Previous results have shown that the adult human masseter muscle contains myosin isoforms that are specific to early stages of development in trunk and limb muscles, i.e. embryonic and fetal (neonatal) myosin heavy chains (MHC) and embryonic myosin light chain (MLC1emb). We wanted to know if this specific pattern is the result of a late maturation or of a distinct evolution during development.

We show here that the embryonic and the fetal MHC and the MLC1emb are expressed throughout perinatal and postnatal masseter development. Our results also demonstrate that MLC1emb accumulation increases considerably during the postnatal period. In addition, both the slow MLCs and the slow isoform of tropomyosin are expressed later in the masseter than quadriceps and the fast skeletal muscle isoform MLC3 is not detected during fetal and early postnatal development in the masseter whereas it is expressed throughout fetal development in the quadriceps.

Our results thus confirm previous histochemical data and demonstrate that the masseter muscle displays a pattern of myosin and tropomyosin isoform transitions different to that previously described in trunk and limb muscles. This suggests that control of masseter muscle development involves mechanisms distinct from other body muscles, possibly as a result of either its craniofacial innervation or of a possibly different embryonic origin.

     
  • MLC

    myosin light chain

  •  
  • MHC

    myosin heavy chain

  •  
  • MLC1s, MLC2S

    slow myosin light chain

  •  
  • MLC1F, MLC2F, MLC3F

    fast myosin light chain

  •  
  • MLC1emb

    embryonic myosin light chain

  •  
  • MLC1A

    atrial myosin light chain

  •  
  • SDS

    Sodium dodecyl sulfate

  •  
  • 2D

    two dimensional.

The masseter, one of the mandibular elevator muscles, can be divided into a superficial portion and a deep portion.

Histochemical studies have shown that the human masseter muscle has certain unique characteristics that can be linked to its activity. It contains fibers with an ATPase activity intermediate to that of type I (low activity) and that of type II fibers (high activity); the type II fibers are of a much smaller diameter (Ringqvist, 1971; Eriksson el al. 1981; Eriksson et al. 1983); and there is a marked predominance of type I fibers (60–70%) in the adult. In addition, the expression pattern in masseter muscle differs depending upon the species (Rowlerson et al. 1983). The masseter muscles of herbivores that masticate slowly and continuously contain only slow type I fibers, whereas rat and mouse, which present a more rapid mastication activity, have both type I and type IIA fibers. The masseter of guineapig contains only type IIA, and that of rabbit has a mixture of both type I and IIA fibers. In carnivores there is a specific fiber, type IIM, which is associated with the aggressive bite and which contains a super fast type of myosin.

Biochemical studies have shown that skeletal muscles contain specific proteins that are involved in the contractile activity. Among these, the myosin heavy chain (MHC) isoforms have been shown to be encoded by a multigene family (Nguyen et al. 1982), the expression of each gene being regulated in a tissue- and stage-specific manner (Periasamy et al. 1985; Whalen, 1985; Izumo et al. 1986). Protein and mRNA studies have demonstrated that during the early fetal stage, mammalian skeletal muscles mainly express the embryonic and neonatal (fetal) MHCs that are replaced later by the adult fast and slow isoforms (Whalen et al. 1981; Fitszimons et al. 1981; Gambke et al. 1983; Butler-Browne et al. 1984). However, this sequential transition may vary in some muscles.

The other components of the native molecule of myosin, the myosin light chains (MLC), are also encoded by a multigene family which is developmentally regulated according to the following evidence.

  • Early stages of muscle development are characterized by the expression of the embryonic light chain (MLC1emb) (Whalen et al. 1978). Molecular studies have demonstrated that the MLC1emb and the MLC1A found in the atrial muscle are the products of the same gene (Barton et al. 1988).

  • The fast isoform MLC3F is never expressed during embryogenesis.

Previous studies have shown that the embryonic and fetal MHCs and the MLC1emb are expressed in adult human masseter (Butler-Browne et al. 1988). In this paper, we have carried out a study of the human masseter in order to determine whether the developmental myosin isoforms are continuously expressed throughout masseter development or disappear prior to being re-expressed after birth.

Specimens

We have studied 10 fetuses aged between 14 and 37 weeks (14, 19, 24, 30 and 37 week) obtained by spontaneous or therapeutic abortion. The muscles of these fetuses were considered to be normal by histochemical criteria and there was no evidence of any underlying neuromuscular pathology of either the fetus or the family. The gestational age for each fetus was determined by comparing the degree of histological maturation with the clinical, macroscopic and radiological data as described in Barbet et al. (1988). Both sexes were equally represented. The 18 month samples were obtained from a male infant who died from late sudden infant death syndrome. An autopsy was carried out almost immediately after death. No evidence of any neuromuscular disease was found. The adult muscle samples were removed from a 27 year old man during surgery. The atrial muscle was a biopsy of an atrial appendage removed during surgery from a 65 year old man. The quadriceps (vastus lateralis) and masseter (superficial portion) muscles were divided into two parts, one of which was immediately frozen in liquid nitrogen and stored at –80°C for the biochemical analyses and the other was frozen in deep cooled isopentane for immunocytochemical analyses. Masseters and control quadriceps were studied at 24 weeks of gestation, 30 weeks of gestation, 37 weeks of gestation, 18 months postnatal and adult stages. Control adult atrial muscle was kindly provided by M. Yakoub (National Heart and Lung Institute, London).

