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Muscle patterning, differentiation and vascularisation in the chick wing bud.

Adult muscle is highly vascularised, with blood vessels being essential for adequate oxygenation of the tissue and for supporting increased metabolic demands. Whether this is the case during muscle development has not been examined. Resin histology was used to map the muscle splitting process and conventional transmission electron microscopy to examine early muscle differentiation at the midlimb level or later at the mid radius/ulna level in the chick wing bud from stages 24 (4.5 d) to 36 (10 d) (Hamburger & Hamilton, 1951). Microinjection of India Ink into the extra-embryonic vasculature was used to visualise the patent muscle microcirculation. The results showed that the premuscle masses are present at stage 24 and initial splitting of the muscle masses commences at stage 28. The final muscle pattern is not established until stage 36. At stage 26 the cells within the premuscle masses exhibited a mesenchymal morphology, but at stage 28 overt muscle differentiation was evident with myofibrils present within myoblasts. Undifferentiated mononucleated cells were interspersed with the differentiating myoblasts. The ratio of mononucleated cells:myoblasts decreased and the myoblasts became plumper and increasingly packed with myofibrils with age. There was no evidence of secondary myotube formation at any of the stages examined. Vascular invasion of the limb occurred at stage 35 just prior to the establishment of the final muscle pattern. This was surprising as it was assumed that myogenic differentiation would be both oxygen and nutrient dependent. The results of this study provide descriptions of the splitting of the premuscle masses through to the establishment of the final muscle pattern at the midlimb or mid radius/ulna level of the chick wing bud together with the differentiation of the myogenic cells within the developing muscles. However, the relationship between muscle patterning at the tissue level and muscle differentiation at the cellular level with vascularisation remains unclear. It is hoped that the results of the study may provide the basis for future investigations into mechanisms involved in muscle patterning and the signalling mechanisms for vascular invasion.

Animals↗

derailed is required for muscle attachment site selection in Drosophila.

During development, muscles must form and attach at highly stereotyped positions to allow for coordinated movements. In Drosophila, muscles grow towards and attach to specifically positioned cells within the epidermis. At the molecular level, very little is known about how muscles recognize these attachment sites. The derailed gene encodes a receptor tyrosine kinase family member that is essential for the pathfinding ability of expressing neurons. Here we show that the Drl RTK is also expressed by a small subset of developing embryonic muscles and neighboring epidermal cells during muscle attachment site selection. In drl mutants, these muscles often fail to attach at appropriate locations although their epidermal attachment cells appear unaffected. These results show that, similar to its role in neuronal pathway recognition, the Drl RTK participates in a mechanism required for muscle attachment site selection. The data suggest that both neurons and muscles use common mechanisms to recognize their paths or targets, and that Drl plays an analogous role in both developing systems.

Animals↗

Molecular expression of myostatin and MyoD is greater in double-muscled than normal-muscled cattle fetuses.

Excessive muscling in double-muscled cattle arises from mutations in the myostatin gene, but the role of myostatin in normal muscle development is unclear. The aim of this study was to measure the temporal relationship of myostatin and myogenic regulatory factors during muscle development in normal (NM)- and double-muscled (DM) cattle to determine the timing and possible targets of myostatin action in vivo. Myostatin mRNA peaked at the onset of secondary fiber formation (P < 0.001) and was greater in DM (P < 0.001) than in NM. MyoD expression was also elevated throughout primary and secondary fiber formation (P < 0.001) and greater in DM (P < 0.05). Expression of myogenin peaked later than MyoD (P < 0.05); however, it did not differ between NM and DM. These data show that myostatin and MyoD increase coincidentally during formation of muscle fibers, indicating a coordinated role in the terminal differentiation and/or fusion of myoblasts. Myostatin mRNA is also consistently higher in DM than NM, suggesting that a feedback loop of regulation is also disrupted in the myostatin-deficient condition.

Animals↗

Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis.

