Types of troponin components during development of chicken skeletal muscle

DEVELOPMENTAL

BIOLOGY

82, 11-19 (1981)

Types of Troponin Components during Development of Chicken Skeletal Muscle RYOICHI *Department

of Biology,

MATSUDA,*

Chiba University,

TAKASHI

OBINATA,*

Chiba 260, and tDepartment

Received March

AND YUTAKA

SHIMADA?

of Anatomy,

Chiba University,

Chiba 280, Japan

31, 1980; accepted in revised form July 1, 1980

Changes in troponin components during development of chicken skeletal muscles have been investigated by using electrophoretic, immunoelectrophoretic, and immunoelectron microscopic methods. Previous reports (S. V. Perry and H. A. Cole, 1974, Biochem. J. 141, ‘733-743; J. M. Wilkinson, 1978, Biochem. J. 169,229-238) pointed out that breast and leg muscles of adult chicken contain different types of troponin-T (TN-T), i.e., breast- and leg-type TN-T, respectively. However, the present paper indicates that the embryonic breast muscle contains leg-type TN-T. As development progresses two types of TN-T, i.e., breast- and leg-type TN-T, are found, and finally breast-type TN-T becomes the only species of TN-T present in the breast muscle. This change is well coordinated with the change of tropomyosin in the breast muscle. In contrast, the leg muscle contains leg-type TN-T through all the developmental stages. Legtype TN-T is present in myogenic cells in vitro, irrespective of their origin, whether from the breast or leg muscle. The types of troponin-I and troponin-C in both breast and leg muscles do not change during development. The significance of the changes in the types of TN-T is discussed in terms of differential gene expression during development of chicken breast and leg muscles. INTRODUCTION

proteins exist in It has been shown that myofibrillar several different forms in vertebrate skeletal muscles. As the most obvious case, it is well known that myosin exists in multiple forms, which are distinguishable electrophoretically as well as immunologically, in vertebrate striated muscles (Sarkar et al., 19’71; Lowey and Risby, 1971; Masaki, 1974; Hoh et al., 1976; Obinata et al., 1979a). Furthermore, polymorphic forms of tropomyosin (Cummins and Perry, 1973; Hayashi et al., 1977; Roy et al., 1979) and troponin (cf. Dhoot and Perry, 1979) in skeletal muscle have been demonstrated. Perry and Cole (1974) and Wilkinson (1978) showed that the breast and leg muscles of chicken contain different types of troponin-T (TN-T) which are distinguishable by molecular weights. Wilkinson (1978) suggested that TN-T of breast muscle and that of leg muscle are the products of different genes, based on the sequence analysis of both proteins. Changes in types of myofibrillar proteins, e.g., myosin (Streter et al., 1972; Masaki and Yoshizaki, 1974; Pelloni-Muller et al., 1976; Rubinstein et al., 1977; Obinata et al., 1980), actin (Whalen et al., 1976), and tropomyosin (Roy et al., 1979) during development of skeletal muscle have been pointed out. However, troponin in developing muscle tissue has not been investigated. In this work, we have studied the developmental changes in troponin components by using electrophoretic and immunoelectrophoretic methods to compare with the changes in

other contractile and regulatory proteins, and then analyzed the types of troponin located on thin filaments of developing chicken skeletal muscle by immunoelectron microscopy. MATERIALS

Preparation

AND METHODS

of Troponin and Tropom yosin

By applying a slight modification of the method of Ebashi et al. (1971), troponin was extracted from the breast (m. pectoralis major) and thigh muscle (m. iliotibialis) of various developmental stages of chicken: 18-day-old embryos and l- to 14-day posthatched and adult chickens. According to this method, muscle mince was first extracted with Guba-Straub solution (0.3 M KCl, 0.09 M KH2POI, 0.06 M K2HPOI, pH 6.5) to remove myosin, and then the residue was extracted with 0.4 M LiCl containing 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM NaHC03, 0.05% NaNa, and 5 pg/ml pepstatin (an inhibitor of acid protease, catepsin) for 5 to 10 hr at 0°C. Tropomyosin was precipitated from the extract isoelectrically at pH 4.5. For further purification of tropomyosin, the isoelectric precipitation in the presence of 0.4 M LiCl, 2 mM EDTA, 0.05% NaN3, and 5 pg/ml pepstatin was repeated and ammonium sulfate fractionation at 40-50% saturation was performed. Troponin was obtained from the supernatant at pH 4.5 by ammonium sulfate fractionation at 50-60% saturation. Both proteins were dialyzed against 1 mM NaHC03 containing 2 mM2-mercaptoethanol and stored 11 0012-1606/81/030011-09$02.00/O Couvrinht All rights

