of fiber types in the sartorius, semitendinosus, and vastus lateralis muscles was not uniform but typically arranged in the form of a gradient (Fig. 5). To minimize sampling errors in the fiber counts from these muscles, each was divided into three segments with the cuts defining those segments being made perpendicular to the gradient of slow myosin-containing fibers. Tissue fragments from each of these segments were processed for immunocytochemistry and the fibers in these sections counted and typed. For the superficial biceps, lateral, and medial adductors muscles, in which no gradient of fiber types was observed, tissue segments were taken without respect

CROW

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Development

to location. For each adult muscle, a total of at least 1500 fibers representing muscle fragments from three different adult chickens were counted and typed. For embryonic muscles, cross sections of the entire thigh from four different embryos were cut and all the fibers present within the entire crosssection of individual Day8 embryonic muscles were counted and typed. For both adult and embryonic muscles, tissue sections were cut at a level in which the cross-sectional area of the muscle was the greatest. The fiber types composition of the adult and embryonic muscles are reported in Table 2. Administration of curare. Curare (D-tubocurarine; Sigma Chemical Co., St. Louis, MO.) was administered twice daily to embryos from the fourth day of incubation until they were sacrificed. Each dose (1.5 mg in 0.1 ml PBS, pH 8.0) was injected onto the chorioallantoic membrane through holes drilled through the egg shell into the air sac. Control embryos were injected on the same time schedule with 0.1 ml of PBS, pH 8.0. Between injections, the eggs were sealed with cellophane tape and returned to the incubator. Stock solutions of curare were made fresh daily. Muscles in embryos treated with curare were markedly paralyzed and did not contract as a result of stimulation of the sciatic nerve. RESULTS

MHC Antibody

Specificity

The specificities of the fast and slow MLC monoclonal antibodies (McAbs) used in this study have been documented in an earlier report (Crow et al., 198313). McAb F310 reacts with the fast alkali MLCs, L& and LCBI, while McAb S21 is specific for the slow alkali MLC, LC1,. The specificities of the fast and slow MHC McAbs that were generated for this study were demonstrated by immunoblotting and immunocytochemistry and are summarized in Table 1. Figure 1 shows the immunoblotting pattern of the two MHC antibodies with crude myosin preparations from thigh muscles composed predominantly of fast (superficial biceps, lane l), slow (medial adductor, lane 2), and a mixture of fast and slow fiber types (sartorius, lane 3). McAb F59 is a fast MHC antibody and reacts with MHC(s) from both fast (Fig. lb, lane 1) and mixed (lane 3) muscles. It also reacts, to a lesser extent, with the MHCs present in the slow muscle (lane 2). This pattern of reactivity was the same as that exhibited by the fast MLC McAb F310 (Crow et al., 1983b). McAb S58 is a slow MHC antibody and reacts with the MHCs in both slow (Fig. lc; lane 2) and mixed (lane 3) muscles, but not in fast muscles (lane 1). This pattern of reactivity is the same as that exhibited by the slow MLC McAb S21 (Crow et ah, 1983b). The immunoblots in Fig. 2 show the reaction of both McAbs with various subfragments of the myosin mol-

of

ANTIBODY

Muscle

Fiber

SPECIFICITY

241

Types TABLE 1 AND STAINING CHARACTERISTIC MUSCLE FIBER TYPES Fiber

staining

characteristics

Adult Monoclonal antibody F310 F59 s21 S58

OF CHICKEN

Embryonic”

Specificity

Fa

F/&*

F/S2

S

Femb

MLGm MHG..t MLC1, MHC.,,,

+ + -

+ + + -

+ + + +

+ +

+ + -

F%nb + + + +

a Fiber type nomenclature described in Crow et al. (1983a,b). bType F/S fibers that do not stain with anti-slow MHC are designated as type F/S1, while the type F/S that stain with both anti-slow MLC and anti-slow MHC are designated type F/S2. ‘Embryonic fibers are those in the period of 6 to 8 days of limb development.