The specification and morphogenesis of slow and fast twitch muscle fibers are crucial for muscle development. In zebrafish, Hedgehog is required for slow muscle fiber specification. However, less is known about signals that promote development of fast muscle fibers, which constitute the majority of somitic cells. We show that when Hedgehog signaling is blocked, fast muscle cell elongation is disrupted. Using genetic mosaics, we show that Hedgehog signal perception is required by slow muscle cells but not by fast muscle cells for fast muscle cell elongation. Furthermore, we show that slow muscle cells are sufficient to pattern the medial to lateral wave of fast muscle fiber morphogenesis even when fast muscle cells cannot perceive the Hedgehog signal. Thus, the medial to lateral migration of slow muscle fibers through the somite creates a morphogenetic signal that patterns fast muscle fiber elongation in its wake.

Animals↗

Comparison of a Modified Atmosphere Stunning-Killing System to conventional electrical stunning and killing on selected broiler breast muscle rigor development and meat quality attributes.

Two experiments were conducted to compare the effects of a 70:30 argon:carbon dioxide Modified Atmosphere Stun-Kill (MASK) system to both a low voltage (LV) and a high current (HC) electrical stunning and conventional killing system on broiler breast rigor development and meat quality. In Experiment 1, the effects on breast muscle pH and meat tenderness of the MASK system and the LV system were compared. In Experiment 2, the MASK system was compared to both HC and LV stunning for pH, R-value, C.I.E. L*, a*, b*, cooked yield, and tenderness of broiler breast meat. In Experiment 1, there were no significant differences in breast muscle pH between LV and MASK, except at 24 h. Only at 5 h post-mortem did the MASK system result in significantly tougher breast meat. In Experiment 2, there were no significant differences between treatments in breast muscle color or cooked yield at any sampling time. The breast muscle from birds subjected to the HC stun had consistently higher pH values until 5 h post-mortem, at which time the breast meat from birds subjected to the MASK system had higher pH. R-value data showed a similar trend, with the HC stunned birds having the lower R-values until 5 h post-mortem. Breast meat tenderness values for the three treatments were significantly different only at 3 and 5 h post-mortem, when birds stunned with HC had the highest shear values. The MASK system did not exhibit any rigor accelerating benefits when compared to LV stunning and conventional killing, and only minimal rigor acceleration when compared to HC killing.

Abattoirs↗

Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle.

To examine how nascent myosin heavy chains associate with the cytoskeletons of developing muscle cells, we used pulse labeling, cell fractionation, and immunoprecipitation. More than 80% of nascent myosin heavy chains associate with the cytoskeleton. More than one-third of these nascent chains are not released by puromycin and/or RNase. The fraction of nascent heavy chains that resists release increases during development of muscle cells in culture. Treatment with cytochalasin D but not nocodazole decreases myosin heavy chain cotranslational assembly. These results indicate that (i) cotranslational assembly of myosin heavy chains is developmentally regulated, (ii) structures containing actin and not microtubules may mediate initial association of the heavy chains with the cytoskeleton, and (iii) the site of translation dictates where a significant fraction of the heavy chains will be inserted into the cytoskeleton.

Animals↗

Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock-down of myostatin-1.

Myostatin, a secreted growth and differentiation factor (GDF-8) belongs to transforming growth factor (TGF-beta) superfamily that plays as a negative regulator of skeletal muscle development and growth. Recently, myostatin has been isolated from fish; however, its role in muscle development and growth remains unknown. Here, we present the expression of myostatin during development and the effects of its knock-down on various genes such as muscle regulatory transcription factors (MRFs), muscle-specific proteins (MSP), and insulin-like growth factors (IGFs). The myostatin expression was found to be maternal as it starts in one-cell stage onward. The reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Southern and Northern blots demonstrated that the myostatin expression is not only restricted to skeletal muscle, but it expressed all the tested tissues. Expression of myostatin was effected by using antisense morpholinos resulted in significant phenotypic difference in stages 18 and 20 hours postfertilization (hpf). To confirm the specificity of myostatin morpholino, furthermore, a rescue experiment was conducted. The length as well as width of somites was increased with almost no gap in between the somites. In addition, it deserves to mention that this is a first animal model that shows changes in the size of the somites. Moreover, analyses of MRFs, MSP, and IGFs in the knock-down embryos by RT-PCR revealed the up-regulation of MyoD, Myogenin, and Mck transcription, whereas IGF-2 transcription showed mild response with no effect on IGF-1, Desmin, and Myf5. In situ hybridization showed that there was an increase in the number of somites from 3 to 4 at 13 and 22 hpf. Taken together, these data suggest that myostatin plays a major role during myogenesis, apart from inhibition of proliferation as well as differentiation.