Two-dimensional gel electrophoresis

Muscle samples, weighing between 20 and 50 mg, were scissor minced in two volumes (w/v) of sample buffer: 9.95M-urea, 4% NP40, 2% ampholines LKB pH 5–7, 100mM-DTT, 1 mg of solid urea per mg of tissue was then added. The muscles were extracted for 15–20 min at room temperature and were vortexed several times during the extraction. Samples were then stored either at –20° or –80°C. Prior to electrophoresis the samples were centrifuged for 5 min in a microfuge to remove any tissue fragments.

Electrophoresis was carried out according to O’Farrell (1975), as modified by Garrels (1979).

Elution of proteins from Coomassie-blue-stained gels

At each stage of development the light chain spots were eluted from the Coomassie-blue-stained 2D gels in order to quantify the amount of embryonic light chain present. Equalsized pieces of the gel were excised with a scalpel and were put into 1 ml of a solution containing 50 % isopropanol, 3 % SDS. The tubes were capped and incubated overnight at 37 °C. The following day the supernatants were collected and their optical density at 595 nm was measured. The blanks were made by eluting pieces of gel of an equivalent size but devoid of any protein.

Northern blot analysis

RNA was prepared from muscle samples according to Chirg-win et al. (1979). 10 μg of total RNA was heat denatured, run on a 1 % agarose 2.5 % formaldehyde gel and transfered by blotting to diazotized Pall Biodyne (Pall Corp.) membranes as described by Barton et al. (1985). Following baking, filters were hybridised with 37×106 ctsmin-1 of heat-denatured 32P-labelled probe (plasmid LCHemp 4, kindly provided by Anna Starzinski-Powitz and Katrina Zimmerman, University of Küln, West Germany) in 50% formaldehyde, 5×SSC, 10× Denharts, 10μgml-1 heat denatured salmon sperm DNA. Following hybridisation, filters were washed in 0.1×SSC, 0.1 % SDS at 50 or 65°C and exposed to Kodak X-Omat film.

Native pyrophosphate gel electrophoresis

Fetal masseter and quadriceps muscles were extracted in two volumes and adult muscles in four volumes of sample buffer; 300mM-sodium chloride, lOOmM-dihydrogen phosphate, 50 mM-disodium phosphate, 1 mM-magnesium chloride, lOmM-sodium pyrophosphate, IOITIM-EDTA, 0.1% 2-mer-captoethanol (pH 6.5). The tissue was scissor minced and extracted for 30 min on ice, the extract was clarified by centrifuging for 15 min in a microfuge. The supernatants were diluted tenfold into diluting buffer 50% glycerol, 40mM-sodium pyrophosphate, 0.1% 2-mercaptoethanol (pH6.5) and were stored at –20°C. Electrophoresis was carried for 20–22 h at 2°C according to d’Albis et al. (1979).

Immunohistochemistry

Immunohistochemistry was carried out on 5–10 μm frozen sections. These sections were incubated for 2 h at 37°C with a polyclonal antibody against embryonic or with monoclonal antibodies against fetal, slow and fast MHCs (Butler-Browne and Whalen, 1984) and the antibody binding was revealed by the peroxidase-anti-peroxidase technique (the slow monoclonal antibody was a kind gift from W. Brown).

Note that all antibodies directed against the MHC isozymes used in this paper (immunotransfer and immunochemistry) have been tested for their specificity on human myosin isozymes by Western blot and by immunocytochemistry on defined muscle blocks.

(1) Expression of MLC1emb

During the early stages of development (14–24 weeks of gestation), equivalent amounts of MLC1emb are expressed in both the masseter and the quadriceps (Fig. 1A, D; E is MLC1emb) F1 is MLClfast and F2 is MLC2fast). In the masseter, this light chain continued to be expressed throughout the whole period of fetal development (Fig. 1B and C). In addition in the masseter more MLC1emb appeared to be present after birth (Fig. 2A and B) than during fetal development (Fig. 1A,B). This is in contrast to the quadriceps where MLC1emb is no longer detected after 30 weeks (Fig. 2C,D). The amount of MLC1emb present at each stage of development in the masseter was quantified and this value has been expressed as a percentage of the total amount of the MLCs (Fig. 3). Our results clearly confirm that instead of being repressed in the masseter, the MLC1emb gradually increases during fetal and postnatal development so that it represents 19 % of the total light chains expressed in the adult.

Fig. 1.

Two-dimensional gel electrophoresis of muscle extracts during prenatal development. Total extracts from human masseter (A,B,C) and quadriceps (D,E,F) muscles at 24 (A,D), 30 (B,E) and 37 weeks of gestation (C,F). Isoelectrofocusing in the first dimension (IEF) was carried out between pH 5 and pH 7. Proteins were separated in the second dimension on 12.5% SDS-polyacrylamide slab gels which were stained with Coomassie blue. The various myofibrillar proteins are indicated as follows: tropomyosin (tm); slow myosin light chains (s1, s2); fast myosin light chains: (f1, f2 and f3); embryonic light chain (e). Note the presence of MLClemb and the absence of LC3 fast throughout fetal development of the masseter muscle.

Fig. 1.