0 1981 bv Academic Press. Inr. of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGYV0~~~~82.1981

at -20°C until use. In some experiments, proteins including troponin were directly extracted from muscle with a SDS solution. In this method, small pieces of muscle were quick-frozen by liquid nitrogen immediately after dissecting, transfered into an SDS solution at 95°C containing 1% SDS, 1% 2-mercaptoethanol, and 20 mM phosphate buffer, pH 7.0, and incubated for 5 min. Dissolved proteins were separated by centrifuging at 15,000~ for 20 min. Preparation of Antisera Antisera against TN-T and tropomyosin from chicken breast muscle were prepared using Freund’s complete adjuvant (Difco) as previously described (Obinata et al., 1979b). The specificities of the antisera in all cases were established by Ouchterlony’s immunodiffusion test, immunoelectrophoresis, and the demonstration of specific staining of isolated myofibrils (Obinata et al., 1979b). Electrophoresis and Disc Immunoelectrophoresis Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed in 10 or 15% acrylamide gel containing 1/37th of the amount of methylene bisacrylamide according to Weber and Osborn (1969). Electrophoresis was carried out in a buffer system containing 0.1 A4 Na-phosphate buffer (pH 7.0) and 0.1% SDS. The gels were fixed with 50% methanol containing 5% acetic acid, stained with 0.25% Coomassie brilliant blue R-250 in the presence of 7% acetic acid, and destained electrophoretically. Immunodiffusion combined with SDS-acrylamide gel electrophoresis (referred to here as disc immunoelectrophoresis) was performed as described previously (Obinata et al., 1979a). According to this method, SDS-acrylamide gel electrophoresis was first carried out through cylindrical gels of 10% acrylamide and, immediately after the completion of electrophoresis, the gels were embedded in a 1.5% agarose plate containing 0.05% SDS and phosphate-buffered saline (PBS). Immunoreactions were brought about by applying the antisera against troponin components or tropomyosin in troughs parallel to the acrylamide gels. Muscle Cultures Suspensions of mononucleated myogenic cells were obtained by the standard procedure of dissociation with trypsin from breast muscles of 12-day-old chick embryos and 3-day posthatched chickens (Shimada et al., 1967). Cell suspensions enriched in myogenic cells and prepared by a differential adhesion procedure (Yaffe, 1968) were used for the present study. Cells were plated at a concentration of 5 x lo5 in 3 ml of culture medium

consisting of Eagle’s minimal essential medium with glutamine, 15% horse serum, 4% embryo extract, and penicillin-streptomycin in a concentration of 50 U/ml and 50 kg/ml each. The culture medium was exchanged every 3 days. After 6 days in vitro, the cultures were treated with 50% glycerol containing 0.1 M KCl, 1 mM MgClz, and 20 mM Na-phosphate buffer, pH 7.0, and stored at -20°C until use. For electrophoretic analysis of cellular proteins, the cells were rinsed with PBS and homogenized in 1% SDS containing 1% 2-mercaptoethanol and 10 mM Na-phosphate buffer, pH 7.0, with a Dounce tissue grinder, and then incubated at 95°C for 3 min. The dissolved proteins were used as material for electrophoresis. Negative Staining The embryonic skeletal muscles (19 days of incubation) and the muscle cells in culture were immersed in 50% glycerol in standard buffer (0.1 M KCl, 1 mM MgClz, and 10 mM Na-phosphate buffer, pH 7.0) and left at 0°C for 24 hr. They were then stored at -20°C until use. Glycerinated muscles were rinsed with a standard buffer and disrupted in the same buffer with a homogenizer of the blade-type or a Dounce homogenizer (Kontes Co., Vineland, N. J.). The homogenate was centrifuged at 12,000g for 20 min. The resultant precipitates were rehomogenized extensively by 20-30 strokes in a tight-fitting Dounce homogenizer in a relaxing buffer containing 0.1 M KCl, 10 mM Na-phosphate buffer (pH 7.0), 0.5 mM ethylene glycol bis(a-aminoethyl ether) N,N,N’N’-tetraacetic acid (EGTA) and 5 mMadenosine triphosphate (ATP). This myofibrillar suspension was then mixed with antibody (l-2 mg/ml), incubated for 1 hr at room temperature, and then centrifuged at 6,000g for 20 min. The precipitates formed were washed with the relaxing buffer and resuspended in the same solution. The materials were placed on carbon-coated, collodion, or Forvar films on 400 mesh copper grids, washed twice in 0.1 M KC1 containing 0.3 mM NaHC03, and stained with 2% uranyl acetate. Specimens were examined with a Hitachi H-700 electron microscope operated at 200 kV. RESULTS