Animals↗

Do the corticospinal and corticobulbar tracts mediate functions in the human newborn?

Unlike the numerous dispersed bulbospinal pathways that are already well myelinated at term, the more compact corticospinal and corticobulbar tracts are only beginning their myelination cycle in late gestation and do not complete it until two years of age. During this same period, these pathways also develop extensive ramification of terminal axonal segments, growth of collateral axons, and proliferation of synapses. Despite their immaturity in the full-term human newborn, several proposed functions may be attributed to the descending pathways from the neonatal cerebral cortex: a) a contribution to the differential development of passive muscle tone and resting postures; in general they function as an antagonist to the "subcorticospinal pathways" in mediating proximal flexion and distal extension, except for the rubrospinal tract which is probably synergistic with the corticospinal tract; b) enhancement of tactile reflexes originating in the brainstem and spinal cord, including suck and swallow; c) relay of epileptic activity of cortical origin; d) inhibition of complex stereotyped motor reflexes including many phenomena formerly termed "subtle seizures"; e) a possible influence on muscle maturation, particularly in relaying cerebellar impulses that modify the histochemical differentiation of myofibres. However, the bulbospinal tracts are probably more influential on muscle development. The corticospinal and corticobulbar tracts subserve different needs in the newborn than at older ages, but are functionally important pathways even at birth.

Cerebral Cortex↗

Effect of a single dose of ethanol on developing skeletal muscle of chick embryos.

Fetal alcohol syndrome is a condition occurring in some children of mothers who have consumed alcohol during pregnancy. Many of these affected children show retarded physical growth in the postnatal period despite adequate nutrition. On the basis of findings from studies with animals, it has been proposed that this is due to allometric retardation of growth of skeletal muscle, although the exact reasons for this are not known. The aim of the current study was to examine the structural changes in skeletal muscle in fetal alcohol syndrome in an attempt to understand the mechanisms of growth retardation in fetal alcohol syndrome. Chick embryos were exposed to single doses of 5%, 10%, and 15% ethanol, and the effects on the general growth and development, as well as on the skeletal muscle, of these chicks were studied. There was a significant retardation in crown rump length, head circumference, and body weight in ethanol-exposed chicks when these parameters were compared with findings for appropriate control groups. This retardation was associated with significant and proportionate reductions in the weights of skeletal muscles. Microscopic examination of skeletal muscle showed areas of neutrophil infiltration and necrosis, suggestive of muscle damage, in chicks exposed to 10% and 15% ethanol. Thus, findings of the current study demonstrate the direct toxic effects of a single dose of ethanol on developing embryos in general and skeletal muscle in particular. The pathologic changes seen in skeletal muscle could account for the failure in postnatal growth in fetal alcohol syndrome.

Animals↗

Adult human masseter muscle fibers express myosin isozymes characteristic of development.

Masseter muscle biopsies were obtained from nine patients undergoing orthognatic surgery or surgery for parotid tumors. A detailed enzyme-histochemical and immunocytochemical study of these muscles was performed using antibodies specific to the various isozymes of the myosin heavy chain (MHC) in order to identify the MHC isozymes that were present in the different fiber types. The contractile proteins in these same biopsies were analyzed by two-dimensional gel electrophoresis, native pyrophosphate gel electrophoresis, and by an immunopolypeptide mapping approach. These studies have shown that there is a very heterogeneous distribution of the myosin isozymes, with many fibers containing more than one myosin type. We also present evidence that in addition to adult fast and slow myosin, the human masseter muscle contains two proteins, neonatal MHC and embryonic myosin light chain, that are characteristic of developing muscle.