Two-dimensional gel electrophoresis of muscle extracts during prenatal development. Total extracts from human masseter (A,B,C) and quadriceps (D,E,F) muscles at 24 (A,D), 30 (B,E) and 37 weeks of gestation (C,F). Isoelectrofocusing in the first dimension (IEF) was carried out between pH 5 and pH 7. Proteins were separated in the second dimension on 12.5% SDS-polyacrylamide slab gels which were stained with Coomassie blue. The various myofibrillar proteins are indicated as follows: tropomyosin (tm); slow myosin light chains (s1, s2); fast myosin light chains: (f1, f2 and f3); embryonic light chain (e). Note the presence of MLClemb and the absence of LC3 fast throughout fetal development of the masseter muscle.

Fig. 2.

Two-dimensional gel electrophoresis of muscle extracts during postnatal and adult development. Total extracts from human masseter (A, B) and quadriceps (C, D) from an 18-month-old child (A, C) and from an adult (B, D). First and second dimensions were carried out in the same way as in Fig. 1. Note the presence of MLC1emb in the masseter muscle (e).

Fig. 2.

Two-dimensional gel electrophoresis of muscle extracts during postnatal and adult development. Total extracts from human masseter (A, B) and quadriceps (C, D) from an 18-month-old child (A, C) and from an adult (B, D). First and second dimensions were carried out in the same way as in Fig. 1. Note the presence of MLC1emb in the masseter muscle (e).

Fig. 3.

Histogram of the evolution of MLClemb in the masseter. The MLC spots were eluted from the Coomassie-blue-stained two-dimensional gels and their optical density at 595 nm was measured. MLC1emb is represented as a percentage of the total myosin light chains.

Fig. 3.

Histogram of the evolution of MLClemb in the masseter. The MLC spots were eluted from the Coomassie-blue-stained two-dimensional gels and their optical density at 595 nm was measured. MLC1emb is represented as a percentage of the total myosin light chains.

In order to confirm the persistence of the MLC1emb during postnatal development at the RNA level, we have carried out a Northern blot hybridization of total RNA extracted from human masseter (18 months old), quadriceps (18 months old) and adult atrial muscles. The probe used is a cDNA (Anna Starzinski-Powitz and Katrina Zimmerman, unpublished results) isolated from a cDNA library derived from a human muscle cell line, and is identical with the published sequence of the atrial/embryonic light chain (Kurabayashi et al. 1988).

This Northern blot analysis clearly demonstrates the presence of an mRNA coding for MLC1emb in the masseter muscle but not in the quadriceps (Fig. 4). The mRNA of MLC1emb is the same size as that of the mRNA present in atrial muscle (track A). Washing of the Northern blot at high stringency (65 °C) made no difference to the relative intensities of the band seen in these two tracks.

Fig. 4.

Northern blot analysis. 10 μg of total RNA prepared from adult human atrial muscle (A); an 18-month-old human masseter (M) and quadriceps (Q) were separated by electrophoresis, transfered to membranes and hybridized with a probe specific for MLC1emb.

Fig. 4.

Northern blot analysis. 10 μg of total RNA prepared from adult human atrial muscle (A); an 18-month-old human masseter (M) and quadriceps (Q) were separated by electrophoresis, transfered to membranes and hybridized with a probe specific for MLC1emb.

Together these data demonstrate that the masseter muscle contains MLC1emb mRNA. The fact that the abundance of mRNA in the masseter is less than in atrial muscle is consistent with the observation that the masseter contains a mixture of MLC isoforms (Fig. 2A) and would thus be expected to contain proportionally less MLC1emb mRNA.

(2) Appearance of the adult myosin light chain and tropomyosin profile

During the first weeks of muscle development (14–24) virtually no difference is seen in the pattern of proteins expressed in the masseter and quadriceps muscles. In both muscles, three light chains, MLC1emb, MLC1F and MLC2F, are expressed. However, a small amount of MLC3F is seen in the quadriceps between 20 and 24 weeks.

In the masseter during the subsequent period of fetal development (24–40 weeks), MLC1emb, MLC1F and MLC2F remain the major light chains expressed, although small amounts of the slow light chains, especially MLC2S, begin to be detected at 37 weeks (Fig. 1C). There is a postnatal evolution in the expression of the MLCs so that by 18 months all the MLCs, including MLC3F and MLC1emb, are expressed (Fig. 2A). This pattern consequently evolves, with the elimination of MLC2F and MLC3F, to give the predominantly slow phenotype characteristic of the adult masseter (Fig. 2B).

This is in contrast to what occurs in the quadriceps, where the MLC3F is already expressed at 24 weeks (Fig. ID) and by 30 weeks of gestation all five adult light chains are expressed and the embryonic light chain is absent (Fig. IE). Between 30 and 37 weeks there is a gradual increase in the amounts of MLC1S, MLC2S and MLC3F (Fig. 1E,D), so that by 37 weeks in the quadriceps an adult profile is obtained and there is no further evolution in the pattern of myosin light chains (Fig. 2C,D).

We have also followed the evolution of the tropomyosin isoforms during masseter development (Fig. 5). Between 23 and 30 weeks we detected only the fast tropomyosin isoforms (Fig. 5A,B) and from 37 weeks the slow isoform is observed (Fig, 5C,D). The appearance of this slow form of tropomyosin occurs later in the masseter than in the quadriceps muscle (Fig. 1,2). However, it seems to accumulate before the slow MLCs in the same muscle (Fig. 1C).

Fig. 5.