Troponin Components of Developing Embryonic Skeletal Muscle The electrophoretic patterns of troponin components of developing chicken skeletal muscle are shown in Fig. 1. As previously demonstrated by Perry and Cole (1974) and Wilkinson (1978), troponin-I (TN-I) and troponinC (TN-C) of adult chicken breast muscle are undistinguishable from those of adult leg muscle, while TN-T

MATSUDA, OBINATA, AND SHIMADA

Troponin

Components during Muscle Development

13

A-

a

bcde

f

ghi

j

FIG. 1. SDS-acrylamide gel electrophoresis of troponin components from breast (A) and leg (B) muscles of embryonic or posthatched chicken. About 50 pg of protein was applied in each electrophoretic run. a and f, 18-day embryo; b and g, 2-day posthatched; c and h, g-day posthatched; d and i, ll-day posthatched; e and j, adult. Tb, Breast-type TN-T; T,, leg-type TN-T; I, TN-I; C, TN-C.

of adult breast muscle (regarded here as breast type TN-T) has a larger molecular weight by 4000 daltons than that of adult leg muscle (termed here as leg-type TN-T) (gels e and j, Fig. 1). During development, the electrophoretic patterns of troponin components of leg muscle do not change. In contrast, in developing breast muscle, although the sizes of TN-I and TN-C stay constant, there is a marked difference in the pattern of TN-T. As seen in Fig. 1 (gels a and b), troponin from the breast muscle of embryos and younger chickens was found to contain two components (Tb and T,) in the molecular weight range of TN-T. As judged by their electrophoretic mobilities, it seems that the faster migrating TN-T component of younger chicken is identical with leg-type TN-T of adult and the slower migrating component is the same as the breast-type TN-T of adult. In order to establish whether these two components are varieties of TN-T, an immunochemical method was applied. The antibody formed against breast TN-T reacted not only with breast-type TN-T but also with legtype TN-T to form an immunoprecipitin line in the Ouchterlony’s immunodiffusion test (data not shown). However, breast- and leg-type TN-T were clearly distinguishable from each other by disc immunoelectrophoresis, because each TN-T formed a precipitin line against the antibody at a specific position corresponding to its electrophoretic mobility. When the mixture of breast and leg troponin was tested, two precipitin lines derived from each TN-T were clearly observed (Fig. 2A). Troponin from the breast muscle of 2-day posthatched

chickens formed two immunoprecipitin lines against anti-TN-T antibody at the positions where two types of TN-T-like components (Tb and TJ were present, as in the mixture of breast and leg troponin of adult; in contrast, only one precipitin line was raised against the troponin from the leg muscle of the young chicken (Fig. 2B). These results indicate that both of the TN-T-like components (Tb and TJ observed in the breast muscle of young posthatched chickens are immunologically identical with TN-T. However, it has been known that TN-T is highly sensitive to proteolytic enzymes (Yosogawa et al., 1978; Ishiura et al., 1979). Therefore, the lower molecular weight component of TN-T observed in the extract from the breast muscle of young chickens might be derived from the larger components as the result of partial proteolysis during the preparation procedure. To exclude this possibility, the following experiments were carried out. In order to minimize the degradation of troponin by cellular proteolytic enzymes during preparation, the protein was directly dissolved from quick-frozen breast and leg muscles of 20-day-old embryos with a SDS solution at 95”C, and the types of TN-T in the extracts were examined by disc immunoelectrophoresis. As seen in Fig. 3, the main immunoprecipitin line formed by the breast extract and anti-TN-T antibody was located at a similar level to that of the leg extract (T,). The extract of breast muscle formed an additional faint precipitin line, which is probably due to breast-type TN-T (T,, in Fig. 3). These results strongly suggest that the breast

