Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.     

Induction of zygote formation by ethylene during the sexual development of the cellular slime mold Dictyostelium mucoroides

Immunochemical studies have identified a distinct myosin heavy chain (MHC) in the chicken embryonic skeletal muscle that was undetectable in this muscle in the posthatch period by both immunocytochemical and the immunoblotting procedures. This embryonic isoform, identified by antibody 96J, which also recognises the cardiac and SM1 myosin heavy chains, differs from the embryonic myosin heavy chain belonging to the fast class described previously. Although the fast embryonic isoform is a major species present in the leg and pectoral embryonic muscles, slow embryonic isoform was present in significant amounts during early embryonic development. Immunocytochemical studies using another monoclonal antibody designated 9812, which is specific for SM1 MHC, showed this isoform to be restricted to only presumptive slow muscle cells. From these studies and those reported on the changes in SM2 MHC, it is proposed that as is the case for the fast class, there also exists a slow class of myosin heavy chains composed of slow embryonic, SM1 and SM2 isoforms. The differentiation of a muscle cell involves transitions in a series of myosin isozymes in both presumptive fast and slow skeletal muscle cells.     

Cell diversification within the myogenic lineage: in vitro generation of two types of myoblasts from a single myogenic progenitor cell

We show that a single myogenic progenitor cell in vitro generates two types of myoblasts committed to two distinct myogenic cell lineages. Using fast and slow myosin heavy chain isoform content to define myotube type, we found that myogenic cells from fetal quail (day 10 in ovo) formed two types of myotubes in vitro: fast and mixed fast/slow. Clonal analysis showed that these two types of myotubes were formed from two types of myoblasts committed to distinct fast and fast/slow lineages. Serial subcloning demonstrated that the initial myoblast progeny of an individual myogenic progenitor cell were in the fast lineage, whereas later progeny were in the fast/slow lineage. Fast and slow myosin expression within particular myotubes reflects the genetic processes underlying myoblast commitment to diverse myogenic lineages.     

Myosin types in cultured muscle cells

Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins.     

Diversity of myosin heavy chain expression in satellite cells from mouse soleus and EDL muscles.

Postnatal myoblasts, the satellite cells, originating from slow and fast skeletal muscle fibres differentiate and fuse into myotubes expressing different phenotype of myosin heavy chain (MyHC) isoforms. Little is known, however, of factors which establish and maintain this phenotypic diversity. We used immunofluorescent labelling and Western blotting to examine the expression of slow and fast MyHC isoforms in myotubes formed in vitro from satellite cells isolated from mouse fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles. Satellite cells were cultured in serum-rich growth medium promoting myoblast proliferation until cross-striated and self-contracting myotubes were formed. We report that in both cultures myotubes expressed slow as well as fast MyHC isoforms, but the level of slow MyHC was higher in soleus culture than in EDL culture. Hence, the pattern of expression of slow and fast MyHC was characteristic of the muscle fibre type from which these cells derive. These results support the concept of phenotypic diversity among satellite cells in mature skeletal muscles and suggest that this diversity is generated in vitro irrespectively of serum mitogens.     

Fiber-type compositions in postnatal chicken muscle spindles with low intrafusal fiber counts and their developmental significance

The fiber-type composition of postnatal chicken leg muscle spindles with from one to four intrafusal fibers was examined in sections incubated with monoclonal antibodies against fast and slow myosin heavy chains. In monofibral spindles the lone intrafusal fiber was almost always fast. In duofibral spindles usually one slow and one fast fiber were present. Trifibral spindles most often displayed two fast and one slow fiber, whereas quadrofibral receptors characteristically contained two slow and two fast fibers. Earlier results showed that the primary intrafusal myotube in nascent spindles has almost always a fast myosin heavy chain profile and that the proportion of slow myotubes and fibers increases as intrafusal fiber bundles grow in size. Data from postnatal chicken leg muscles collected here suggest that up to the first four fibers this proportional increase can be largely accounted for if consecutive intrafusal fibers arise in a fast-slow-fast-slow sequence. The late recognition during myogenesis of primary intrafusal myotubes and their fast myosin heavy chain profiles warrant exploring if nascent chicken muscles spindles are first seeded by fast fetal myoblasts. © 1995 Wiley-Liss, Inc.     