ecule. Figure 2a shows the protein staining pattern of myosin containing both fast and slow isoforms (lane 1) and the subfragments derived from it, including the 90,000-Da Sl subfragment (lane 2), the complementary 120,000-Da rod subfragment (lane 3), and the 77,000-Da light meromyosin (LMM) subfragment (lane 4). McAbs F59 (Fig. 2b, lane 2) and S58 (Fig. 2c, lane 2) reacted only with the Sl subfragment. Figure 3 shows the pattern of staining for these MLC and MHC antibodies with serial cross sections of the adult sartorius muscle. These antibodies reacted with this and other muscles to identify four muscle fiber types. These were: (1) fibers which contained only fast MLCs and fast MHC(s) which we have designated as type F fibers; (2) fibers which contained only slow MLCs and slow MHC(s) and are designated type S; (3) fibers which contained only fast MHC(s) but both fast and slow MLCs and are designated type F/S,; and (4) fibers which contained both fast and slow MLCs and MHCs and are designated type F/&. A summary of the distinguishing staining characteristic of these different fiber types is listed in Table 1. All type F and most type F/S1 fibers exhibited histochemical staining characteristics typical of type II fibers (Barnard et ah, 1982), while the majority of type S fibers were similar to type I fibers (Barnard et al., 1982; MacLennan, 1983a). Type F/S2 fibers which comprised between 30 and 40% of the fibers in the “slow” muscles of the chicken (e.g., medial adductor and anterior latissimus dorsi) exhibited histochemical staining properties characteristic of both type I and II fibers. Such fibers have been separately classified by others as type IIIB (Barnard et aZ., 1982). The adult fiber type compositions of the five thigh muscles whose development was followed in this study are listed in Table 2.

242

DEVELOPMENTAL

a

b

C

123

12

BIOLOGY

MHC+ C Protein,

Actin

*

-

-.) -

2’:=

Ir -7

2-.--z-

3

FIG. 1. Specificity of MHC monoclonal antibodies. Crude extracts of the adult superficial biceps (lane l), medial adductor (lane 2), and sartorius (lane 3) muscles were prepared as described under Materials and Methods and subjected to SDS-electrophoresis on a 10% polyacrylamide gel. (a) Protein staining pattern (Coomassie blue R250) of 40 fig of each crude extract with the positions of some of the contractile proteins indicated to the left. (b) Autoradiogram of duplicate gel loaded with 10 pg of each extract, electrophoretically transferred to nitrocellulose, and reacted with a 1:50 dilution of McAb F59 followed by 3 X lo6 cpm/ml rEI-sheep anti-mouse immunoglobulin. (c) Autoradiogram of a duplicate gel loaded with 10 pg of each extract, electrophoretically transferred to nitrocellulose, and reacted with a 1:25 dilution of McAb S58 followed by 3 X lo5 cpm/ml radiolabeled sheep anti-mouse immunoglobulins. MHC, myosin heavy chain; MLC, myosin light chain.

VOLUME

113, 1986

MLC or MHCs (Fig. 4d). These slow myosin-containing fibers were localized within the segregating masses into specific areas which would become the predominant regions containing slow myosin fibers in the adult. In Fig. 4, for example, the regions corresponding to the future locations of slow myosin-containing sartorius (SA) and semitendinosus (ST) muscles reacted with McAb to slow MHCs, whereas the region in which the future fast superficial biceps (SB) was to be located displayed little or no reaction (Fig. 4d). The separation and delineation of the muscles in the thigh was completed before Day 8 in uvo (stage 32). Fibers containing different myosin isoforms were recognizable in such embryos either after staining with McAbs to fast and slow MHCs (Figs. 5a, b) or with McAbs to fast and slow MLCs (Figs. 5c, d). As was the case during cleavage of the premuscle masses, the different fiber types were not randomly distributed within the early hindlimb but occupied unique and characteristic positions within most muscles. In many of these

myosin from

Fiber Diversijcation

in the Embryonic

a

b

Hindlimb MLCs

The individual muscles of the developing hindlimb arise from the progressive splitting of the primary muscle masses (Romer, 1927). These muscle masses can be seen in the Day-5 in ovo (stage-26) embryo after their reaction with antibodies to skeletal muscle myosin. Figure 4 shows the reaction of the premuscle masses in a Day-5 embryo with McAbs to fast (Fig. 4a) and slow (Fig. 4b) MHCs. It was apparent that the reaction with the fast antibody was much greater than that with the slow antibody and that the slow antibody exhibited some specificity with respect to its binding. Similar results were obtained with McAbs to the MLCs. By Day 6 in ovo (stage 29), both the splitting and cleavage of the premuscle masses to form the individual muscles of the thigh and the diversification of fibers within these masses had begun. Most, if not all, the fibers at this stage reacted with McAbs to fast MLCs or MHCs (Fig. 4c), while only some reacted with McAbs to slow

2s+ 2F-h 3F+

I)

-

1

2

3

4

2

3

4

2

3

4

FIG. 2. Reaction of MHC monoclonal antibodies with myosin subfragments. Subfragments were prepared by cu-chymotryptic cleavage of myosin from thigh muscles containing a mixture of fast and slow muscle fiber types and then subjected to SDS-electrophoresis on 12.5% gels. (a) Protein staining pattern (Coomassie blue R250) of 40 pg of crude myosin extract (lane l), 25 pg of the purified Sl subfragment (lane 2), 25 pg of purified rod subfragment (lane 3), and 25 fig of purified LMM subfragment (lane 4) with the positions of the myosin subunits and subfragments indicated to the left. (b) Autoradiogram of a duplicate gel loaded with 5 pg of each subfragment, electrophoretically transferred to nitrocellulose, and reacted with a 1:50 dilution of McAb F59 followed by 3 X 10’ cpm/ml izI-sheep anti-mouse immunoglobulin. (c) Autoradiogram of a duplicate gel loaded with 5 pg of each subfragmerit, electrophoretically transferred to nitrocellulose, and reacted with a 1:25 dilution of McAb S58 followed by the iodinated second stage antibody. MHC, myosin heavy chain; Sl, subfragment 1; LMM, light meromyosin subfragment; MLC, myosin light chains.