Evolution of tropomyosin isoforms during masseter development. Total extracts of masseter muscles were run on two-dimensional gels. Ten times less protein was loaded than in Figs 1 and 2. The proteins were silver stained. αf: a fast isoform; α3: a slow isoform; β: b isoform. (A) 30 weeks; (B) 37 weeks; (C) 18 months after birth; (D) adult.

Fig. 5.

Evolution of tropomyosin isoforms during masseter development. Total extracts of masseter muscles were run on two-dimensional gels. Ten times less protein was loaded than in Figs 1 and 2. The proteins were silver stained. αf: a fast isoform; α3: a slow isoform; β: b isoform. (A) 30 weeks; (B) 37 weeks; (C) 18 months after birth; (D) adult.

(3)Appearance of the adult MHCs and the persistence of embryonic and fetal MHCs throughout the development of the masseter

The non-denaturing pyrophosphate gels of the masseter (M) and quadriceps (Q) show that the predominant forms of MHC accumulated in both muscles between 14 and 30 weeks of gestation are the embryonic and fetal forms (Fig. 6A–D). By 37 weeks, the fetal myosin is the predominant isoform present in the masseter, the embryonic isoform is greatly reduced and small amounts of adult fast and slow myosins are present (Fig. 6E). This is in contrast to the quadriceps where fast and slow myosins are already predominant at 37 weeks, embryonic myosin is nearly absent and fetal myosin is a minor species. After birth in the masseter the fetal MHC is gradually replaced by the adult fast and slow isoforms which are the major forms at 18 months. Between 18 months and adulthood, the fast isoform is gradually replaced by the slow isoform (Fig. 6F–H). In the adult, a predominantly slow pattern is finally established. If these gels are run with 10-20 times as much extract, bands corresponding to the fast, embryonic and fetal isoforms can be demonstrated, suggesting a persistence of these isoforms during the postnatal development of the masseter muscle (Fig. 6H). These results were confirmed by Western blot analysis (results not shown) and immunocytochemistry.

Fig. 6.

Pyrophosphate gel analysis. Electrophoresis of total muscle extracts from developing human masseter (M) and quadriceps (Q) muscles in non-dissociating conditions. The stages studied were: 14 weeks of gestation (A); 19 weeks of gestation (B); 24 weeks of gestation (C); 30 weeks of gestation (D); 37 weeks of gestation (E) ; 18 months postnatal (F) and adult (G). The stages were noted: weeks of gestation (W); months postnatal (mpn); adult (A). The bands corresponding to slow, fast and fetal myosin are indicated as: fast myosin (Fast), slow myosin (Slow), fetal myosin (f) and embryonic myosin (e). In order to visualize the minor myosin species, in the masseter the amount of protein loaded onto the gels was increased (H).

Fig. 6.

Pyrophosphate gel analysis. Electrophoresis of total muscle extracts from developing human masseter (M) and quadriceps (Q) muscles in non-dissociating conditions. The stages studied were: 14 weeks of gestation (A); 19 weeks of gestation (B); 24 weeks of gestation (C); 30 weeks of gestation (D); 37 weeks of gestation (E) ; 18 months postnatal (F) and adult (G). The stages were noted: weeks of gestation (W); months postnatal (mpn); adult (A). The bands corresponding to slow, fast and fetal myosin are indicated as: fast myosin (Fast), slow myosin (Slow), fetal myosin (f) and embryonic myosin (e). In order to visualize the minor myosin species, in the masseter the amount of protein loaded onto the gels was increased (H).

When sections of quadriceps muscle were stained with antibodies against embryonic and fetal MHC, by 6 months no staining was seen with the embryonic antibody and only a few scattered fibers stained with the fetal antibody (data not shown). In the masseter, immunocytochemistry has confirmed the biochemical results, and there is a slow decrease in the fetal and embryonic MHCs and a slow appearance of the adult isoforms. However, although there is a gradual diminution in the number of fibers that contained these myosin isoforms in the masseter (Fig. 7 and 8), there is never a stage when these isoforms were absent, as one can still find fibers expressing them.

Fig. 7.

Immunocytochemistry using polyclonal anti-embryonic MHC antibody. Transverse sections of masseter muscles at different developmental ages: (A) 30 weeks of gestation; (B) 37 weeks of gestation; (C) 18 months after birth and (D) adult. The antibody binding on the sections was revealed by the peroxidase-anti-peroxidase technique.

Fig. 7.

Immunocytochemistry using polyclonal anti-embryonic MHC antibody. Transverse sections of masseter muscles at different developmental ages: (A) 30 weeks of gestation; (B) 37 weeks of gestation; (C) 18 months after birth and (D) adult. The antibody binding on the sections was revealed by the peroxidase-anti-peroxidase technique.

Fig. 8.

Immunocytochemistry using monoclonal anti-fetal MHC antibody. Tranverse sections of the masseter muscles at different developmental ages (A) 30 weeks of gestation; (B) 37 weeks of gestation; (C) 18 months after birth; (D) adult were stained with anti-fetal MHC and antibody binding was revealed by the peroxidase-anti-peroxidase technique.

Fig. 8.

Immunocytochemistry using monoclonal anti-fetal MHC antibody. Tranverse sections of the masseter muscles at different developmental ages (A) 30 weeks of gestation; (B) 37 weeks of gestation; (C) 18 months after birth; (D) adult were stained with anti-fetal MHC and antibody binding was revealed by the peroxidase-anti-peroxidase technique.