14

DEVELOPMENTAL BIOLOGY

TN B

VOLUME 82, 1981

and breast-type TN-T are located in the electrophoresis, respectively, while troponin from the breast muscle of l-day posthatched chickens formed two marked precipitin lines due to leg- and breast-type TN-T (c in Fig. 4). At g-day posthatched or later stages, troponin from breast muscle formed only one immunoprecipitin line which was derived from breast-type TN-T (b in Fig. 4). Thus, it is obvious that the type of TN-T present in the breast muscle is drastically reversed within a week after hatching. With the troponin extract from embryonic and young posthatched leg muscles, anti-TN-T antibody formed an immunoprecipitin line only at the region corresponding to leg-type TN-T. This result suggests that breast-type TN-T is scarcely present in the developing leg muscles. By Ouchterlony’s immunodiffusion as well as immunoelectrophoresis experiments, TN-I and TN-C present in the embryonic skeletal muscles were found to be immunologically identical with the proteins in the adult muscles. Types of Troponin-T in Muscle Cultures

“TN ‘=

.(

.

FIG. 2. Immunodiffusion combined with SDS-acrylamide gel electrophoresis of troponin from adult (A) and 2-day posthatched chicken skeletal muscle(B). The immunoreactions of TN-T varieties with antibreast TN-T antibody were tested. Electrophoretic acrylamide gels were embedded in agarose gel plate immediately after electrophoresis and the antibody was applied in troughs parallel to the electrophoretic gels to cause immunoreactions. The quantities of proteins applied in the eleetrophoresis were in the range of 60 to 80 pg. Ab, anti-TN-T antibody; TNb, breast -troponin; TN,, leg troponin. The arrow indicates the direction of electrophoresis. Tb, breast-type TN-T, T,, leg-type TN-T.

muscle of chick embryos or young posthatched chickens contains TN-T which is the same in size as that in adult leg TN-T in situ. As judged by electrophoretic patterns, the relative proportions of the two types of TN-T in breast muscle change with development (Fig. 1). The leg-type TN-T exists predominantly in the breast muscle of H-day-old embryos or l-day posthatched chickens, but at 8 days after hatching the breast muscle contains predominantly breast-type TN-T, and leg-type TN-T is present only in a trace amount. The changes in the type of TN-T during development were further demonstrated by the analysis using disc immunoelectrophoresis. In Fig. 4, the immunoelectrophoretic patterns of troponin from breast muscle of various developmental stages against anti-TN-T antibody are shown. As mentioned above, troponin from embryonic breast muscle formed one marked and one faint immunoprecipitin line at the position where leg-

Because of the limitation in quantity for analyzing the troponin components from muscle cells in culture,

a

Tb

b

d

TI

FIG. 3. Disc immunoelectrophoresis of whole SDS extracts from the muscle. Proteins were extracted from the muscle, which had been frozen by liquid nitrogen immediately after dissection, with the SDS solution containing 1% SDS and 1% 2-mercaptoethanol at 95°C. The quantities of troponin in each gel were arranged to be comparable with those in Fig. 2 by performing electrophoretic runs preliminarily to estimate protein concentrations in the extracts beforehand. a, The extract from adult chicken breast muscle; b, the extract from 2-day posthatched chicken breast muscle; c, the mixture of the extracts from 2-day posthatched chicken breast and leg muscles; d, the extract from 2-day posthatched chicken leg muscle. Symbols are as in Fig. 2.

MATSUDA, OBINATA, AND SHIMADA

Troponin Components during Muscle Development

the total extracts of cultured muscle cells were used for electrophoresis. The electrophoretic gels were embedded in an agarose plate, and anti-TN-T antibody was applied in one side of the gels and anti-tropomyosin antibody in the other side. The positions of immunoprecipitin lines formed were compared with those of the electrophoretic bands of TN-T and tropomyosin of adult muscles. Since the mobility of tropomyosin in SDS-acrylamide gel electrophoresis is close to that of leg-type TN-T but considerably lower than that of breast-type TN-T, it was possible to identify the type of TN-T by comparing the mobility of TN-T with that of tropomyosin. Figure 5 shows disc immunoelectrophoresis of the extract from the culture of myogenic cells from the breast muscle of S-day-old posthatched chickens. The electrophoretic mobility of TN-T from the cultured cells was almost comparable with that of tropomyosin; in contrast, there was a marked difference in mobilities between adult chicken breast TN-T and tropomyosin. Therefore, the type of TN-T of the cultured cells was identified as leg type. Similarly, only leg-type TN-T was detectable in cultures of cells derived from breast or