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Types

243

FIG. 3. Muscle fiber types in the adult chicken sartorius muscle. Serial frozen sections (10 pm) of adult sartorius muscle reacted with fast MHC McAb F59 (a); slow MHC McAb S58 (b); fast MLC McAb F310 (c); and slow MLC McAb S21 (d). The arrows identify the same fibers (type F, type S, and type F/Sr) in each of the sections. The (X) identifies a group of three intrafusal fibers that display differential staining with fast and slow myosin antibodies. Indirect immunoperoxidase procedure. Bar in (d) represents 20 pm.

muscles, slow myosin-containing fibers were distributed along a gradient from one side of the muscle to the other. In the sartorius (SA; Fig. 5), slow myosin-containing fibers were concentrated along the medial aspect of the muscle, while in the semitendinosus (ST; Fig. 5) the concentration of such fibers was greatest at the anterior end. In the vastus lateralis (VL, Fig. 5), slow myosincontaining fibers exhibited a radial distribution with the highest concentration of fibers adjacent to the femur. This unique spatial distribution of fibers within the muscles of the hindlimb was characteristic of all 6- to &day embryos that were studied (>lOO). Furthermore, the basic features of this pattern or “blueprint” were conserved throughout development. The data in Fig. 6 illustrate this point by showing that the distribution of slow myosin-containing fibers in cross sections of different muscles from the Day-8 (Fig. 6b), Day-12 (Fig. 6d), and Day-16 (Fig. 6f) hindlimbs was the same throughout this developmental period. Diversification of myosin expression among the first fibers, therefore,

resulted in the establishment expression of a unique pattern or “blueprint” of fiber types which was conserved throughout the remainder of development. The distinction between presumptive fast and slow muscles and regions of muscles in terms of myosin content is quantitative. Presumptive fast muscles do contain slow myosin, although the amount is much less than that present in presumptive slow or mixed muscle. Slow myosin-containing fibers among presumptive fast muscles such as the superficial biceps (SB) and lateral adductor (LA) were evident only after staining with slow MHC McAb S58 (see arrow in Fig. 5b). Based upon their time of appearance and morphology, the fibers occupying the early hindlimb were identified as primary generation fibers and were classified into two staining groups (type Femb and type F/S,,,,,, Fig. 7). Fibers of the group designated type Femb reacted with McAbs to the fast MHCs and MLCs, while those of the group designated type F/E!&, reacted with McAbs to both the fast and the slow MHCs and MLCs. The relative

244

DEVELOPMENTAL

FIBER

COMPOSITION

TABLE 2 OF ADULT AND EMBRYONIC

7% Adult Muscle

MUSCLES % Embryonic fiber

types

F”

F/S*

S

Femb

Sartorius (SA) [sartorius]”

48.5 (1.7)

33.7

17.7

41.8

(2.7)

0.2)

(3.6)

58.2 (4.1)

Superficial [iliotibialis

99.0

0.1 (0.3)

0

95.7 (2.3)

(0.8)

35.6 (3.2)

24.9 (3.3)

25.1

Semitendinosus [iliofibularis]

types

fiber

THIGH

BIOLOGY

biceps (SB) posterior] (ST)

(0.2) 39.5

(2.6)

F&nb

6.3

(2.6)

75.0 (3.2)

Medial adductor (MA) [adductor externus]

0

38.7d (3.5)

61.7 (2.7)

0.1 (0.1)

99.9

Lateral adductor (LA) [adductor internus]

28.0 (5.1)

53.0

18.7

51.8

42.3

(7.9)

(2.8)

(4.2)

(3.6)

Note. Percentages given as mean (standard deviation). named according to mammalian homologs. Abbreviations in parentheses. a Fiber classification based on myosin isofirm content 1983a,b). *Includes both type F/S1 and F/S2 fibers. ‘Muscle synonym according to Romer (1927). d Type F/S2 fibers.

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113. 1986

the basic topographical distribution of slow myosincontaining fibers established by the primary generation fibers remained unchanged. By Day 16 in ovo (stage 42), the difference in fiber size which had served to distinguish primary from secondary generation fibers at earlier stages was less apparent. In addition, changes in isoform expression among some of the fibers were already evident (Fig. 10). These changes included a reduced reaction of the type F/S,,b fibers of the medial and lateral adductor muscles (MA and LA; Figs. lOc, d) with fast myosin antibodies and differential staining with the slow MLC and slow MHC antibodies (SA, Figs. lOa, b).

