Anthea Rowlerson

Comparative study of myosins present in the lateral muscle of some fish: species variations in myosin isoforms and their distribution in red, pink and white muscle

SummaryMyosin isoforms and their distribution in the various fibre types of the lateral muscle of eight teleost fish (representing a wide range of taxonomic groups and lifestyles) were investigated electrophoretically, histochemically and immunohistochemically.Polyclonal antisera were raised against slow (red muscle) and fast (white muscle) myosins of the mullet, and used to stain sections of lateral muscle. Antisera specific for fast and slow myosin heavy chains only (anti-FHC and anti-SHC respectively) and for whole fast and slow myosins (anti-F and anti-S respectively) were obtained, and their specificity was confirmed by immunoblotting against electrophoretically separated myofibrillar proteins.The ATPase activity of myosin isoforms was examined histochemically using methods to demonstrate their acid- and alkali-lability and their Ca-Mg dependent actomyosin ATPase.As expected, the predominant myosin (and fibre) type in the red muscle showed an alkali-labile ATPase activity, reacted with the anti-S and anti-SHC sera (but not anti-F or anti-FHC) and contained two ‘slow’ light chains, whereas the predominant myosin (and fibre) type in the white muscle showed an alkali-stable ATPase activity, reacted with anti-F and anti-FHC sera (but not anti-S or anti-SHC) and contained three ‘fast’ light chains. However, superimposed upon this basic pattern were a number of variations, many of them species-related.On analysis by two-dimensional gel electrophoresis fish myosin light chains LC1s, LC2s and LC2f migrated like the corresponding light chains of mammalian myosins, but fish LC1f consistently had a more acidic pI value than mammalian LC1f. Fish LC3f varied markedly inMr in a species-related manner: in some fish (e.g. eel and mullet) theMr value of LC3f was less than that for the other two light chains (as in mammalian myosin), whereas in others it was similar to that of LC2f (e.g. cat-fish) or even greater (e.g. goldfish). Species differences were also seen in the relative intensity of LC1f and LC3f spots given by the fish fast myosins.In most of the fish examined the red muscle layer showed some micro-heterogeneity, containing (in addition to the typical slow fibres) small numbers of fibres with a histo- and immunohistochemical profile typical of white muscle (fast) fibres. However, other immunohistochemically distinct minority fibres were found in the red muscle of the goldfish.Three types of pink muscle were distinguished: (1) a mosaic of immunohistochemically typical red and white fibres (e.g. grey mullet); (2) a ‘transition zone’ with properties intermediate between those of red and white muscle (e.g. guppy); and (3) a layer of fibres which appeared on the basis of their myosin and actomyosin ATPase activities to contain a distinct myosin type, although this could not be distinguished from the white muscle fast myosin by any of the antisera used (e.g. goldfish, cat-fish, carp).Four of the fish examined had a mosaic white muscle consisting of fibres of a very wide range of diameters. The larger sized fibres always had the histo- and immunohistochemical profile of fast (white) fibres, but the characteristics of the small fibres varied according to the species. In the trout no histo- or immunohistochemical difference between large and small fibres could be detected, and in the mullet there was a histochemical difference only. In the eel some of the smallest fibres reacted with the anti-S serum as well as with anti-fast sera, and in the carp the small fibres reacted with both anti-S and anti-SHC in addition to the anti-fast sera, but in neither species could any trace of slow light chains be found in this muscle. In these two cases the small fibres may contain a cross-reacting form of myosin analogous to mammalian embryonic myosin. The significance of the small fibres of mosaic white muscles in the context of postlarval hyperplastic growth mechanisms in fish muscle is discussed.

DIFFERENTIATION AND GROWTH OF MUSCLE IN THE FISH SPARUS AURATA (L). II: HYPERPLASTIC AND HYPERTROPHIC GROWTH OF LATERAL MUSCLE FROM HATCHING TO ADULT

