Cytoplasmic activation of human nuclear genes in stable heterocaryons

We have induced the stable expression of muscle-specific genes in human nonmuscle cells. Normal diploid human amniocytes were fused with differentiated mouse muscle cells by using polyethylene glycol. The fusion product, a stable heterocaryon in which the parental cell nuclei remained distinct, did not undergo division and retained a full complement of chromosomes. This is in contrast with typical interspecific hybrids (syncaryons), in which the parental nuclei are combined and chromosomes are progressively lost during cell division. The human muscle proteins, myosin light chains 1 and 2, MB and MM creatine kinase and a functional mouse-human hybrid MM enzyme molecule were detected in the heterocaryons. Synthesis of these proteins was evident 24 hr after fusion and increased in a time-dependent manner thereafter. Our results indicate that differentiated mouse muscle nuclei can activate human muscle genes in the nuclei of a cell type in which they are not normally expressed, and that this activation occurs via the cytoplasm. The activators are still present in cells which have already initiated differentiation, are recognized by nuclei of another species, and do not diffuse between unfused cells. The reprogrammed amniocyte nuclei of stable heterocaryons provide a unique system in which to study the mechanisms regulating gene expression during cell specialization.     

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro

Summary Cultures of embryonicDrosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. InDrosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in post-embryonic insect or vertebrate material. Mrs. Pilar Toribio-Fiorio provided excellent technical assistance, and Patricia Minter, the secretarial expertise. This investigation was supported, in part, by NIH Grant NS9330 and the James Douglas Research Fund to Robert L. Seecof and NIH Grant No. 1 RO1 CA17223-01 to Raymond L. Teplitz.     

An ultrastructural study of regeneration of minced smooth muscle in the vas deferens of the guinea-pig

Summary Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3–5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.This work was supported by grants from the Wellcome Trust and the Medical Research Council     

THE EFFECTS OF ACTINOMYCIN D ON MUSCLE CELLS OF ASCIDIAN EMBRYO*

The effect of actinomycin D on muscle cells development of the ascidian, Herdmania momus was studied ultrastructurally. No myofilament was formed when the drug was given at any stage before early tail-bud stage (stage 3). Some aggregates of myofilaments in various size were formed when the treatment was started at stage 4 (4.5 hr after fertilization at about 28°C). Above 60% of myofibrils of fully differentiated muscle cells were formed when the treatment was initiated at stage 5 (5 hr after fertilization). Muscle cells of the tadpoles treated from stage 7 (6 hr after fertilization), at which myofilaments were first detectable in normal development, differentiated almost normally. It is therefore suggested that most mRNAs for muscle proteins are synthesized preceding the onset of myofilament formation and are relatively stable. It is also suggested that mRNAs for myosin, actin and Z band materials are almost simultaneously synthesized. One of the characteristic features of the muscle cell development of the ascidian embryo is almost synchronous differentiation of relatively small numbers of cells (about 54–60 cells). The significance of these results is discussed in relation to mosaic natures of the muscle development.     

Quantitative studies on the distribution of myofilaments in intrafusal muscle fibres

Summary Muscle spindles contain two types of intrafusal muscle fibre, nuclear bag fibres and nuclear chain fibres. The intrafusal fibres of rabbit and guinea pig spindles have been studied using quantitative stereological techniques at the ultrastructural level. The crosssectional areas occupied by myofilaments have been measured in the polar and equatorial regions of both types of intrafusal fibre. There are considerably fewer myofilaments in the equatorial regions of both types of fibre compared with their polar regions.This work was carried out with the aid of grants from the Medical Research Council and the Science Research Council of Great Britain.     

Initial stages of the myocardial differentiation of the lymph hearts of frog tadpoles. A study by the methods of electron microscopy and electron microscopic autoradiography

Using electron microscope autoradiography, a study was made of the ultrastructure of early stages of muscle differentiation and 3H-thymidine (3H-T) labelled cells in the wall of the developing lymph heart of larvae of Rana temporaria L. The mononucleated postmitotic myoblasts with small bundles of thin and thick myofilaments deprived of Z-bodies were found in the lymph heart wall. No thin or intermediate-sized subsarcolemmal filaments were detected in the cytoplasm of these myoblasts. Myosatellites occurred under the basal lamina of muscle cells at stages 41-42. The primitive muscle-nerve junction was found at stages 44-45. Four hours after a single 3H-T administration only mononuclear cells without myofilaments were labelled. If the fixation was made 72 hours after a single 3H-T administration, the label was found, in addition, on the muscle cell nuclei. These data evidence that at the early stages of muscle differentiation in the developing lymph heart wall DNA synthesis and muscle specific protein synthesis are incompatible.     

