Human Muscle Development Research Articles

Expression of β-Amyloid Precursor Protein Gene Is Developmentally Regulated in Human Muscle Fibers in Vivo and in Vitro

Regenerating muscle fibers in 25 human muscle biopsies, obtained from patients with a variety of neuromuscular diseases, manifested increased mRNA for the β-amyloid precursor protein (βAPP) that contains the Kunitz-type protease inhibitor (KPI) motif, whereas adult human muscle fibers were negative in their vast non-extrajunctional region. Aneurally cultured normal human muscle fibers also expressed strong KPI-βAPP mRNA signal, which became significantly down-regulated during muscle differentiation. Our study demonstrates that in human muscle KPI-βAPP mRNA is developmentally regulated, and it suggests that βAPP may play a role in human muscle development.

Expression of dystrophin-associated glycoproteins during human fetal muscle development: A preliminary immunocytochemical study

An immunocytochemical study was performed on quadriceps muscle from eight fetuses ranging from 12 weeks of gestation to term, using antibodies against the dystrophin-associated proteins, in order to evaluate the developmental expression of these proteins. For comparison, antibodies against dystrophin and utrophin were also used. The expression of the 59 kDa dystrophin-associated protein was simultaneous with that of dystrophin, which is also a subsarcolemmal protein. The extracellular glycoprotein of 156 kDa (α-dystroglycan) and the transmembrane glycoprotein of 43 kDa (β-dystroglycan) appeared to be expressed later. The transmembrane glycoproteins of 50 kDa (adhalin) and 35 kDa were fully expressed at an even later stage of fetal muscle development. This study suggests that the subsarcolemmal proteins may have an essential role in the assembly of the transmembrane and extracellular components of the dystrophin-glycoprotein complex during fetal muscle development. The knowledge obtained from observing the developmental expression of these proteins may contribute to the understanding of the molecular mechanism of their different involvement in muscle disorders.

Expression of the low‐affinity NGF receptor during human muscle development, regeneration, and in tissue culture

The expression of the low-affinity NGF receptor (LNGFR) during human muscle development, regeneration, and in tissue culture was analyzed using a murine monoclonal antibody to human LNGFR (MAb ME 20.4). Muscle cells from 12-22-week fetuses stained strongly for LNGFR. In adult normal muscle, only intramuscular nerve endings showed immunoreactivity with MAb ME 20.4, but no staining was detected in muscle fibers. In Duchenne muscular dystrophy, immunohistologically demonstrable LNGFR was present in regenerating muscle fibers. In these fibers, LNGFR gene expression was also demonstrated at the transcriptional level using in situ hybridization with riboprobes coding with human LNGFR. Cultures from human fetal and adult muscle were studied by double label indirect immunofluorescence microscopy with MAb ME 20.4 and antisera against human fetal myosin. Most myosin-positive cells, both at the myoblast and myotube stages, displayed surface LNGFR immunostaining. In cells from fetal muscle, LNGFR was detected during the first 2 weeks in vitro, whereas in cells from adult muscle the expression of LNGFR was observed for up to 7 weeks. These findings suggest a potential involvement of LNGFR in human muscle development and regeneration.

Histochemical and contractile property changes during human muscle development.

When the histochemical and contractile properties of infant muscles change postnatally, and what influence muscle function has on these changes, were the focus of this study. Contractile properties were measured in the plantaflexor (PF) and dorsiflexors (DF) of 19 newborns and 36 infants aged 5-16 months. Infants were tested between one and four times at monthly intervals. Measurements included maximal twitch tension (Pt), time to peak tension (TPT), and half-relaxation times (1/2RT). TPT was similar in PF (77 ms) and DF (73 ms) at birth, remained unchanged in DF, and slowed in PF to 110-120 ms between 9 and 12 months. Type I distributions were determined at autopsy in fetal through to adult muscles. Completion of differentiation occurred in soleus at about the age that contractile properties slowed and infants started to use these muscles more. A trend of higher percentages of type I distributions was also noted in children than either newborns or adults in other muscles. The implications of these findings and the clinical use of these methods for evaluating peripheral neuromuscular function is discussed.

Proliferation and differentiation of human fetal myoblasts is regulated by PDGF-BB

Abstract A myoblast clone, G6, was obtained from thigh muscle of an 11 week old human fetus, and used to examine the effect of platelet-derived growth factor (PDGF) on cell multiplication and differentiation. G6 myoblasts showed extensive fusion, and expressed creatine phosphokinase activity and muscle specific gene mRNA (myosin heavy chain, α-actin) when switched to a differentiation medium. The cells expressed PDGF β-receptor mRNA, and bound 125I-PDGF-BB specifically. Expression of PDGF β-receptors declined during in vitro differentiation. Relative levels of transcripts for the myogenic regulatory factors Myf4 (myogenin). Myf5, and Myf6 (MRF4) increased during the differentiation process, whereas Myf3 (MyoD1) was preferentially expressed in undifferentiated myoblasts. Treatment of the myoblasts with PDGF-BB increased DNA synthesis and cell density. Myogenic differentiation, analyzed as number of nuclei present in myotubes and expression of creatine phosphokinase and myosin heavy chain, was partly inhibited by the presence of PDGF-BB in the differentiation medium. PDGF-BB may, therefore, have the potential of regulating human muscle development and muscle regeneration.