During muscle development at 20 weeks a population of fibers with a large diameter and containing exclusively slow MHC (Wohlfart b fibers) can be distinguished in both the quadriceps and the masseter muscles. All the remaining fibers in these muscles stain predominantly with the antibodies against fetal and embryonic MHC. During the remaining period of muscle development there is a transition of these fibers with a decrease in the amounts of fetal and embryonic myosin and an increase in the amount of fast myosin. Certain of these fibers will also begin to express slow MHCs and there is a gradual increase in both their number and size so that at 37 weeks a homogeneous population of slow fibers is detected (Fig. 9). In the masseter, this evolution is slower and, as can be seen in Fig. 9 at 37 weeks, two populations of fibers containing slow MHC are still evident, one of a larger diameter (Wohlfart b fibers) and one of a smaller diameter.

Fig. 9.

Immunocytochemistry using a monoclonal anti-slow MHC antibody. Transverse sections of masseter (A) and quadriceps (B) at 37 weeks of gestation were stained with an anti-slow MHC.The antibody binding was revealed by the peroxidase-anti-peroxidase technique.

Fig. 9.

Immunocytochemistry using a monoclonal anti-slow MHC antibody. Transverse sections of masseter (A) and quadriceps (B) at 37 weeks of gestation were stained with an anti-slow MHC.The antibody binding was revealed by the peroxidase-anti-peroxidase technique.

In the mature quadriceps, the fast and slow fibers have the same diameter and contain exclusively fast or slow myosin. However, this is not the case in the masseter since the embryonic and fetal myosin never disappear and by 18 months two distinct populations of fibers can be distinguished (Fig. 10). One of these is a population of small diameter fibers that contain in a variable manner embryonic, fetal and fast myosin but never slow myosin. The second is a population of much larger diameter, which in the adult will become the predominant fiber type. These fibers all contain slow MHC, which can be associated with any combination of the other myosin isoforms. The mature profile gradually emerges in the masseter muscle over a long period of time and the exact age at which a stable mature profile is established has not yet been determined.

Fig. 10.

Immunocytochemistry with monoclonal anti-slow and anti-fast MHC. Transverse sections of masseter during postnatal development: 18 months after birth (A,C); adult (B,D). A and C were stained with anti-slow and B and D with anti-fast antibody. The antibody binding was revealed by the peroxidase-anti-peroxidase technique.

Fig. 10.

Immunocytochemistry with monoclonal anti-slow and anti-fast MHC. Transverse sections of masseter during postnatal development: 18 months after birth (A,C); adult (B,D). A and C were stained with anti-slow and B and D with anti-fast antibody. The antibody binding was revealed by the peroxidase-anti-peroxidase technique.

The temporalis and masseter muscles differ from limb muscles in a number of properties (Rowlerson et al. 1981; Rowlerson, 1983; Mabuchi et al. 1984). These muscles belong to a group of so-called specialised muscles found in the head region which are involved in mastication, eye movements and in the production and reception of sound. Although there is some controversy as to the exact origin of these muscles, it is classically thought that they differ from limb muscles in their embryonic origin, being derived from the mesoderm of the first paired branchial arch, except the extraocular muscles, which are of somitic mesoderm origin (Hamilton et al. 1972; Ontell, 1982; Rowlerson et al. 1983). In contrast to the skeletal muscles, which are innervated by the spinal nerves, all of the specialised muscles are innervated by cranial nerves.

One of these specialised muscles, the masseter, shows species differences in its histochemical profile and hence differences in its myosin isoform content. These differences may have evolved to meet the functional requirements of each animal. This is especially striking in predators such as the cat where the jaw muscles contain a special super fast myosin which is used for the aggressive biting reflex required for predators to kill their prey (Rowlerson et al. 1983).

Histochemical studies of the human masseter (Ringqvist et al. 1982; Eriksson et al. 1983) have shown that it contains basically the same fiber types as the limb muscles, but has a predominance of type I fibers. There is a gradient of fiber types in the masseter from the superficial portion, which contains a large number of small diameter type II fibers, and the deep masseter, which contains in many cases almost exclusively type I fibers.

Previous studies have shown that the adult human masseter contains isoforms of both myosin light- and heavy-chains, which are characteristic of developmental stages in the trunk and limb muscles, i.e. embryonic and fetal MHCs and MLC1emb (Butler-Browne et al. 1988).

In this study, we have demonstrated that these isoforms are continuously expressed throughout the prenatal and postnatal development of the masseter muscle.

The MLC1emb is characteristic of the early stages of skeletal muscle development (Whalen et al. 1978; Biral et al. 1984; Pons et al. 1987). By two-dimensional gel electrophoresis, we have shown that the MLC1emb is present in perinatal, postnatal and adult masseter muscles whereas in the quadriceps it is already eliminated by 30 weeks of gestation (Figs 1,2). Moreover, quantitative analysis of this light chain in the masseter shows that, instead of being repressed early in development, it is accumulated in the last trimester of pregnancy and after birth, so that, in the adult, it represents almost 20% of the total light chain content. This is confirmed at the RNA level by Northern blot analysis, since we have shown that in infants the MLC1emb transcript is present in the masseter but not in the quadriceps. This light chain has only been detected in two other normal adult muscles, the atria (Barton et al. 1985; Cummins, 1982) and the extraocular muscle (personal data not shown). The precise function and localisation of this light chain in the masseter muscle are not yet known.