(1.1)

Muscles are are given (Crow

et al.,

abundance of these two primary fiber types and their locations in specific regions of the thigh muscles were similar to the relative abundance and position of fast and slow myosin-containing fibers in these same muscles in the adult. Table 2 compares the fiber type composition of five muscles from Day 8 and adult thigh to illustrate this point. Between Day 8 (stage 32) and Day 12 in ovo (stage 38), the size of the hindlimb increased dramatically as a result of the hypertrophy of existing fibers and the formation of new fibers beginning around Day 10 in ovo (Kelly and Zachs, 1969; MacLennan, 1983a). These additional fibers belong to the secondary generation of muscle fibers. At Day 12 in ova, primary and secondary fibers differed in fiber diameter and antibody staining (Fig. 8). The larger-diameter, “doughnut-shaped,” primary generation fibers continued to exhibit two different staining types with the fast and slow McAbs, while the smaller-diameter secondary generation fibers reacted only with the McAbs to fast myosin. The formation of secondary generation fibers occurred predominantly in presumptive fast and mixed fiber type muscles such as the superficial biceps (SB; Figs. 9e, f) and lateral adductor (LA; Figs. 9b, and c); presumptive slow muscles such as the medial adductor (MA, Figs. 9b, c) appeared to contain few such fibers. The result of this was that

Fiber Diversification

in Curarized

Embryos

Experiments were performed to determine if the appearance of different fiber types in the early embryo was dependent on functional neuromuscular connections. Embryos were paralyzed through the administration of D-tubocurarine (curare) injected daily onto the chorioallantoic membrane beginning at Day 4 in ovo (stage 22) a time prior to the onset of spontaneous movements (Hamburger and Balaban, 1963) and the innervation of the hindlimb by pioneering motorneurons (Landmesser, 1978). In contrast to saline-injected controls, curaretreated embryos exhibited no spontaneous movements. Contraction of muscles in such embryos could be elicited only by direct stimulation of the muscle; repeated stimulation of the sciatic nerve resulted in no visible contractions of the hindlimb. Figure 11 shows the profile of thigh muscles from Day 8 control (Figs. lla, b) and curare-treated (Figs. llc, d) embryos after staining with the fast and slow MHC McAbs. (Identical results were observed with the MLC McAbs.) The fact that no observable differences were detected between control and experimental sections suggests that the formation and early growth of muscles in the thigh, the differential expression of fast and slow myosin isoforms among the primary generation fibers, and the distribution of the resulting fiber types into the unique spatial patterns characteristic of normal development were all unaffected by the administration of curare. While the early stages of muscle development were unaffected by drug-induced paralysis of the limb, later stages of development (after Day 12 in ouo) were markedly altered by such treatment. Figure 12 shows the profile of Day 16 thigh muscles from control and curaretreated embryos after staining with fast and slow MHC antibodies. In the curare-treated embryos, both the size of the limb and many of its individual muscles were

CROW AND

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Development

of hhscl~ Fiber

Types

245

dorsal dorsal ventral a

b

C

FIG. 4. Myosin antibody staining of the muscle fibers in the early limb. Serial frozen sections (10 pm) of Day 5 (stage 26) limb buds stained with MHC McAbs F59 (a) and S58 (b). “Dorsal” refers to the dorsal muscle mass and “ventral” to the ventral muscle mass. Serial frozen sections of Day 6 (stage 29) hindlimbs stained with MHC McAb F59 (c) and S58 (d) showing the splitting of muscles from the dorsal and ventral muscle masses. The presumptive muscles in the dividing masses are indicated by the following abbreviations: SA, sartorius; MA, medical adductor; LA, lateral adductor; SB, superficial biceps; and ST, semitendinosus. ABC peroxidase method. Bar in (b) and (d) represents 150 pm.

reduced in comparison to saline-injected controls (Figs. 12c, d). Atrophy was more pronounced in some muscles than in others. For example, muscles such as the sartorius (SA), medial (MA) and lateral (LA) adductors underwent large reductions in size, while muscles such

as the superficial biceps (SB) and semitendinosus (ST) were hardly affected at all. On the other hand, loss of slow myosin staining was evident among all slow myosin-containing muscles. These effects of chronic curarization are similar to those described for surgical

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SB J

b

FIG. 5. Antibody staining profile of Day 8 (stage 32) hindlimbs. Serial frozen sections of the entire Day 8 (stage 32) hindlimb F59 (a); slow MHC McAb S58 (b); fast MLC McAb F310 (c); and slow MLC McAb S21 (d). Sart, sartorius; SB, superficial biceps; ST, semitendinosus; MA, ABC peroxidase procedure. Bar in (d) represents 400 pm medical adductor; LA, lateral adductor; VL, vastus lateralis; F, femur

denervation and chemical-induced paralysis (Drachman, 1964; Rubinstein and Kelly, 1978). DISCUSSION