Proliferation and differentiation of human fetal myoblasts is regulated by PDGF-BB

Abstract A myoblast clone, G6, was obtained from thigh muscle of an 11 week old human fetus, and used to examine the effect of platelet-derived growth factor (PDGF) on cell multiplication and differentiation. G6 myoblasts showed extensive fusion, and expressed creatine phosphokinase activity and muscle specific gene mRNA (myosin heavy chain, α-actin) when switched to a differentiation medium. The cells expressed PDGF β-receptor mRNA, and bound 125I-PDGF-BB specifically. Expression of PDGF β-receptors declined during in vitro differentiation. Relative levels of transcripts for the myogenic regulatory factors Myf4 (myogenin), Myf5, and Myf6 (MRF4) increased during the differentiation process, whereas Myf3 (MyoD1) was preferentially expressed in undifferentiated myoblasts. Treatment of the myoblasts with PDGF-BB increased DNA synthesis and cell density. Myogenic differentiation, analyzed as number of nuclei present in myotubes and expression of creatine phosphokinase and myosin heavy chain, was partly inhibited by the presence of PDGF-BB in the differentiation medium. PDGF-BB may, therefore, have the potential of regulating human muscle development and muscle regeneration.

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.

Growth and development of human muscle: a quantitative morphological study of whole vastus lateralis from childhood to adult age.

The mechanisms underlying the increase in volume of muscle tissue, and the functional development of muscle fibers from childhood through adolescence to adult age, have been studied. Cross sections of autopsied whole vastus lateralis muscle from 22 previously physically healthy males, 5 to 37 years of age, were prepared enzyme histochemically (myofibrillar ATPase) and examined morphometrically. The data obtained on muscle cross-sectional area, size, total number, and proportion of type 1 (slow-twitch) and type 2 (fast-twitch) fibers were analyzed using linear regression techniques. The results show that the increase in muscle cross-sectional area from childhood to adult age is caused by an increase in mean fiber size. This is accompanied by a functional development of the fiber population: the proportion of type 2 fibers increases significantly from the age of 5 (approx. 35%) to the age of 20 (approx. 50%), which, in the absence of any discernible effect on the total number of fibers, is most likely caused by a transformation of type 1 to type 2 fibers.

Transition of myosin isozymes during development of human masseter muscle: Persistence of developmental Isoforms during postnatal stage

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.

Monoclonal antibody ALD 180: a reagent specific for a human prenatal skeletal muscle myosin isoform.

We report the characterization of monoclonal antibody (MAb) ALD 180, prepared against the myosin of slow avian muscle, for studies of human muscle development and disease. With the use of radioimmunoassays, Western immunoblots of native and denatured myosins, and epifluorescent indirect immunocytochemistry, we show that ALD 180 is specific for an epitope in human prenatal skeletal muscle myosin heavy chain (MHC), which is expressed in diminishing abundance in fetal fibers from at least 19-22 weeks' gestation to term and also in regenerating muscle fibers seen in diseased muscles from both children and adults. ALD 180 recognizes an epitope apparently different from those reacting with anti-prenatal human myosin MAb previously described, and therefore affords a complementary reagent for use in future studies of human myosin isoform expression and regulation.

Isolation of full-length cDNAs encoding abundant adult human skeletal muscle mRNAs

We have used a method [Gunning et al., Mol. Cell. Biol. 3 (1983) 787–795] of cDNA clone isolation from a cDNA library that selects for clones corresponding to abundant mRNAs and simultaneously yields a large number of different cDNA clones containing a high fraction of nearly full-length inserts. We screened an adult human skeletal muscle (skm) cDNA library and have isolated 46 cDNA clones which correspond to different mRNAs expressed at significant levels in adult skm. Of these cDNA clones 17 appear to be muscle-specific. Eleven are expressed in both cardiac muscle and skm but six are expressed primarily in skm. The remainder are expressed in muscle as well as in human fibroblasts. Comparison of cDNA insert size with mRNA size for the 17 clones expressed only in skeletal plus cardiac muscle revealed that eight are full-length, five are not and four recognize multiple transcripts which prevent a definitive conclusion. These cDNA clones will greatly facilitate the characterization of genes which are regulated during human muscle development.