Previous studies by Ringqvist et al. (1982) demon strated that limb muscles (biceps brachii) differentiate earlier than the masticatory muscles, and that even at birth the masseter was less differentiated than the biceps. Our observations demonstrate that this later histochemical maturation is coupled with a later biochemical differentiation of the human masseter muscle compared to that observed in the quadriceps. MLC1S, MLC2S, slow MHC, fast MHC and slow tropomyosin (TMs) are all expressed later in the masseter than in the quadriceps. The evolution of the MLCs in these two muscles is shown in Table 1. MLC3F is not expressed in the masseter during fetal development. This absence of MLC3F seems to be correlated with the later expression of the adult fast MHC during fetal development which only begins to be detected at 37 weeks. At 18 months, when adult fast MHC is one of the major isozymes observed, MLC3F can also be detected. These results are in agreement with those of d’Albis et al. (1986) who reported a similar delay in the induction of the adult type myosins in the masseter of rodents.

Table 1.

Schematic representation of MLC evolution during human muscle development in the masseter compared to the quadriceps

Schematic representation of MLC evolution during human muscle development in the masseter compared to the quadriceps
Schematic representation of MLC evolution during human muscle development in the masseter compared to the quadriceps

With antibodies directed against the different MHC isozymes, it is possible to demonstrate by immunocytochemistry that the masseter at 37 weeks of gestation is less mature than other skeletal muscles at the same stage. In addition to larger amounts of the embryonic and fetal MHCs and smaller amounts of the adult fast and slow MHCs present in the muscle, there is also a difference in the morphological maturation. The masseter seems to be subjected to a very prolonged period of maturation as demonstrated by the biochemical and immunocytochemical data. A similar prolonged period of maturation was observed in the rat soleus (Butler-Browne and Whalen, 1984), a muscle which is also predominantly slow in the adult, but which at birth contains only 50% slow fibers, the remaining 50% of which contain neonatal (fetal) myosin. During a period of several months there is a gradual diminution in the number of fibers containing neonatal myosin which are replaced by fast and slow myosin so that in the adult there are usually between 80–90% slow type I fibers in the soleus muscle.

Finally we have also demonstrated a persistence of the embryonic and fetal MHC throughout development in the masseter. It thus seems that the masseter develops at a different rate and in a different way from other skeletal muscles, as previously proposed by Maxwell et al. (1980).

In skeletal muscle the biochemical specialisation is correlated with the segregation of the specific isoforms of myosin, tropomyosin, troponin, M protein, C protein and sarcoplasmic proteins in the muscle cells, and the different biochemical properties of these proteins account for the physiological properties of the motor units. It now seems evident that there is both a neuronal and a hormonal control of this specialisation (Butler-Browne et al. 1982; Gambke et al. 1983; Izumo et al. 1986; Butler-Browne et al. 1987).

In a fast muscle during development, there is a transition of the MHCs: embryonic→fetal→adult fast. In these muscles, thyroid hormone is critical for a correct MHC expression; it activates fast MHC synthesis and stimulates the fetal-fast transition, independently of the nerve. In a slow muscle, thyroid hormone does not seem to have any effect on the fetal-slow transition, and it is the nerve that is essential for the expression of a slow phenotype. Neonatal denervation of the rat soleus muscle results in the production of an atrophied muscle, which contains fast MHC (Gambke et al. 1983). The same results is observed during the regeneration of a denervated soleus (Whalen et al. 1985).

The masseter is a predominantly slow muscle and, if the human muscle behaves in the same way as the rat, it will be insensitive to thyroid hormone, and so would be expected to follow the pathway of expression that we have previously defined for the rat soleus. This seems to be true for the developmental evolution of the slow-myosin-containing fibers in the human masseter. But there is a persistence of the embryonic and fetal MHC isoforms. This is not typical and could be the result either of its innervation or of its embryological origin. The extensive study of the skeletal muscles of different species by Rowlerson et al. (1981) proved that there was no correlation between origin and fiber type distribution. Rowlerson suggested that fiber type profile is a result of an adaptation to particular contractile characteristics. The persistence of developmental isoforms in both the masseter, which is of branchial origin, and in the extraocular muscle, which is of somitic origin, both of which are innervated by cranial nerves, suggest a common neuronal regulation of the expression of the isoforms. However, in order to prove this hypothesis, we will have to extend our studies to the other muscles of branchiomeric origin that are innervated by craniofacial nerves.

Since the masseter coexpresses isoforms that are usually exclusive, it is an interesting model to study the factors involved in the control of these isoforms during maturation.

We thank Dr Anna Starzinski-Powitz and Katrina Zimmerman for providing us with plasmid LCHemp 4.

This work is supported by the Institut National de la Santé et de la Recherche Médicale and the Association Française contre les Myopathies.