15

FIG. 5. The determination of the type of TN-T in cultured muscle cells by disc immunoelectrophoresis using anti-TN-T antibody. The total extracts of g-day culture of myogenic cells from 12-day embryo and 3-day posthatched chicken breast muscle and the extract of adult breast muscle were applied to immunoelectrophoresis. The electrophoretic mobilities of TN-T and tropomyosin of adult breast muscle and the culture were determined by the formation of immunoprecipitin lines against anti-TN-T or anti-tropomyosin antibody. The type of TN-T in the muscle culture was determined as leg-type (TJ based on the comparison of the mobility of TN-T with that of tropomyosin. a, the extract from adult breast muscle; b, the extract from the culture of myogenic cells from 12-day embryo breast muscle; c, the extract from the culture of myogenic cells from 3-day posthatched chicken breast muscle. Ab-T, antibody against TN-T; Ab-TM, antibody against tropomyosin; Tb, breast-type TN-T; T,, leg-type TN-T; TM, tropomyosin. The arrow indicates the direction of electrophoresis.

leg muscles of 12-day embryos or the breast muscles of 14-day-old posthatched chickens. The mobilities of TNT from chicken muscles of various developmental stages, in comparison with that of tropomyosin, were collectively shown in Table 1. Only leg-type TN-T was detectable in muscle cultures irrespective of the origin of the cells, i.e., embryonic or posthatched, or breast or leg. Changes in Tropomgosin Subunits during Development

FIG. 4. The analysis of the types of TN-T by disc immunoelectrophoresis using anti-TN-T antibody. Troponin from the breast muscle of adult (a), ‘I-day posthatched (b), e-day posthatched chicken (c), 18day embryo (d), and troponin from leg muscle of 18-day embryo (e) were tested. The amounts of proteins applied were as in Fig. 2. The direction of electrophoresis was shown by the arrow.

The changes in the tropomyosin subunit patterns during the development of skeletal muscles are shown in Fig. 6. As previously demonstrated by Roy et al. (1979), young chicken breast muscles contain (Y- and P-tropomyosin as do adult leg muscles, while as development progresses, the proportion of a-tropomyosin gradually increases so that in adult breast muscles (Ytropomyosin is the only species present. Through the developmental stages, leg muscles contain both a- and P-tropomyosin. Our present observation, while confirming the previous report (Roy et al. 19’79), further indicates that the change in the type of TN-T occurs at

16

DEVELOPMENTAL BIOLOGY

TABLE 1 THERELATIVEELECTROPHORETICMOBILITIESOFTROPONIN-T(TNT), THE MOBILITY OF TN-T/THE MOBILITY OF TROPOMYOSIN,IN THE DEVELOPING SKELETALMUSCLE AND DETERMINATIONOFTHETYPES OFTN-T Relative mobility (TN-T/tropomyosin) In vivo Adult breast Adult leg 20-Day embryo breast 20-Day embryo leg

Type of TN-T

0.92 1.03 1.00 1.03

Breast type Leg type Leg type Leg type

1.02 1.02 1.02 1.03

Leg Leg Leg Leg

vOLUME82,1%1 DISCUSSION

The present electrophoretie and immunoelectrophoretie investigations have demonstrated that, during the posthatching period, the types of TN-T and tropomyosin in the breast muscle change markedly, while the types of TN-T and tropomyosin present in the leg muscle scarcely change throughout the developmental stages. These changes in TN-T and tropomyosin are well eoordinated. Considering that TN-T is a protein functionally connected with tropomyosin, the correlated

In vitro (B-Day culture of myogenic cells from) 12-Day embryo breast 12-Day embryo leg 3-Day posthatched breast ll-Day posthatched breast

type type type type

Note.The relative mobilities were calculated based on the comparison of the positions of immunoprecipitin lines formed by the cell extracts against anti-TN-T or anti-tropomyosin antibodies.