Fiber Specialization

in Developing

Chicken Muscles

We have used monoclonal antibodies specific to the MLC and MHC subunits of chicken skeletal muscle to study the expression of fast and slow isoforms within individual muscle fibers of the developing chick hindlimb and to ascertain whether the cellular heterogeneity of adult muscle tissue can be traced to a similar heterogeneity in expression of myosin in the embryo. Our data show that embryonic fibers differing in both MLC and MHC content were present among the primary generation fibers from the outset of the formation of the hindlimb. Two types of fibers were present in the early muscles: type Femb, which reacted only with antibodies to the fast myosin isoforms (MLCs and MHCs) and type Fhmb, which reacted with antibodies to both fast and

slow isoforms. From their origin, these primary generation fiber types were distributed within each muscle of the developing hindlimb according to a specific spatial pattern or “blueprint.” This pattern, which formed independently of functional innervation, remained essentially unchanged throughout the remainder of development. By Day 8 in o~o (stage 32), when many muscles contained less than 10% of their final adult numbers, it was possible to accurately define and locate specific embyronic muscles not only from their shape and location within the hindlimb but also from their distinctive and characteristic distribution of fiber staining types. Increases in muscle size that occurred as a result of additional rounds of fiber formation did not change the basic topographical distribution of fast and slow fibers established during the early period of muscle formation. The data we report here and the recent analyses using histochemical staining (ATPase) have considerably clarified the early events in avian muscle development. Diversification of primary generation fibers is one of the earliest events in the differentiation of the avian limb

CROW

AND

STOCKDALE

Dewlopm~nt

oj’ Muscle

FIG. 6. Antibody staining of embryonic hindlimbs at different stages in development. (a and b), Day 12 (stage 38) (c and d), and Day 16 (stage 42) (e and f) embryonic hindlimbs slow MLC McAb S21 (b, d and f). Abbreviations as in Fig. 5. Indirect peroxidase method. 1.2 mm; bar in (f) represents 1.6 mm.

musculature. Our observations using antibody probes of defined specificity are consistent with recent histochemical data for chicken skeletal muscle development which indicate that two fiber types differing in the pH stability of their ATPases can be distinguished among the first fibers to populate the muscles of the wing and leg (Butler and Cosmos, 1981; McLennan, 1983a; Phillips and Bennett, 1984). From their locations within specific muscles, it is likely that the histochemical fiber type designated by these investigators as type Iemb represents the same

Fiber

Types

247

Serial frozen sections (10 pm) of Day 8 (stage 32) stained with fast MLC McAb F310 (a, c and e) or Bar in (b) represents 500 pm; bar in (d) represents

fibers that react with both fast and slow myosin McAbs (type F/S,,& and that those fibers designated as type IIemb represent fibers that react only with fast myosin McAbs (type Femb). The data reported here are an important biochemical adjunct to the histochemical studies since they allow for the identification of these differences in terms of defined molecular components. Other studies, particularly on mammals, have addressed the issue of the development of different muscle fiber types. Gauthier et al. (1978, 1982) have reported

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f Femb

b

FIG. 7. Antibody staining of Day 8 (stage 32) limb muscles. Serial frozen section from the vastus lateralis (a and b) sartorius (c and d), and medial and lateral adductor (e and f) muscles stained with fast MHC McAb F59 (a, c and d) and slow MHC McAb S58 (b, d and f). Arrows identify the same type Fe,,+, and type F/Z&, fibers in appropriate serial sections. Abbreviations as in Fig. 5. ABC peroxidase method. Bar in (f) represents 30 pm.

that fibers from developing rats and chickens react uniformly with antibodies to adult fast and slow myosin and that the differential expression of these proteins does not occur until just before hatching (chicken) or just after birth (rat). The appearance of fiber diversity late in development has been attributable by these authors to changes in polyinnervation. Data on other mammals indicate that fiber type diversification may occur earlier and result from differences in isoform expression by fibers of different generations. In the fetal

lamb, for example, Ashmore et ah (1972) have shown that primary and secondary generation fibers differ, as a group, in histochemical staining characteristics so that differences in the fiber composition of different muscles are due to differences in the relative proportion of fibers from these various generations. Rubinstein and Kelly (1981) using antisera to adult myosin have presented immunocytochemical data for developing rat muscle that is consistent with this proposal. By way of contrast, we have shown that the blueprint for predicting future

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Development

of MWCIP

Fiber

Types

249

bu

Femb FIG. 8. Antibody sartorius muscle generation fibers, 20 urn.