D’albis
,
A.
,
Pantaloni
,
C.
and
Bechet
,
J. J.
(
1979
).
An electrophoretic study of native myosin isozymes and of their subunit content
.
Eur. J. Biochem.
99
,
261
272
.
D’albis
,
A.
,
Janmot
,
C.
and
Bechet
,
J. J.
(
1986
).
Comparison of myosin from the masseter muscle of adult rat, mouse and guineapig. Persistence of neonatal-type isoforms in the murine muscle
.
Eur. J. Biochem.
156
,
291
296
.
Barbet
,
J. P.
,
Houette
,
A.
,
Barres
,
D.
and
Durigon
,
M.
(
1988
).
Histological assessment of gestational age in human embryos and fetuses
.
Am. J. of Forensic Medicine and Pathology.
9
,
40
44
.
Barton
,
P.
and
Buckingham
,
M.
(
1985
).
The myosin alkali light chain proteins and their genes
.
Biochem. J.
231
,
249
261
.
Barton
,
P.
,
Robert
,
B.
,
Cohen
,
A.
,
Garner
,
I.
,
Sassoon
,
D.
,
Weydert
,
A.
and
Buckingham
,
M.
(
1988
).
Structure and sequence of the myosin alkali light chain gene expressed in adult cardiac atria and fetal striated muscle
.
J. biol. Chem.
263
,
12669
12676
.
Barton
,
P.
,
Robert
,
B.
,
Fiszman
,
M. Y.
,
Leader
,
D. P.
and
Buckingham
,
M. E.
(
1985
).
The same alkali light myosin chains gene is expressed in adult cardiac atria and in foetal skeletal muscle
.
J. Mus. Res. Cell Motil.
6
,
461
—475.
Biral
,
D.
,
Diamiani
,
E.
,
Margreth
,
E.
and
Scarpini
,
E.
(
1984
).
Myosin subunit composition in human developmental muscle
.
Biochem. J.
224
,
923
931
.
Butler-Browne
,
G. S.
,
Bugaisky
,
L. B.
,
Cuenoud
,
S.
,
Schwartz
,
K.
and
Whalen
,
R. G.
(
1982
).
Denervation of newborn rat muscle does not block the appearance of adult fast myosin
.
Nature
229
,
830
833
.
Butler-Browne
,
G. S.
,
Eriksson
,
P. O.
,
Laurent
,
C.
and
Thornell
,
L. E.
(
1988
).
Adult human masseter muscle fibers express myosin isozymes characteristic of development
.
Muscle and Nerve
11
,
610
620
.
Butler-Browne
,
G. S.
and
Whalen
,
R. G.
(
1984
).
Myosin isozyme transitions occuring during the post-natal development of the rat soleus muscle
.
Devi Biol.
102
,
324
334
.
Butler-Browne
,
G. S.
,
Pruuere
,
G.
,
Cambon
,
N.
and
Whalen
,
R. G.
(
1987
).
Influence of the dwarf mutation on skeletal and cardiac myosin isoforms
.
J. biol. Chem.
262
,
15188
15193
.
Chirgwin
,
J. M.
,
Przybyla
,
A. E.
,
Macdonald
,
R.
and
Rutter
,
W. J.
(
1979
).
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease
.
Biochemistry
18
,
5294
5299
.
Cummins
,
P.
(
1982
).
Transitions in human atrial and ventricular myosin light-chain isoenzymes in response to cardiac presure overload induced hypertrophy
.
Biochem. J.
205
,
195
204
.
Eriksson
,
P. O.
,
Eriksson
,
A.
,
Ringqvist
,
M.
and
Thornell
,
L. E.
(
1981
).
Special histochemical muscle-fibre characteristics of the human lateral pterygoid muscle
.
Archs Oral Biol.
27
,
207
215
.
Eriksson
,
P. O.
and
Thornell
,
L. E.
(
1983
).
Histochemical and morphological muscle fibre characterisation of the human masseter, the medial pterygoid and the temporal muscles
.
Archs Oral Biol.
28
,
781
795
.
O’farrel
,
P. H.
(
1975
).
High resolution two-dimensional electrophoresis of proteins
.
J. biol. Chem.
250
,
4007
4021
.
Fitzsimons
,
R. B.
and
Hoh
,
J. F. Y.
(
1981
).
Embryonic and fetal myosins in human skeletal muscle
.
J. Neurol. Sci.
52
,
367
384
.
Gambke
,
B.
,
Lyons
,
G. E.
,
Haselgrove
,
J.
,
Kelly
,
A. M.
and
Rubenstein
,
N.
(
1983
).
Thyroidal and neural control of myosin transitions during the development of rat fast and slow muscles
.
FEBS Letts.
156
,
335
339
.
Garrels
,
J. I.
(
1979
).
Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines
.
J. biol. Chem.
254
,
7961
7977
.
Hamilton
,
W. J.
,
Boyd
,
J. D.
and
Mossman
,
H. W.
(
1972
).
Human Embryology, ed. Hamilton, and Mossman
.
Izumo
,
S.
,
Nadal-Ginard
,
B.
and
Mahdavi
,
V.
(
1986
).
All members of the myosin heavy chain multigene family respond to thyroid hormone in a highly tissue specific manner
.
Science
231
,
597
600
.
Kurabayashi
,
M.
,
Komuro
,
I.
,
Tsuchimochi
,
H.
,
Takaku
,
F.
and
Yazaki
,
Y.
(
1988
).
Molecular cloning and characterisation of human atrial and ventricular myosin alkali light chain clones
.