Satellite cells from slow rat muscle express slow myosin under appropriate culture conditions

Abstract. Satellite cells were isolated at high yields from slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles of adult male Wistar rats. The number of satellite cells isolated from soleus muscle exceeded that from TA muscles by a factor of three. A comparison of satellite cells grown on gelatin- or Matrigel-coated dishes revealed that Matrigel greatly enhances the maturation of the satellite-cell-derived myotubes. As judged from immunohistochemistry, myosin heavy chain electrophoresis and immunoblot analyses, only cells grown on Matrigel, but not on gelatin, expressed adult myosin isoforms. Slow myosin expression was only detected in Matrigel cultures. Soleus cultures contained, in addition to the majority of myotubes expressing fast myosin, a small fraction (maximally 10%) of myotubes coexpressing fast and slow myosins. The number of fast/slow myosin-containing myotubes was negligible in TA cultures. The expression of slow myosin increased with age. Slow myosin was nonuniformly distributed along the length of specific myotubes and accumulated around some myonuclei. These results point to the existence of myotubes with a heterogeneous population of myonuclei, probably resulting from fusion of differently preprogrammed satellite cells. We suggest that the patch-like expression of slow myosin results from local accumulation of myonuclei of slow-type satellite cells.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle

Monoclonal antibodies were prepared to stage-specific chicken pectoral muscle myosin heavy chain isoforms. From comparison of serial sections reacted with these antibodies, the myosin heavy chain isoform composition of individual myofibers was determined in denervated pectoral muscle and in regenerating myotubes that developed following cold injury of normal and denervated muscle. It was found that the neonatal myosin heavy chain reappeared in most myofibers following denervation of the pectoral muscle. Regenerating myotubes in both innervated and denervated muscle expressed all of the myosin heavy chain isoforms which have thus far been characterized in developing pectoral muscle. However, the neonatal and adult myosin heavy chains appeared more rapidly in regenerating myotubes compared to myofibers in developing muscle. While the initial expression of these isoforms in the regenerating areas was similar in innervated and denervated muscles, the neonatal myosin heavy chain did not disappear from noninnervated regenerating fibers. These results indicate that innervation is not required for the appearance of fast myosin heavy chain isoforms, but that the nerve plays some role in the repression of the neonatal myosin heavy chain.     

A combined histochemical and immunohistochemical study on the dynamics of fast-to-slow fiber transformation in chronically stimulated rabbit muscle

Summary Chronically stimulated fast-twitch muscles of the rabbit were histochemically and immunohistochemically analyzed in serial cross sections (1) for percentages of fiber types, and (2) for the presence of myosin heavy chain isoforms during fast-to-slow transformation. By four weeks of stimulation the number of type-I fibers had increased more than fourfold, while only about 6% of the original IIB fibers remained. Type-IC and -IIC fibers transiently rose to 20% of the total fiber population. After 16 weeks, the number of type-I fibers had increased to 42%. With prolonged stimulation fewer fibers reacted with antibodies against embryonic and neonatal myosins and more with the antibody against slow myosin. The reaction for embryonic myosin was most often detected in the C fibers (IC, IIC). Immunohistochemical subtypes were observed for each fiber type in the stimulated muscles. The greatest number was seen in type-IIC fibers, which, in addition to their reaction for fast/neonatal and slow myosins, might also react with the antibodies against neonatal/embryonic and embryonic myosins. These findings indicated that the transforming fibers temporarily expressed myosin heavy chain isoforms normally not detectable in adult skeletal muscle. Myotubes reacted strongly with the antibodies against fast/neonatal and embryonic myosins, and some of them also with the antibody against slow myosin. Thus, it appears that under the influence of the low frequency stimulus pattern some of the newly formed myotubes developed into type-I fibers.     