Electrophoresis↗

Basal lamina development in chicken muscle spindles.

The development of basal laminas was examined in immunohistochemical sections of chicken leg muscle spindles from embryonic day (E) 13 to 8 weeks postnatal. Fragments of basal laminas as seen with immunostaining for isoforms of laminin were already observed in E6 muscles. When clusters of intrafusal myotubes were first recognized at E13-14, they were surrounded by basal laminas which were incomplete both in terms of coverage and molecular composition. More mature basal lamina tubes individually enclosed young myofibers at E18. After afferents made contact with myotubes, synaptic portions of basal laminas at myosensory junctions reacted strongly with antibodies against s-laminin and chondroitin sulfate proteoglycan, while extrasynaptic portions were negative or reacted only weakly. At synaptic basal laminas of neuromuscular junctions heparin sulfate proteoglycan and s-laminin became prominent after E16. Contrary to the early presence of basal lamina proteins around intrafusal fibers, initial deposition of basal lamina proteins in the outer spindle capsule was not recognized until E17-18, and significant amounts were not detected until postnatal week 1. Unlike intrafusal basal laminas, capsular basal laminas developed no distinct specialized regions; however, molecular compositions of intrafusal and capsular basal laminas were similar.

Animals↗

Cardiac fibroblasts: function, regulation of gene expression, and phenotypic modulation.

Cardiac fibroblasts constitute the majority of the non-myocyte cell population in the ventricular myocardium. These cells are located in the interstitium, in areas between and surrounding cardiac myocytes. Cardiac fibroblasts are responsible for the synthesis of extracellular matrix proteins such as fibrillar collagen types I and III, basement membrane type IV collagen, fibronectin, and laminin. In addition to its role in muscle development and myoblast differentiations, extracellular matrix consisting primarily of fibrillar collagen is an intricate and highly organized structure that serves to support cardiac myocytes and to maintain functional integrity of the myocardium. Balanced synthesis and degradation of this matrix is the key to normal development of cardiac muscle and perfect myocardial function. Collagen remodeling and accumulation has been demonstrated in several experimental models of cardiac hypertrophy. To gain insights into molecular and cellular mechanisms that affect cardiac fibroblast behavior, cardiac fibroblasts from rat and rabbit ventricular myocardium were cultured and the impact of neurotransmitters and growth factors such as norepinephrine and transforming growth factor--beta (TGF-beta 1), to which cardiac fibroblasts are exposed in vivo, was studied. Results of these studies, with regards to gene expression, proliferation and differentiation of cardiac fibroblasts in culture, and their biological implications are discussed.

Animals↗

Development of masticatory muscles and oral behavior from suckling to chewing in dogs.

1. At the age of 20 postnatal days, more than a half of the dogs (13/20) showed incisor eruption. At the age of 25 postnatal days, half of the dogs (10/20) showed molar eruption. 2. At the age of 24 postnatal days, half of the dogs (10/20) started chewing behavior. 3. EMG amplitudes of temporal muscle were larger than that of masseter muscle until 26.5 days after birth, but they were reversed at 26.5 days after birth. 4. This suggests that in dogs the earlier teeth erupted, the earlier the dominant oral behavior was changed from suckling to mastication, and concomitantly the dominant muscle for the oral behavior was changed from the temporal to the masseter muscle.

Animals↗

Protease inhibitors reduce the loss of nerve terminals induced by activity and calcium in developing rat soleus muscles in vitro.