SummaryPost-hatching growth of lateral muscle in a teleost fish, Sparus aurata (L) was studied morphometrically to identify and quantify muscle fibre hyperplasia and hypertrophy, and by in vivo nuclear labelling with 5-bromo-deoxyuridine to identify areas of myoblast proliferation. Muscle fibre types were identified principally by myosin ATPase histochemistry and immunostaining, and labelled nuclei were identified at light and electronmicroscope level by immunostaining with a specific monoclonal antibody. Hyperplastic growth was slow at hatching, but then increased to a maximum at the mid-point of larval life. Larval hyperplastic growth occured by apposition of new fibres along proliferation zones, principally just under the lateral line and in the apical regions of the myotome, but also just under the superficial monolayer at intermediate positions. The first of these zones gave rise to slow and pink muscle fibres, in a process which continued through into postlarval life. The other zones added new fibres to the fast-white muscle layer in a process which was exhausted by the end of larval life. Post-larvally, between 60 and 90 days posthatching, a new hyperplastic process started in the fast-white muscle as nuclei proliferated and new muscle fibres were formed throughout the whole layer. This process resulted in a several-fold increase in the number of fast-white fibres over a few weeks, and then waned to very low levels in juveniles. Hyperplasia by apposition continued for some time postlarvally on the deep surface of the superficial monolayer, but at this stage gave rise to slow fibres only. Hypertrophic growth occurred at all ages, but was the dominant mechanism of muscle growth only in the juvenile and adult stages. Mechanisms giving rise to these different growth processes in fish muscle are discussed, and compared with muscle development in higher vertebrates.

No classical type IIB fibres in dog skeletal muscle

SummaryTo analyse the fibre type composition of adult dog skeletal muscle, enzyme histochemistry, immunohistochemistry for type I, IIA and IIB myosins, and peptide mapping of myosin heavy chains isolated from typed single fibres were combined. Subdivision of type II fibres into two main classes according to the activity of the m-ATPase after acidic and alkaline preincubation proved to be rather difficult and was only consistently achieved after a very careful adjustment of the systems used. One of these sub-classes of type II fibres stained more strongly for m-ATPase activity after acidic and alkaline preincubation, was oxidative-glycolytic and showed a strong reaction with an anti-type IIA myosin. The other one, however, although unreactive with anti-IIA myosin, was also oxidative-glycolytic, and only showed a faint reaction with an anti-type IIB myosin. Peptide mapping of the myosin heavy chains of typed single fibres revealed two populations of heavy chains among the type II fibre group. Thus, in dog muscle, we are confronted with the presence of two main classes of type II fibres, both oxidative-glycolytic, but differing in the structure of their myosin heavy chains. In contrast to some reports in the literature, no classical type IIB fibres could be detected.

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.)

SummaryIn this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers.In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance.Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life.The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a “flattened” profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle.

The fibre-type composition of the first branchial arch muscles in Carnivora and Primates.

SummaryA combination of standard histochemical techniques and immunohistochemical staining using myosin type-specific antisera was used to determine the fibre-type composition of the muscles of first branchial arch origin (that is, masseter, temporalis, pterygoideus medialis and lateralis, tensor veli palatini, tensor tympani, anterior digastricus and mylohyoideus) in a wide range of the Carnivora and the Primates.The rare IIM fibre type was found in the first branchial arch muscles of most of the species examined, but never in the limb muscles used as controls for this study. The jaw-closer muscles (masseter, temporalis and pterygoideus medialis) were found to contain IIM fibres in all the Carnivora except the lesser panda and in all the Primates except man. When present, the IIM fibres were usually the predominant fibre type, and the only other fibre types present were types I, II or IIC. The presence of IIM fibres in the jaw-closer muscles of most of the Carnivora and the Primates seems to be associated with an aggressive bite which is required for predation by the former and defence by the latter. In both groups of species there was one member which does not have an aggressive bite, the lesser panda and man, respectively, and these (like all other orders of mammals such as Lagomorpha, Rodentia, etc.) were found to have no IIM fibres in the jaw-closer muscles.The two muscles of the first branchial arch group which are derived from the ventral constrictor muscles of the (phylogenetically) original mandibular arch never contained IIM fibres, and were composed of type I and II fibres similar to those found in the control muscles of the limb.Tensor veli palatini and tensor tympani showed species-dependent variations in fibre-type composition and did not always reflect the composition of the jaw-closer muscles. Thus their common origin with the jaw-closers cannot be responsible for the occurrence of IIM fibres in tensor veli palatini and tensor tympani in some species. Furthermore, in tensor tympani but not in tensor veli palatini, the presence of IIM fibres was always accompanied by immunohistochemically slow-tonic fibres.Finally, with regard to the association of oxidative activity with the fibre type as defined by the myofibrillar ATPase method and by the isoform of myosin present, we suggest that in the first branchial arch muscles this is probably not directly comparable to the situation in the typical limb muscle.