staining of primary and secondary generation fibers stained with fast MHC McAb F59 (a) and slow MHC while the smaller arrows identify a group of secondary

muscle composition is present with the primary generation fibers and visible from its antibody staining profile. The reasons for the differences among these various studies may be due to differences in the program of fast and slow isoform expression among different animal species, to differences in the affinity of various antibody preparations, or to differences in the ability of antibodies to discriminate between fast and slow isoforms at different developmental stages. In the case of the MHCs, for example, it is clear that changes in the structure of this protein occur within individual fibers during the course of development (Whalen et al., 1981; Bandman et al., 1982; Winkelmann et al., 1983). MHC antibodies that

in Day 12 (stage 38) hindlimb muscle. Serial frozen sections of Day 12 McAb S58 (b). The large arrows identify type f and type f/s primary generation fibers. Indirect peroxidase method. Bar in (b) represents

are capable of distinguishing fiber types at one stage of development may not be able to do so at another stage since the antigenic structure of the protein may change as a function of developmental time. In this study we have taken advantage of the fact that the fast and slow alkali MLCs expressed in the embryo are the same as those expressed in the adult (Crow et al., 1983a,b). Using McAbs specific to these MLCs, we can identify whether a fiber is “fast,” “slow,” or “fast and slow” and determine whether the MHC McAbs react in a similar fashion. The fact that MHC McAbs F59 and 558 exhibited specificities of reaction that were, for the most part, the same as the fast and slow MLC McAbs

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FIG. 9. Antibody staining of Day 12 (stage 38) embryonic limb muscles. Serial frozen sections (10 pm) of Day 12 (stage 38) sartorius (a and b), medial and lateral adductors (c and d), and superficial biceps and semitendinosus (e and f) muscles stained with fast MHC McAb F59 (a, c and e) and slow MHC McAb S58 (b, d and f). Arrows identify type f and types f/s primary generation fibers. Abbreviations as in Fig. 5. Indirect peroxidase method. Bar in (f) represents 60 pm.

strongly suggests that these antibodies recognize epitopes that are common to the various developmental isoforms of fast and slow MHCs. While we have made no attempt in this study to determine if the MHC isoforms recognized by these antibodies are the same as those classified by other investigators as “embryonic,” the different staining profiles of early embryonic fibers with antibodies F59 and S58 indicate that there are at least two MHCs present in the early stages of limb and muscle development. Whether

these isoforms occur in addition to the “embryonic” MHC or represent two different “embryonic” MHCs cannot be determined from immunocytochemistry alone. Innervation

and Muscle Fiber Specialization

The biochemical transformations that accompany myogenesis in vivo also occur when myoblasts differentiate in vitro, indicating that at least some of the information necessary for muscle differentiation may be

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FIG. 10. Antibody staining of Day 16 (stage 42) embryonic limb muscles. Serial frozen section (10 pm) of stage 42 (Day 16) semitendinosus, medial adductor, and lateral adductor stained with fast MLC McAb F310 (a), slow MCL McAb S21 (b), fast MHC McAb F59 (c), and slow MHC McAb S58 (d). Arrows identify type s fibers. Abbreviations as in Fig. 5. Indirect peroxidase method. Bar in (d) represents 90 +m.

preprogrammed within the myoblasts. It is often assumed, however, that the information necessary for directing the development of specific fast and slow fiber types is derived from factors outside the myoblast. The motor nervous system is a particularly attractive candidate for such a factor. Motorneurons innervate muscles early in development (Day 5 in ouo) prior to overt expression of different muscle fiber types (Landmesser, 1978). In mature muscles, denervation often results in atrophy and loss of fiber staining characteristics (Drachman, 1964; Rubinstein and Kelly, 1978) while changes in the pattern of innervation often result in changes in the pattern of fast and slow isoform expres-

sion (Streter et al., 1976). The relevance of these latter observations to the early events in muscle development and fiber types specializations is not clear. The results presented here demonstrate that fiber diversification among early limb muscles is unaffected by chronic neuromuscular paralysis induced by curare. It is recognized that curare has multiple effects on neuromuscular interactions in addition to the postsynaptic blocking of neural transmission, including a delay or failure of motorneuronal cell death (Pittman and Oppenheim, 19X%), the prevention of secondary fiber formation (Harris, 1981; McLennan, 1983b), and the prevention of the normal regression of polyneuronal inner-

FIG. 11. Antibody staining of Day 8 limb muscles from (a and b) and curarized (c and d) Day 8 embryos stained as in Fig. 5. Indirect immunoperoxidase procedure. Bar

curarized embryos. Serial frozen sections (IOpm) of the entire hindlimbs from control with fast MHC McAb F59 (a and c) and slow MHC McAb S58 (b and d). Abbreviations in (d) represents 1.2 mm.