about the same time as that in tropomyosin during the development of breast muscle. In contrast, a marked replacement of tropomyosin species as well as that of TN-T does not occur during development of leg muscle, although the change in relative proportions of cx-.and P-tropomyosin takes place. Immunoelectron Microscopy By treating the homogenate of glycerinated muscles with the antibody against TN-T, cross-striations with a periodicity of 38 nm were invariably formed along the whole filament bundle. In confirmation with the previous work (Ohtsuki, 19’75,1979), the striations formed in adult breast muscles were wide (20-30 nm), and sometimes split into a pair of substriations (Fig. 7A). However, the striations formed in adult leg muscles were found to be narrow (lo-20 nm) (Fig. 7B). In contrast to adult breast muscles, many of the striations formed in embryonic breast muscles were narrow (lo-20 nm) (Fig. 8A) and similar to those seen in adult leg muscles. The striations formed in embryonic leg muscles were narrow (8-12 nm) (Fig. 8B) and indistinguishable from those observed in adult leg muscles. These results are summarized in Fig. 10. Bundles of thin filaments from embryonic breast and leg muscle cells cultured for 6 days were treated with anti-TN-T. The striations formed in cultures of both muscles (width: 8-12 nm) (Figs. 9A, B) were similar to those seen in embryonic muscle in vivo; no change in the width of striations was found in breast muscles cultured for up to 6 days.

L

B+L B

FIG. 6. SDS-acrylamide gel electrophoresis of tropomyosin from breast and leg muscle of embryonic and posthatched chicken. The quantities of proteins applied were 20 pg in a and about 10 pg in b-d. a, 13-day embryo; b, g-day posthatched; c, &day posthatched; d, adult. B, breast tropomyosin; B + L, the mixture of breast and leg tropomyosin; L, leg tropomyosin. LY,a subunit of tropomyosin; 8, fl subunit of tropomyosin.

MATSUDA, OBINATA, AND SHIMADA

Troponin Components during Muscle Development

17

FIG. 7. Bundles of thin filaments precipitated with antibody against TN-T from the adult breast (A) and leg muscle (B). The regular crossstriations of 38-nm intervals are formed along the thin filaments. Striations formed in the breast muscle (double arrows) are wider than those seen in the leg muscle (arrows), and are sometimes split into a pair of substriations. Negative staining with 2% uranyl acetate. Bar, 0.5 Wm. X105,000.

change in both proteins seems to be pertinent, although the physiological significance of the change is unknown. Recently, it was also shown in the chicken breast muscle that the patterns of myosin light chains and the myosin isozymes change during the same period (Roy et al., 1979; Hoh, 1979).

Immunoelectron microscopic investigations have demonstrated that the types of TN-T present in myofibrils can be characterized by the patterns of the striations formed along the thin filaments by anti-TN-T antibody. The difference in the striation pattern between the breast and leg muscles may be interpreted as fol-

FIG. 8. Bundles of thin filaments precipitated with antibody against TN-T from the embryonic breast (A) and leg muscle (19th day of incubation) (B). Striations with regular 38-nm intervals are formed in both muscles (arrows). In contrast to the adult breast muscle (Fig. ‘7A), the width of the striations in the embryonic breast muscle is narrow. The striations formed in embryonic leg muscles are also narrow. Negative staining with 2% uranyl acetate. Bar, 0.5 pm. X105,000.

18

FIG. 9. Bundles of thin filaments from embryonic cell cultures of breast (A) and leg muscles (B) precipitated by treating with antibody raised against TN-T. Striations with the same intervals as those seen in muscles in vivo (Figs. 7 and 8) are observed (arrows). The width of striations in the embryonic breast muscle cultured for 6 days is narrow. As expected, that in the leg muscle cultured for 6 days is also narrow. Bar, 0.5 pm. X105,000.

lows: since the leg-type TN-T has a shorter polypeptide chain than the breast-type TN-T, the part of the antigenic site which is relevant to the formation of striations by the antibody could be missing in the case of leg-type TN-T, so that the striations formed in leg muscles are narrow. In connection with this observation, the recent report by Ohtsuki (1979) demonstrated that when a part of the breast TN-T was cleaved by chymotrypsin, the major fragment of TN-T raised the antibody which forms the narrow striations. The immunoelectron microscopic observations in the present work have shown that embryonic breast muscles and myotubes formed in vitro, as well as leg muscles of all the developmental stages, contain mainly leg-type TNT, while matured breast muscles in vivo contain breasttype TN-T. These results are in accordance with the results by electrophoretic and immunoelectrophoretic experiments, and further indicate that leg-type TN-T area& hk?cle

FIG. 10. Schematic illustration tibody along the filaments.