Differential Expression of PTP1D, a Protein Tyrosine Phosphatase with Two SH2 Domains, in Slow and Fast Skeletal Muscle Fibers

We show that PTP1D, a protein tyrosine phosphatase that contains two SH2 domains, is preferentially expressed in slow skeletal muscle fibers. Immunohistochemical staining using polyclonal antibodies against PTP1D demonstrated that PTP1D was expressed in a subpopulation of rodent muscle fibers. These fibers were identified as slow Type I fibers based on histochemical ATPase assays and slow myosin heavy chain expression. Northern and Western analyses showed that PTP1D levels were higher in predominantly slow muscles than in predominantly fast muscles. This differential expression of PTP1D in slow muscle fibers appeared by birth. In cultures of mouse myogenic cells, PTP1D was expressed after MyoD and myogenin and appeared in myotubes derived from embryonic, fetal, and postnatal myoblasts. Remarkably, PTP1D was organized into sarcomeres in a pattern coincident with myosin heavy chain, suggesting that PTP1D associates with a component of the thick filament. These results show that PTP1D is preferentially expressed in slow muscle fibers. We speculate that PTP1D may play a role in slow muscle fiber function and differentiation.     

Differential expression of adult type MHC in satellite cell cultures from regenerating fast and slow rat muscles.

The local anaesthetic (Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) has been used to induce myofiber damage (and thus satellite cells proliferation) and thereby represents a tool for increasing the yield of myoblasts from adult muscles. Replicating satellite cells were isolated by enzymatic dissociation from soleus (slow type) and tibialis anterior (fast type) muscles of adult rats, and categorized by the isoform (embryonic, fast and slow) of myosin heavy chain (MHC) expressed following myotube formation in a similar in vitro environment. According to light microscopic criteria, no morphological differences exist between the satellite cell cultures obtained from adult fast and slow muscles after Bupivacaine injection. On the other hand the derived myotubes express, beside the embryonic type, the peculiar myosin heavy chains which characterize the myosin pattern of the donor muscles.     

Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.     

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.     

Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.     

Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.     

Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles

The expression of myosin isoforms was studied during development of calf muscles in foetal and neonatal rats, using monoclonal antibodies against slow, embryonic and neonatal isoforms of myosin heavy chain (MHC). Primary myotubes had appeared in all prospective rat calf muscles by embryonic day 16 (E16). On both E16 and E17, primary myotubes in all muscles with the exception of soleus stained for slow, embryonic and neonatal MHC isoforms; soleus did not express neonatal MHC. In earlier stages of muscle formation staining for the neonatal isoform was absent or faint. Secondary myotubes were present in all muscles by E18, and these stained for both embryonic and neonatal MHCs, but not slow. In mixed muscles, primary myotubes destined to differentiate into fast muscle fibres began to lose expression of slow MHC, and primary myotubes destined to become slow muscle fibres began to lose expression of neonatal MHC. This pattern was further accentuated by E19, when many primary myotubes stained for only one of these two isoforms. Chronic paralysis or denervation from E15 or earlier did not disrupt the normal sequence of maturation of primary myotubes up until E18, but secondary myotubes did not form. By E19, however, most primary myotubes in aneural or paralyzed tibialis anterior muscles had lost expression of slow MHC and expressed only embryonic and neonatal MHCs. Similar changes occurred in other muscles, except for soleus which never expressed neonatal MHC, as in controls. Paralysis or denervation commencing later than E15 did not have these effects, even though it was initiated well before the period of change in expression of MHC isoforms. In this case, some secondary myotubes appeared in treated muscles. Paralysis initiated on E15, followed by recovery 2 days later so that animals were motile during the period of change in expression of MHC isoforms, was as effective as full paralysis. These experiments define a critical period (E15-17) during which foetuses must be active if slow muscle fibres are to differentiate during E19-20. We suggest that changes in expression of MHC isoforms in primary myotubes depend on different populations of myoblasts fusing with the myotubes, and that the normal sequence of appearance of these myoblasts has a stage-dependent reliance on active innervation of foetal muscles. A critical period of nerve-dependence for these myoblasts occurs several days before their action can be noted.     

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5'-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1-2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.