SB ST LA

FIG. 12. Antibody staining of Day 16 limb muscles from curarized embryos. Serial frozen sections (10 nm) (a and b) and curarized (c and d) Day 16 embryos stained with fast MHC McAb F59 (a and c) and slow MHC as in Fig. 5. Indirect immunoperoxidase procedure. Bar in (d) represents 1.6 mm,

of the entire hindlimb from control McAb S58 (b and d). Abbreviations

CROW

AND

STOCKDALE

Development

vation (Srihari and Vrbova, 1978). Any or all of these factors might have affected the pattern of myosin expression among the primary muscle fiber types, but did not. It has been shown that diversification among early embryonic muscle fibers and the establishment of the unique spatial pattern of muscle fiber types were not dependent on functional contact with motorneurons. These results corroborate the findings of Butler et al. (1982) and Phillips and Bennett (1984), who observed that the diversification of fibers in terms of histochemical staining was unaffected by removal of the brachial spinal cord or the administration of curare, and of Laing and Lamb (1983), who noted that diversification of ATPase staining occurred normally in limbs that received inappropriate innervation as a result of transplantation to different regions of the embryo. However, our results differ from those of McLennan (1983b), who has reported loss of type Iemb fibers (type F/S,,,,) at early stages of development following curare treatment. The neural independence of early limb development and primary fiber diversification fits well with the conclusions gathered from grafting of the limb to the chorioallantoic membrane (Eastlick, 1943; Bradley, 1970) and from chemical and surgical denervation of the limb (Shellswell, 19’77). These studies have shown that neither the formation nor early growth of the muscles in the hindlimb require interaction with motorneurons. In contrast, it is well documented that later stages of embryonic development are affected by disruption of neuromuscular contacts. In the absence of innervation, the fibers present in muscles at later embryonic stages atrophy, slow myosin-containing fibers become more like fast fibers (Rubinstein and Kelly, 1978; Gauthier et al., 1984), and secondary generation fibers fail to appear (McLennan, 1983b; Harris, 1981). Many of these effects are evident in the curare-treated embryos in Fig. 12. The fact that developing muscles display a stage specificity for neural dependence indicates that the initial specialization and the subsequent maintenance of fiber types are independent processes and that it is possible to identify two major phases or muscle development prior to newborn life. The first of these phases is the embryonic phase, the period prior to completion of morphogenesis, during which individual primary fiber types are formed and specific muscles are assembled. The events of this phase are dependent upon intrinsic properties of the muscle fibers and local factors within the environment of the fibers, with innervation playing no obvious role. The second phase is the fetal phase, occuring after morphogenesis is complete, during which the major growth in size of a muscle occurs by new fiber (secondary generation) formation and hypertrophy of existing fibers. Innervation plays a major role in initi-

of Muscle

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253

ating and sustaining the fetal phase of growth. During the embryonic phase, the expression of both the fast and slow classes of myosin isoforms occurs independently of innervation, whereas during the fetal phase the continued synthesis of the slow class of myosin isoforms requires functional innervation. We gratefully acknowledge the technical assistance of Sandra Conlon and the helpful comments of Dr. Jeff B. Miller. This study was supported by grants from the NIH (AG 02822) and the Muscular Dystrophy Association of America. Michael T. Crow was a NIH postdoctoral fellow during the the tenure of this study. REFERENCES ARNDT, T., and PEPE, F. A. (19’75). Antigenic specificity of red and white muscle myosin. J. Histochem. Cytochem. 235, 159-168. ASHMORE, C. R., ROBINSON, D. W., RATTRAY, P., and DOERR, L. (1972). Biphasic development of muscle fibers in the fetal lamb. Exp. Neural. 37,241-255. BANDMAN, E., MATSUDA, R., and STROHMAN, R. C. (1982). Developmental appearance of myosin heavy and light chain isoforms in uivo and in uitro in chicken skeletal muscle. Dew. Biol. 93, 508518. BARNARD, E. A., LYLES, J. M., and PIZZEY, J. A. (1982). Fiber types in chicken skeletal muscles and their changes in muscular dystrophy. J. Physiol. (London) 331, 333-354. BRADLEY, S. J. (1970). An analysis of self-differentiation if chick limb buds in chorioallantoic grafts. J. Anat. 107, 479-490. BURKE, R. E., and EDGERTON, V. R. (1975). Motor unit properties and selective involvement on movement. Exercise Sports Med. Review 3, 31-81. BURNETTE, W. N. (1981). “Western blotting:” Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203. BUTLER, J., and COSMOS, E. (1981). Differentiation of the avian latissimus dorsi primordium: Analysis of fiber type expression using the myosin ATPase histochemical reaction. J. Exp. 2001. 218,219-232. BIJTLER, J., COSMOS, E., and BRIERLEY, J. (1982). Differentiation of muscle fiber types in aneurogenic brachial muscles of the chick embyro. J. Exp. Zool. 224, 65-80. CROW, M. T., OLSON, P. S., CONLON, S. B., and STOCKDALE, F. E. (1983a). Myosin light chain expression in the developing avian hindlimb. 17~ “Limb Development and Regeneration,” pp. B417-428. A. R. Liss, Inc., New York. CROW, M. T., OLSON, P. S., and STOCKDALE, F. E. (198313). Myosin light chain expression during avian muscle development. J. Cell Biol. 96, 736-744. CROW, M. T., and STOCKDALE, F. E. (1983). Myosin isoforms and the cellular basis of skeletal muscle development. Exp. Biol. Med. 9,165174. DRACHMAN, D. B. (1964). Atrophy of skeletal muscle in chick embryos treated with botulinum toxin. Science (Washington, D. C.) 145, 719721. EASTLICK, H. L. (1943). Studies on transplanted embryonic limbs of the chick. I. The development of muscle in nerveless and in innervated grafts. J. Exp. Zool. 93, 27-45. GAUTHIER, G. F., and LOWEY, S. (1977). Polymorphism among skeletal muscle fiber types. J. Cell Biol. 74, 769-779. GAUTHIER, G. F., LOWEY, S., BENFIELD, P. A., and HOBBS, A. W. (1982). Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers. J. Cell Biol. 92, 471-484. GAUTHIER, G. F., LOWEY, S., and HOBBS, A. W. (1978). Fast and slow myosin in developing muscle fibers. Nature (London) 274.25-29.