The end-plate of a mammalian skeletal muscle fibre is innervated by several axons at the time of birth but by only one axon in the adult. In the rat soleus muscle the transition from polyneuronal to single innervation occurs during the first 2-3 weeks after birth. While it is evident that the loss of the excess nerve terminals depends to some extent on neuromuscular activity, the mechanism involved is not known. In the present experiments neonatal rat soleus muscles were stimulated in vitro in the presence of a variety of combinations of calcium, the cholinesterase inhibitor edrophonium and the proteolytic enzyme inhibitors leupeptin, pepstatin and Ep-475. Electron microscopical examination revealed that stimulation alone had little effect on the morphology of the end-plate region but stimulation in the presence of raised levels of calcium caused severe disruption of the nerve terminals and a marked reduction in the number of intact nerve terminal profiles contacting each end-plate. Contraction measurements showed that, in spite of this, the muscles were not functionally denervated to any large extent. The addition of edrophonium potentiated the morphological alterations but caused no further reduction in the number of profiles. Conversely, the protease inhibitors wholly or partially (in the case of Ep-475) prevented the effects of stimulation and calcium on the nerve terminals. These results are consistent with the idea that neuromuscular activity induces the secretion of proteolytic enzymes into the end-plate region, where they digest the immature nerve terminals. The importance of calcium suggests that the calcium-dependent neutral protease may be involved, and is also consistent with a secretory mechanism. The possibility that the nerve terminals are digested by their own proteases is also discussed.

Acetylcholine↗

Cellular and cis-regulation of En-2 expression in the mandibular arch.

Investigations into early muscle development have focused primarily on somite derived cells. Cranial mesoderm does not undergo somitogenesis, and muscle formation in this region is less well understood. In the present study, we have focused upon the expression of engrailed in mandibular arch myoblasts. We demonstrate that En-2 is expressed in mandibular arch myoblasts of the mouse. The activity of the En-2 enhancer is maintained in several functionally related muscles that arise from the first arch. Through the use of reporter transgenics, we demonstrate that local cell-cell interactions are important in maintaining En-2 expression in the mandibular arch cells. En-2 enhancer activity in the first arch requires a combination of cis-acting sequences that includes a motif which is identical to one found in the Otx2 enhancer and which is sufficient to direct expression in the first arch. These data support the notion that cranial muscle development is regulated by local cell-cell interactions which distinguish distinct anatomical and functional muscle groups.

Animals↗

Postmortem proteome changes of porcine muscle related to tenderness.

Proteome analysis was used to investigate the relation between changes in postmortem proteome of porcine muscle and tenderness development. Muscle samples were taken at slaughter and 72 h postmortem, and the registered changes in the proteome were related to Warner-Bratzler shear force. One hundred and three protein spots were found to change significantly (P < 0.01) over time, and of these the 27 most pronounced changes were identified. Eleven out of the 27 changes were fragments of actin. Other identified myofibril proteins or fragments included myosin heavy chain, titin, myosin light chain I, myosin light II, CapZ, and cofilin. Correlation analysis revealed significant correlations between three of the identified actin fragments and the myosin heavy chain fragment to shear force. Moreover, myosin light chain II and triose phosphate isomerase I were also found to correlate significantly to shear force. The results clearly demonstrate that postmortem degradation of actin and myosin heavy chain is related to meat tenderness.

Animals↗

Polysialic acid and mucin type o-glycans on the neural cell adhesion molecule differentially regulate myoblast fusion.

Polysialic acid attached to the neural cell adhesion molecule (NCAM) is thought to play a critical role in development. NCAM in muscle tissue contains a muscle-specific domain (MSD) to which mucin type O-glycans are attached. In the present study, using the C2C12 myoblast system, we show that NCAM containing MSD is increasingly expressed on the cell surface as myotubes form. Polysialic acid is primarily attached to N-glycans of NCAM, and polysialylated NCAM is expressed on the outer surface of myotube bundles. By transfecting cDNAs encoding wild type and mutant forms of NCAM, we found that NCAM containing MSD facilitates myoblast fusion, and this effect is diminished by mutating O-glycosylation sites at MSD. By contrast, forced expression of polysialic acid in early differentiation stages reduces myotube formation and delays the expression of NCAM containing the MSD domain. Strikingly, inhibition of polysialic acid synthesis by antisense DNA approach induced differentiation in both human rhabdomyosarcoma cells, which overexpress polysialic acid, and C2C12 cells. These results indicate that polysialic acid and mucin type O-glycans on NCAM differentially regulate myoblast fusion, playing critical roles in muscle development.

Base Sequence↗