Ultrastructural analysis of the transdifferentiation of smooth muscle to skeletal muscle in the murine esophagus

The ultrastructure of the mouse esophagus at the level of the diaphragm was studied from embryo day 17 to adult. The transdifferentiation of smooth muscle into skeletal muscle was categorized into seven ultrastructural stages: during phase I normal smooth muscle myogenesis was observed. In phase II subpopulations of cells changed into aggregates of myoblast-like cells. At the center of these cell aggregates, phase III cells appeared that contained condensed myofilaments. Dense bodies and dense bands appeared enlarged by the accumulation of thin filaments. In phase IV the condensed myofilaments organized into sarcomere pretemplate structures. The dense bodies and dense bands formed rudimentary Z-lines. In phase V the sarcomere templates appeared as more defined structures and began to align. An elaborate perinuclear region appeared. During phase VI, skeletal muscle sarcomeres were apparent and myofilaments were arranged in a typical hexagonal array. Phase VII skeletal muscle fibers were unique with sarcomeric bifurcations and anastomoses between adjacent myofibrils. Non-contractile organelles were less organized in these cells than in skeletal muscles such as rectus and vastus lateralis muscles. During the transdifferentiation process, other cell types remained unchanged, except the number of interstitial cells of Cajal became reduced. Immunocytochemical studies with antibodies against smooth and skeletal muscle myosin were also performed during the process of transdifferentiation. An osmium tetroxide/potassium ferricyanide en bloc mordant enabled the use of ultrathin Unicryl sections for immunocytochemistry. Cells exhibited smooth muscle myosin-like immunoreactivity from the smooth muscle stage through the condensed myofilament stage. Cells were immunopositive for skeletal muscle myosin before the formation of sarcomere templates, during the condensed stage, and after development of mature skeletal muscle cells. We also observed a hybrid muscle cell with properties of both smooth and skeletal muscle cells.     

Effect of attachment to a solid substrate on the structural organization of skeletal muscle symplasts

A study was made of the ultrastructural reorganization of myosymplasts passed from the primary suspension culture to the secondary monolayer culture. The microtubules in myoblasts and myosymplasts from suspension are very rare, the intermediate filaments form a perinuclear network, small bundles of myofilaments are arranged in disorder, often as whorls around nuclei. After attachment to the solid substrate the myosymplasts form pseudopodia to move as non-muscle fibroblast-like cells. On the leading end of moving symplasts some stress fiber-like structures are found. Numerous microtubules appear. The microtubules and intermediate filaments are arranged in parallel along the axis of a lengthening symplast. A stepwise reorganization of the non-muscle type cytoskeleton to sarcomeres of differentiated myotubes is observed later. The role of attachment and mechanical stress in myotube morphogenesis are discussed.     

Morphogenesis of helical fibers in haplotaxids

There are two different muscle fiber types in haplotaxids. The pseudo-circomyarian type is typical of Haplotaxis gordioides and the flattened circomyarian type of Pelodrilus leruthi. The mechanisms of growth in fiber size and in fiber number of the two fiber types in the hindmost region of adult specimens have been studied ultrastructurally. The increase in length and girth of the muscle fiber is always the result of the insertion of new myofilaments in the peripheral zones of the muscle cells. The increase in the number of fibers seems to be due to division of differentiated muscle cells.     

Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization

We have localized myosin heavy chain (MHC) mRNAs in cells of intact embryonic chick muscle using high resolution in situ hybridization. Blocks of muscle were aldehyde-fixed prior to detergent treatment and hybridized with a biotinated cDNA probe, followed by colloidal gold-labeled antibodies, before embedment. Labeling was determined to represent MHC mRNA by extensive quantitative comparisons of electron micrographs from experimental and four different types of control samples. MHC mRNA was localized primarily to peripheral regions of 14-day chick pectoral muscle cells, where the majority of developing myofibrils were found. MHC mRNAs were consistently associated with the nonmyofibrillar cytoskeletal filaments which had diameters ranging from 4 to 10 nm. They were often oriented parallel to the longitudinal axis of the cell. The resolution of the ultrastructural approach allowed us to demonstrate that the mRNA molecules visualized were not directly associated with myofilaments, suggesting that nascent chains read from those messages do not assemble directly into myofilaments simultaneous with translation.     

Further observations by electron microscope of striated muscle in progressive Duchenne muscular dystrophy

Gastrocnemius muscle fragments of children affected by clinically diagnosed progressive muscular dystrophy of Duchenne have been studied. At the light microscope, in the semi-thin sections, the more evident changes are represented by a wide diameter range of the fibers and fatty infiltration. Some fibers show numerous nuclei in their central part, a sarcoplasmic degeneration of vacuolar type and an irregular and tortuous course of the myofibrils. Moreover, the ultrastructural findings have shown characteristic changes in myofilaments and Z bands represented by: streaming of the Z bands, collection of the triads and concentric laminated bodies. These observations have pointed out a certain gradualness of the alterations, starting from focal changes of Z band to the complete disarrangement of myofilaments.     