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types in wing muscles during embryonic development: Effect of neural tube removal. Dev. Biol. 106,457-468. PITTMAN, R., and OPPENHEIM, R. W. (1978). Cell death of motorneurons in the chick embryo spinal cord. J. Camp. Neural. 187,425-446. POLLARD, T. D. (1982). Purification of nonmuscle myosins. In “Methods in Enzymology” (L. Lorand, ed.), Vol. 85, pp. 331-356. Academic Press, New York. ROMER, A. S. (1927). The development of the thigh musculature of the chick. J. Morphol. Physiol. 43, 347-385. RUBINSTEIN, N. A., and KELLY, A. M. (1978). Myogenic and neurogenic contributions to the development of fast and slow twitch muscles in the rat. Dev. Biol. 62, 473-485. RUBINSTEIN, N. A., and KELLY, A. M. (1981). Development of muscle fiber specialization in the rat hindlimb. J. Cell Biol. 90, 128-144. RUBINSTEIN, N. A., PEPE, F. A., and HOLTZER, H. (1977). Myosin types during the development of embryonic chicken fast and slow muscles. Proc. N&l. Acad. Sci. USA 74,4524-4527. SALTIN, B., and GOLLNICK, P. D. (1984). Skeletal muscle adaptability: Significance for metabolism and performance. In “Handbook of Physiology, Skeletal Muscle,” pp. 555-633. American Physiological Society, Bethesda, Maryland. SHAFIG, S., SHIMIZU, T., and FISCHMAN, D. A. (1984). Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibody to slow myosin. Muscle Nerve 7, 380-387. SHELLSWELL, G. B. (1977). The formation of discrete muscles from the chick wing dorsal and ventral muscle masses in the absence of nerves. J. Embryol. Exp. Morphol. 41, 269-277. SRIHARI, T., and VRBOVA, G. (1978). The role of muscle activity in the differentiation of neuromuscular junctions in slow and fast chicken muscles. J. Neurocytol. 7, 529-540. STOCKDALE, F. E., RAMAN, N., and BADEN, H. (1981). Myosin light chains and the developmental origin of fast muscle. Proc. N&l. Acad. Sci. (USA) 7.5,931-935. STRETER, F. A., LUFF, A. R., and GERGELY, J. (1976). Effect of crossinnervation on physiological parameters and on properties of myosin and sarcoplasmic reticulum of fast and slow muscles of the rabbit. J. Gen. Physiol. 66, 811-821. WEEDS, A. G., and TAYLOR, R. S. (1975). Separation of subfragment-l isozymes from rabbit skeletal muscle myosin. Nature (Londolz) 257, 54-56. 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. Nuture (London) 292, 805-809. WHALEN, R. G., SELL, S. M., ERIKSSON, A., and THORNELL, L. E. (1982). Myosin subunit types in skeletal and cardiac tissue and their developmental distribution. Dev. Biol. 91,478-484. WINDER, W., Frrrs, R., HOLLOSZY, J., KAREY, K., and BROOKE, M. (1980). Effects of thyroid hormones on different types of skeletal muscle. h> “Plasticity of Muscle” (D. Pette, ed.), pp. 581-591. Walter de Gruyter, Berlin. WINKELMAN, D. A., LOWEY, S., and PRESS, J. L. (1983). Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis. Cell 34, 295-306.