J. biol. Chem.
263
,
13930
13936
.
Lompre
,
A. M.
,
Nadal-Ginard
,
B.
and
Mahdavi
,
V.
(
1984
).
Expression of the cardiac ventricular a- and b-myosin heavy chain genes is developmentally and hormonally regulated
.
J. biol. Chem.
259
,
6437
6446
.
Mabuchi
,
K.
,
Pinter
,
K.
,
Mabuchi
,
Y.
,
Sreter
,
F.
and
Gergley
,
J.
(
1984
).
Characterisation of rabbit masseter muscle fibers
.
Muscle and Nerve
7
,
431
438
.
Maxwell
,
L. C.
,
Carlson
,
D. S.
and
Brangwyn
,
C. E.
(
1980
).
Lack of ‘acid reversal’ of myofibrillar adenosine triphosphatase in masticatory muscle fibres of rhesus monkey
.
Histochemical Journal
12
,
209
219
.
Nguyen
,
H. T.
,
Gubis
,
R. M.
,
Wydro
,
R. M.
and
Nadal-Ginard
,
B.
(
1982
).
Sarcomeric myosin heavy chain is coded by a highly conserved multigene family
.
Proc. natl. Acad. Sci. U.S.A.
79
,
5230
5234
.
Ontell
,
M.
(
1982
).
The growth and metabolism of developing muscle, in Biochemical Development of the Fetus and Neonate. ed. C. T. Jones
, pp.
213
247
.
Periasamy
,
M.
,
Wieczorek
,
D. F.
and
Nadal-Ginard
,
B.
(
1985
).
Characterisation of a developmentally regulated perinatal myosin heavy chain gene expressed in skeletal muscle
.
J. biol. Chem.
259
,
13573
13578
.
Pons
,
F.
,
Damadei
,
A.
and
Leger
,
J. J.
(
1987
).
Expression of myosin light chains during development of human skeletal muscle
.
Bioch. J.
243
,
425
430
.
Ringqvist
,
M.
(
1971
).
Histochemical fiber type and fiber sizes in human masticatory muscles
.
Scand. J. dent. Res.
79
,
336
368
.
Ringqvist
,
M.
(
1974
).
Fiber type in human masticatory muscles
.
Scand. J. dent. Res.
82
,
333
355
.
Ringqvist
,
M.
,
Ringqvist
,
I.
,
Eriksson
,
P. O.
and
Thornell
,
L. E.
(
1982
).
Histochemical fiber type profile in the human masseter muscle
.
J. Neurol. Sci.
53
,
273
282
.
Rowlerson
,
A.
,
Mascarello
,
F.
,
Veggetti
,
A.
and
Carpene
,
E.
(
1983
).
The fibre type composition of the first branchial arch muscles in carnivora and primates
.
J. Muscle Res. Cell Motil.
4
,
443
472
.
Rowlerson
,
A.
,
Pope
,
B.
,
Murray
,
J.
,
Whalen
,
R. G.
and
Weeds
,
A. G.
(
1981
).
A novel myosin present in cat jaw-closing muscles
.
J. Muscle Res. Cell Motil.
2
,
415
438
.
Schiaffino
,
J.
,
Gorza
,
L.
,
Dones
,
I.
,
Cornelio
,
F.
and
Sartore
,
S.
(
1986
).
Fetal myosin immunoreactivity in human dystrophic muscle
.
Muscle and Nerve
9
,
51
58
.
Schwartz
,
K.
,
Lompe
,
A. M.
,
Bouveret
,
P.
,
Wisnewsky
,
C.
and
Whalen
,
R. G.
(
1982
).
Comparisons of rat cardiac myosins at fetal stages in young animals and in hypothyroid adults
.
J. biol. Chem.
257
,
1412
1418
.
Thornell
,
L. E.
,
Billeter
,
R.
,
Eriksson
,
P. O.
and
Ringqvist
,
M.
(
1984
).
Heterogenous distribution of myosin in human masticatory muscle fibers as shown by immunocytochemistry
.
Archs Oral Biol.
29
,
1
5
.
Whalen
,
R. G.
(
1985
).
Myosin isozymes as molecular markers for muscle physiology
.
J. exp. Biol.
115
,
43
53
.
Whalen
,
R. G.
,
Butler-Browne
,
G. S.
and
Gros
,
F.
(
1978
).
Identification of a novel form of myosin light chain present in embryonic muscle tissue and cultured muscle cells
.
J. molec. Biol.
126
,
415
431
.
Whalen
,
R. G.
,
Butler-Browne
,
G. S.
,
Harris
,
J. B.
and
Herlicoviez
,
D.
(
1985
).
Myosin isozyme transitions in developing and regenerating rat muscle. Proceedings of the MDA colloquium: Gene Expression in Muscle. Advance in experimental Medicine and Biology, cd. Strohman, R. C. and Wolf, S
.
182
,
193
199
.
Whalen
,
R. G.
,
Sell
,
S. M.
,
Butler-Browne
,
G. S.
,
Schwartz
,
K.
,
Bouveret
,
P.
and
Pinset-Harstrom
,
I.
(
1981
).
Three myosin heavy chain isozymes appear sequentially in rat muscle development
.
Nature.
292
,
805
809
.
Wieczorek
,
D. F.
,
Periasamy
,
M.
,
Butler-Browne
,
G. S.
,
Whalen
,
R. G.
and
Nadal-Ginard
,
B.
(
1985
).
Co-expression of multiple myosin heavy chain genes, in addition to a tissuespecific one, in extraocular musculature
.
Cell Biol.
101
,
618
629
.
Yemm
,
R.
(
1977
).
The ordery recruitment of motor units of the masseter and temporal muscles during voluntary isometric contraction in man
.
J. Physiol(Lond).
265
,
163
174
.