Muscle growth and myosin isoform transitions during development of a small teleost fish, Poecilia reticulata (Peters) (Atheriniformes, Poeciliidae): a histochemical, immunohistochemical, ultrastructural and morphometric study

The myosin composition of lateral muscle in Poecilia reticulata from birth to adult was studied by ATPase histochemistry and immunostaining with myosin isoform-specific antibodies. At birth the muscle consists of two layers containing developmental isoforms of myosin. In deep layer fibres the developmental myosin is replaced by the adult fast-white isoform soon after birth. In the epaxial and hypaxial monolayer fibres the myosin composition present at birth (J1) is replaced within 3 days by another (J2). In some fibres, this J2 composition is retained in the adult, but in others it is slowly replaced by the adult slow-red muscle isoform. Close to the lateral line, all monolayer fibres are already in transition between the J2 myosin and the adult slow-red form at birth, and rapidly complete the transition to slow-red form. These fibres, together with others generated de novo in an underlying hyperplastic zone, form the red muscle layer of the adult. The pink muscle develops during the first month after birth, and by 31 days it consists of an outer, middle and inner layer. A few middle layer fibres are already present at birth, while the outer layer fibres first appear 3 days after birth. The thin inner layer is probably a transitional form between the middle pink and adult white types, and appears at about 31 days. A morphometric analysis showed that growth of the white muscle occurs principally by hypertrophy. Even at the magnification level of the electron microscope, no satellite cells or myoblasts which could give rise to new fibres were found in the white muscle, except in the far epaxial and hypaxial regions and only in the first 10 days. A zone of hyperplastic growth was also found lying just under the superficial monolayer close to the lateral line, and this presumably contributes fibres to the red and pink muscle layers.

Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study

Abstract. In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata (sea bream), Solea solea (sole) and Brachydanio rerio (zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).

Differentiation and growth of muscle in the fish Sparus aurata (L): I. Myosin expression and organization of fibre types in lateral muscle from hatching to adult

SummaryPost-hatching development of lateral muscle in a teleost fish, Sparus aurata (L) was examined. At hatching only two fibre types were present; several layers of mitochondria-poor, myofibril-rich deep muscle fibres surrounded the notochord and were covered by a superficial monolayer of mitochondria-rich, myofibril-poor fibres. A third ultrastructurally distinct fibre type first appeared as one or two fibres located just under the lateral line at 6 days post-hatching. This type, which gradually increased in number during larval life, contained a slow isoform of myosin, identified by mATPase staining and immunostaining with myosin isoform-specific antibodies. Deep muscle fibres — the presumptive fast-white type — contained a fast myosin, and superficial monolayer fibres an isoform similar but not identical to that in adult pink muscle fibres. The only fibres present during larval life which showed a clear change in myosin expression were the superficial monolayer fibres, which gradually transformed into the slow type post-larvally. Pink muscle fibres first appeared near the end of larval life. Both slow and pink muscle fibres remained concentrated around the horizontal septum under the lateral line during larval life, expanding outwards towards the apices of the myotomes only after metamorphosis. Between 60 and 90 days very small diameter fibres with a distinct mATPase profile appeared scattered throughout the deep, fast-white muscle layer, giving it a ‘mosaic’ appearance, which persisted into adult life. A marked expansion in the slow muscle layer began at the same time, partly by transformation of superficial monolayer fibres, but mainly by addition of new fibres both on the deep surface of the superficial monolayer and close to the lateral line. The order of appearance of these fibre types, their myosin composition, and the significance of the superificial monolayer layer are discussed and compared to muscle fibre type development in higher vertebrates.

Regeneration of skeletal muscle in two teleost fish: Sparus aurata and Brachydanio rerio.

Abstract.Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5′-bromo-2′-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.

The tensor tympani muscle of cat and dog contains IIM and slow-tonic fibres: an unusual combination of fibre types

SummaryUsing recently developed highly specific antisera to the full range of known adult mammalian skeletal muscle myosins, an immunohistochemical and histochemical examination was made of the middle ear muscle tensor tympani in the dog and cat. Approximately half the fibres were of the IIM type and there was a substantial population of apparently slow-tonic fibres, both these types being rare in mammals. In addition, some type I but no IIA nor IIB fibres were detected. Moreover, as no multiple end-plate innervation, thought to be typical of slow-tonic fibres, could be demonstrated in this muscle by acetylcholinesterase staining or by Ruffini gold impregnation, it is suggested that in tensor tympani the slow-tonic fibres are focally innervated. The very short length of the fibres, only 1–2 mm, is probably sufficient to permit adequate depolarization of a whole fibre by a single centrally situated end-plate. The functional implications of this combination of very rare fibre types in tensor tympani are unclear at present.