A Histochemical Analysis of the Development of Human Fetal Muscles

A Histochemical Analysis of the Development of Human Fetal Muscles

Extracellular matrix antigen of human muscle defined by a monoclonal antibody

A monoclonal antibody (7.1B3) has been produced in mice previously immunised with a glycoprotein fraction prepared from human myotube cells grown in tissue culture. The antibody precipitates an antigen complex of four glycoprotein components from detergent extracted human muscle myotube cultures. These are two chains of about 205K MW and two chains of about 130–140K MW. The antigen complex is trypsin-sensitive collagenase insensitive and bands as a complex of 10S on sucrose gradients in NP40. Indirect immunofluorescence studies on cell cultures of myoblasts and myotubes showed that the antigen existed in a number of locations: as a granular stain on the cell surface of myotubes, and as short streaks of immunoreactivity in the extracellular matrix. Clonal populations of myoblasts and myotubes gave a similar result showing that 7.1B3 is specifically synthesized by cells of the myogenic lineage. Double indirect immunofluorescence staining with 7.1B3 antibody and fibronectin antibody showed no overlap of immunoreactivity indicating that these two antigens are different. 7.1B3 antigen was found during human fetal muscle development in vivo at all time points studied from 15 weeks onwards. Immunoreactivity was localised to the loose extracellular matrix and in the endomysium. Perymysium staining was very prominent at 25 weeks and in adult muscle. Double staining of 7.1B3 antibody and α-bungarotoxin was carried out to determine whether 7.1B3 was present in synaptic junctions. Although 7.1B3 was found in the endomysium it is excluded from synaptic areas. We conclude that 7.1B3 antibody

Extraocular muscles: Morphogenetic study in humans light microscopy and ultrastructural features

The development of human extraocular muscles (EOM) was studied in a series of fetal specimens (12–24 weeks gestation). EOM were evaluated by enzyme histochemistry (EZ) (NADH and ATPase), by differential phase contrast microscopy (DPC) and electron microscopy (EM). In the early fetus (14 weeks), there was no clear-cut sub-division into fibre types. A uniform histochemical reaction was seen with NADH while ATPase showed light and dark myotubes. Myotubes contained large central nuclei, prominent eccentric nucleoli, abundant glycogen granules, free ribosomes, numerous mitochondria, and dense and looser bundles of myofilaments. Mesenchymal cells undergoing mitosis and fibroblasts with prominent stacks of rough endoplasmic reticulum were scattered within endomysium. Mast cells with well formed cytoplasmic granules were found as early as 18–24 weeks. The same specimens by DPC showed differentiation into at least 4 different fibre types at 12 weeks. All the intramuscular nerves at 12–16 weeks were composed of unmyelinated fibres. At 18 weeks, myelinated axons were present. Morphologically immature end-plates devoid of junctional folds were found at 12 weeks. The motor innervation of some EOM appears to be derived from more than one axon (multiple innervated fibres). At 18 weeks gestational age, differentiation into fibre types became apparent by enzyme histochemistry. These histochemical and morphological findings suggest that morphologically mature endplates are not prerequisites for differentiation into muscle fibre types.

Electron microscopic study of the development of human foetal muscle, motor end-plate and nerve. Preliminary report.

Electron microscopic observations were made on muscle and peripheral nerve of human embryos of the period between the 9th and 16th week. These observations showed the presence of motor end-plates at the 10th week of human embryonic life, at a time when the muscle cells were still in the myotube stage. The difference between the structure of nerve fibres of the 9th and those of the 16th week of foetal life consisted in a change from the form of one multiaxon bundle to single axons separated from each other by an intracellular space filled with collagen fibres.

The stages of development of human foetal muscles with reference to some muscular diseases

Oxidative enzyme activity in the centre of the muscle fibres was observed in muscle from human foetus aged from 6 to 14 weeks of embryonal life. The activity was uniform in all muscle fibres independent of their size. ATPase activity was localised in the circumference of the muscle fibre and increased with the age of the foetus. Phosphorylase activity first appeared in foetal muscles at about the 11th week of embryonal life. Areas of ACHE activity were observed at the beginning of the 9th week of embryonal life. From the 10th week of embryonal life, areas of ACHE activity could be observed in the region of the musculo-tendinous junctions. Muscle spindles could be observed from the 10th week of embryonal life. In the period between the 6th and 14th week of intrauterine life, differentiation into metabolic fibre types, as typically seen in mature muscle, had not yet developed. The development of distinctive metabolic types of muscle fibre appears to run parallel with the development of the innervation of the muscle fibres. In the quadriceps femoris muscles of the foetus aged 24 weeks isolated single fibres of one histochemical type, much larger than the others were seen and corresponded to Wohlfart B fibres. There are several similarities between the histochemical pattern of foetal muscle and that seen in diseased muscle in some cases of muscle disease but these similarities should be interpreted with caution.