LegMuscle

of the distribution

of anti-TN-T

an-

is first assembled into thin filaments at the early phase of the development of breast muscles and then gradually replaced by breast-type TN-T as the muscle development progresses. The developmental changes of TN-T and tropomyosin reported here could be the result of either a differential growth of different fiber populations or, alternatively, a switch in the expression of one set of genes to another within the same fiber. According to Marchok and Herrmann (1967), the majority of the cells in the breast muscles of posthatched chicken are in the nonproliferative state but the percentage of proliferating cells in the breast muscles at 2 to 8 days posthatching becomes slightly higher than before or after this stage. In our experiments, when mitotic cells were isolated from the breast muscles of 3 days or a week posthatching and cultured, the myotubes formed in vitro contained legtype TN-T. This observation indicates that the change in the type of TN-T in the developing breast muscles is not due to the rapid growth of the cells which are destined to synthesize breast-type TN-T. Amphlett et al. (1975) reported that types of TN-I present in the skeletal muscles of rabbit are markedly affected by the types of nerves innervated. On the differentiation of myosin during development, Masaki and Yoshizaki (1974) pointed out that multiple genes for myosin are expressed in the same fiber of embryonic muscles at early developmental stages but, as muscle development

MATSUDA, OBINATA, AND SHIMADA

Troponin Components during Muscle

Development

19

MASAKI, T. (1974). Immunochemical comparison of myosins from chicken cardiac fast white, slow red and smooth muscle. J. Biochem. (Tokyo) 76,441-449. MASAKI, T., and YOSHIZAKI, C. (1974). Differentiation of myosinin chick embryos. J. B&hem. (Tokyo) 76,X3-131. OBINATA, T., MASAKI, T., and TAKANO, H. (1979a). Immunochemical comparison of myosin light chains from chicken fast white, slow red and cardiac muscle. J. Biochem. (Tokyo) 86,131-137. OBINATA, T., SHIMADA, Y., and MATSUDA, R. (1979b). Troponin in embryonic chick skeletal muscle cells in vitro. An immunoelectron microscope study. J. Cell Biol. 81, 59-66. OBINATA, T., MASAKI, T., and TAKANO, H. (1980). Types of myosin light chains present during the development of fast skeletal muscle in chick embryo. J. Biochem. (Tokyo) 87,81-88. OHTSUKI, I. (1975). Distribution of troponin components in the thin filaments studied by immunoelectron microscopy. J. Biochem. (Tokyo) 77, 633-639. OHTSUKI, I. (1979). Molecular arrangement of troponin-T in the thin filaments. J. B&hem. (Tokyo) 86,491-497. PELLONI-MUELLER,G., ERMINI, M., and JENNY, E. (1976). Myosin light chains of developing fast and slow rabbit skeletal muscle. FEBS Lett. 67, 68-74. PERRY, S. V., and COLE, H. A. (1974). Phosphorylation of troponin and the effects of interactions between the components of the complex. Biochem. J. 141,733-743. ROY, R. K., SRETER,F. A., and SARKAR, S. (1979). Changes in tropoThis research was supported by grants from the Japanese Ministry myosin subunits and myosin light chains during development of of Education, National Center for Nervous, Mental and Muscular chicken and rabbit striated muscles. Develop. Biol. 69, 15-30. Disorder (NCNMMD) of the Japanese Ministry of Health and WelRUBINSTEIN,N. A., PEPE, F. A., and HOLTZER,H. (1977). Myosin types fare, the Muscular Dystrophy Association, and the Yamada Science during the development of embryonic chicken fast and slow musFoundation. The authors wish to thank Professor K. Maruyama for cles. Proc. Nat. Acad. Sci. USA 74, 4524-4527. his helpful discussions. RUBINSTEIN,N. A., MABUCHI, K., PEPE, F., SALMONS,S., GERGELY,J., and SRETER,F. (1978). Use of type-specific antimyosins to demREFERENCES onstrate the transformation of individual fibers in chronically stimulated rabbit fast muscle. J. Cell Biol. 79, 252-261. AMPHLETT,G. W., PERRY,S. V., SYSKA,G., BROWN,M. D., and