FINE STRUCTURE OF SMOOTH MUSCLE CELLS GROWN IN TISSUE CULTURE

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150–250 A in diameter) and thin (30–80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80–110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.     

Smooth muscle-like cells in ovaries of the hamster and gerbil

Summary Smooth muscle-like cells are present in thecae externae, corpora lutea, and interstitial tissue of hamsters and gerbils. The smooth muscle-like cells, as examined by electron microscopy, are fusiform with central nuclei; the cytoplasm contains numerous myofilaments, dense bodies, micropinocytotic vesicles, and dense accumulations of glycogen-like particles. In addition to the smooth muscle-like cells, fibroblasts and cells that have characteristics of both fibroblasts and smooth muscle are located in the thecae externae of both species. There is no ultrastructural evidence of innervation in the theca folliculi, corpora lutea, or interstitial tissue of either species.A possible function for the smooth muscle-like cells is discussed.     

Effect of developing Sarcocystis muris tissue cysts on ultrastructural change of murine muscle fibers

A study was made of the influence exerted by developing sarcocysts of Sarcocystis muris on the ultrastructural organization of muscle fibres, both harbouring the sarcocysts (HSM) and sarcocyst-free (SFM), from skeletal muscles of experimentally infected mice. Muscle fibres of non-infected mice of the same age served as a control. Mice were sacrificed 6 months following feeding S. muris oocysts (or sporocysts). The developing sarcocysts seriously destroyed HSM: their myofilaments were no hold in register, cross-bridges almost entirely disappeared, M-lines and Z-disks looked as broken structures. The majority of actin myofilaments were arranged along myosin myofilaments as discrete units. The host cell sarcoplasm was packed with numerous vacuoles of different form and size. Compared to muscle fibres in the control, SFM of infected mice also displayed an obvious ultrastructural alteration. On the periphery of SFM, some destroyed sarcomeres with swollen myofilaments were noticed whose cross-bridges were totally lacking. In other extreme areas myosin and actin myofilaments were disintegrated into thin straightened filaments 2.0-2.5 nm in diameter. It is supposed that HSM and SFM of the infected mice may experience different kinds of influence on the part of the developing intracellular parasite (sarcocyst). And it dos not seem unlikely that various biologically active substances, produced by the parasite, may be vesicle transported to SFN through the endomysium space.     

Hypertrophic smooth muscle

Summary The ultrastructure of filaments is studied in the hypertrophic musculature of the small intestine of the guinea pig oral to an experimental stenosis. No structural change is observed in the thin and the thick myofilaments. However, there is a remarkable and consistent increase in the number of intermediate (10 nm) filaments; they are the predominant type of filament in many hypertrophic muscle cells. Experiments, in which the force developed in vitro by strips of control and hypertrophic musculature upon stimulation with carbachol, indicate that the force per unit sectional area is slightly less in the hypertrophic muscle than in the control tissue.The author thanks Miss Eva Franke for excellent technical assistance. This work was supported by grants from the Medical Research Council and the Central Research Funds of the University of London     

Growth of human muscle in tissue culture

Summary A method is described for the culture of normal and diseased human muscle cells. Cell outgrowth was obtained from 63/63 biopsies, and cells differentiated to form myotubes in 57/63 biopsies. The culture technique used readily permitted the growth of both normal and diseased human muscle cells. This work was supported by grants from the Muscular Dystrophy Group of Great Britain and the Medical Research Council.     

Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in ascidian embryos.     

Culture and characterization of fetal human atrial and ventricular cardiac muscle cells

Summary Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses. This work was supported by a postdoctoral fellowship from the American Heart Association, Louisiana Affiliate (JBD) and the National Institutes of Health, Bethesda, MD (HL-35632) (WCC).     

Fine structure of smooth muscle and neuromuscular junctions in the optic tentacles of Helix aspersa and Limax flavus

Summary The smooth muscle cells studied contain a central core of thick and thin myofilaments surrounded by a peripheral layer of myofilament-free cytoplasm. Numerous vesicles, tubules, microfilaments, mitochondria and fine granules are present in the peripheral cytoplasm. Glycogen particles are distributed in large or small groups in both the peripheral cytoplasm and among the myofilaments. In contracted muscle cells the peripheral cytoplasm bulges out at regular intervals into the intercellular connective tissue. Numerous close contacts between single, usually naked, axons and these cytoplasmic protrusions occur. The axons at these contacts contain numerous small (500 Å in diameter) and large vesicles (800–1000 Å in diameter). Sometimes a number of axons simultaneously form close contacts with a muscle cell. These close contacts are considered to be the sites at which transmitter is released and acts on the muscle cell membrane.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.