VRBOVA, G. (1975). Cross innervation and the regulatory protein system of SARKAR, S., SRETER,F. A., and GERGELY,J. (1971). Light chains of myosins from white, red and cardiac muscles. Proc. Nat. Acad. Sci. rabbit soleus muscle. Nature (London) 257, 602-604. USA 68, 946-950. CUMMINS,P., and PERRY,S. V. (1973). The subunits and biochemical activity of polymorphic forms of tropomyosin. Biochem. J. 133, SHIMADA, Y., FISCHMAN,D. A., and MOSCONA,A. A. (1967). The fine structure of embryonic chick skeletal muscle cells differentiated 765-777. in vitro. J. Cell Biol. 35, 445-453. DHOOT, G. K., and PERRY, S. V. (19’79). Distribution of polymorphic forms of troponin components and tropomyosin in skeletal muscle. SRETER,F., HOLTZER,S., GERGELY,J., and HOLTZER,H. (1972). Some properties of embryonic myosin. J. Cell BioL 55, 586-594. Nature (London) 278, ‘714-718. EBASHI, S., WAKABAYASHI, T., and EBASHI, F. (1971). Troponin and SRETER,F., GERGELEY,J., SALMONS,S., and ROMANUL,F. (1973). Synthesis by fast muscle of myosin light chains characteristic of slow its components. J. Biochem. (Tokyo) 69,441-445. muscle in response to long term stimulation. Nature New Biol. 241, GAUTHIER, G. F., LOWEY,S., and HOBBS,A. W. (1978). Fast and slow 17-18. myosin in developing muscle fibers. Nature (London) 274, 25-29. WEBER,K., and OSBORN,M. (1969). The reliability of molecular weight HAYASHI, J., ISHIMODA,T., and HIRABAYASHI, T. (1977). On the hetdeterminations by dodecyl sulfate-polyacrylamide gel electrophoerogeneity and organ specificity of chicken tropomyosin. J. Biochem. resis. J. Biol. Chem. 244, 4406.4412. (Tokyo) 81.1487-1459. WHALEN, R. G., BUTLER-BROWN,G. S., and GROS,F. (1976). Protein HOH, J. F. Y. (1979). Developmental changes in chicken skeletal synthesis and actin heterogeneity in calf muscle cells in culture. myosin isozymes. FEBS Lett. 98, 267-270. Proc. Nat. Acad. Sci. USA 73, 2018-2022. HOH, J. F. Y., MCGRATH, P. A., and WHITE, R. I. (1976). Electrophoretie analysis of multiple forms of myosin in fast-twitch and slow- WILKINSON,J. M. (1978). The components of troponin from chicken fast skeletal muscle. A comparison of troponin-T and troponin-I twitch muscles of the chick. Biochem. J. 157, 87-95. from breast and leg muscle. Biochem. J. 169, 229-238. ISHIURA, S., SUGITA, H., SUZUKI, K., and IMAHORI, K. (1979). Studies of a calcium activated neutral protease from chicken skeletal mus- YAFFE, D. (1968). Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Nat. Acud. Sci. USA cle. J. Biochem. (Tokyo) 86,579-581. 61,477-483. LOWEY, S., and RISBY, D. (1971). Light chains from fast and slow muscle myosins. Nature (London) 234,81-85. YOSOCAWA,N., SANADA, Y., and KATUNUMA, N. (1978). SusceptibiliMARCHOK,A. C., and HERRMANN, H. (1967). Studies of muscle deties of various myofibrillar proteins to muscle serine protease. J. velopment I. changes in cell proliferation. Develop. Biol. 15,129-155. Biochem. (Tokyo) 83, 1355-1360.

proceeds, gene expressions are restricted so that only a single myosin species specific to the fiber type is synthesized. This view was recently supported by Gauthier et al. (1978). Furthermore, recent experiments by Rubinstein et al. (1978) and Sreter et al. (1973) demonstrated that the type of myosin present in the specific muscle fiber could be changed by chronic electric stimulations. Considering together our observations and the other reports mentioned above, we suggest the following sequence of events. The earliest muscle fibers in all muscles synthesize leg-type TN-T. In breast muscle development a process occurs in which the fiber changes its genomic programming: it ceases synthesizing leg-type TN-T and begins synthesizing breast-type TN-T. It appears therefore that the expression of the leg-type gene is an endogenously programmed feature of the development of all muscles. It is possible that expression of the breast-type TN-T gene is dependent upon exogenous factors such as hatching, a particular type of innervation or flapping movement.