A comparative study of embryonic development of Japanese quail selected for different patterns of postnatal growth

Patterns of early embryonic development have traditionally been viewed as invariant within vertebrate taxa. It has been argued that the specific differences which are found arise during the later stages of development. These differences may be a result of allometry, heterochrony or changes in relative growth rates. To test whether early embryonic development is indeed invariant, or whether selection of adult characteristics can alter embryonic growth, we compared embryonic development in birds selected for different patterns of postnatal growth. Using quail lines selected for high and low body mass, we compared somite formation, and muscle and feather development. We obtained data that showed changes in the rate of myotome formation in the brachial somites which contribute to muscle formation in the limbs and thorax. We think these observations are connected with intraspecific changes in adult morphology, ie., breast muscle size. Our findings suggest that selection for late ontogenetic/adult stages affects early embryonic development.     

Immunochemical analysis of troponin T isoforms in adult, embryonic, regenerating, and denervated chicken fast skeletal muscles

Using monoclonal antibodies (McAbs) which can distinguish between breast- and leg-type troponin T (TnT), we studied the spatial distribution of TnT isoforms in adult chicken fast skeletal muscles. The breast (pectoralis major) and leg (iliotibialis posterior) muscles were composed predominantly of homogeneous fibers containing breast- and leg-type TnT, respectively. The posterior latissimus dorsi muscle was composed of heterogeneous fibers of at least two types, namely breast and leg types. In developing and regenerating fast muscles, only leg-type TnT was expressed at early stages, and later breast-type TnT appeared either transiently or permanently. This led ultimately to several distinct adult fast muscle breast/leg TnT isoform profiles. Since both types of TnT were synthesized in embryonic and regenerating muscles with nerves intact as well as in regenerating muscles with nerves resected, the switching on of their expression during fast muscle development appears to be independent of nerves. However, its full development ("fine tuning" of the protein isoform distribution within the fast fiber types) and the maintenance of the adult state are presumed to be dependent on the nerves, since, although regenerating fibers in denervated muscles could exhibit the early and then the later embryonic stainabilities, they again returned to the early embryonic state; further, the denervation of adult muscles caused the replacement of TnT isoform from the adult to the early embryonic state.     

SOME PROPERTIES OF EMBRYONIC MYOSIN

Myosins from the following sources were purified by diethylaminoethyl-Sephadex chromatography: moytubes grown in vitro for 7–8 days, prepared from pectoralis muscles of 10-day old embryos, and breast and leg muscles from 16-day old embryos. The adenosine triphosphatase activities of these myosins were close to that of adult m. pectoralis myosin. The light chains of the embryonic myosins had the same mobilities in sodium dodecyl sulfate electrophoresis as those in adult pectoralis muscle myosin and were clearly distinguishable from those in myosin from tonic muscle m. latissimus dorsi anterior. The fastest light chain in embryonic muscle myosin—apparent mol wt 16,000—was present in smaller amounts than in adult myosin. The negative staining pattern of paracrystals of embryonic light meromyosin (LMM) was indistinguishable from that of adult fast muscle LMM. The significance of these results for differentiation of various muscle types has been discussed.     

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross- striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M- line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'- bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.     

Isoforms of troponin during regeneration of chicken skeletal muscle fibers after cold injury

Summary The regeneration of skeletal muscle fibers of the adult chicken was examined after a focal injury brought about with a liquid-nitrogen cooled brass rod. Immunofluorescence microscopy with antibodies specific for troponin (TN) components (T, I, and C) from adult chicken breast and ventricular muscles showed the presence of different fiber types in both the anterior and posterior latissimus dorsi muscles. New fibers produced in the regions adjacent to the site of injury in both muscles exhibited the same immunoreactivities as those previously seen in embryonic skeletal muscles. As differentiation proceeded, regenerating cells lost their embryonic antigenicities and recovered their characteristic adult reactivities. These results indicate that, during regeneration from cold injury, skeletal muscles apparently pass again through an embryonic stage during which they synthesize embryonic-like TN isoforms.     

Biosynthesis of tropomyosin and troponin during development of the chicken skeletal muscle

The level of functional mRNA coding for myofibrillar proteins was studied during development of the chicken skeletal muscle. RNA isolated from the developing chicken muscle directed protein synthesis in a wheat germ cell-free system. By means of polyacrylamide gel electrophoresis and immunological analysis, tropomyosin subunits and troponin components were identified among the cell-free translation products. The mRNA activities for alpha- and beta-subunit of tropomyosin were prominent in the embryonic breast muscle as well as in the embryonic leg muscle. At the early post-embryonic stage, the mRNA activity for beta-subunit disappeared from the breast muscle, while those for alpha- and beta-subunit were detectable in the leg muscle. Troponin-C and troponin-I synthesized in vitro in response to the muscle RNA formed a binary complex in the presence of calcium ion. Despite the observed difference in molecular weight between troponin-Ts in the breast and leg muscle, RNA preparations from the two muscles encoded identical troponin-Ts whose molecular weights were indistinguishable from that of troponin-T present in the breast muscle of adult chicken. It is suggested from these results that the biosynthesis of tropomyosin is regulated at the pre-translational level during the development of the chicken skeletal muscle, whereas post-translational (or co-translational) events may produce the tissue-specific form of troponin-T.     

Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit striated muscles.

We have selected tropomyosin subunits and myosin light chains as representative markers of the myofibrillar proteins of the thin and thick filaments and have studied changes in the type of proteins present during development in chicken and rabbit striated muscles. The β subunit of tropomyosin is the major species found in all embryonic skeletal muscles studied. During development the proportion of the α subunit of tropomyosin gradually increases so that in adult skeletal muscles the α subunit is either the only or the major species present. In contrast, cardiac muscles of both chicken and rabbit contain only the α subunit which remains invariant with development. Two subspecies of the α subunit of tropomyosin which differ in charge only were found in adult and embryonic chicken skeletal muscles. Only one of these subspecies seems to be common to chicken cardiac tropomyosin. With respect to myosin light chains, embryonic skeletal fast muscle myosin of both species resembles the adult fast muscle myosin except that the LC₃ light chain characteristic of the adult skeletal fast muscle is present in smaller amounts. The significance of these isozymic changes in the two myofibrillar proteins is discussed in terms of a model of differential gene expression during development of chicken and rabbit skeletal muscles.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.     

Ca2+ sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers

Ca⁺ sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca²⁺ sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca²⁺ sparks were rare in these fibers. At 5–7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca²⁺ sparks. The average rate of occurrence of Ca²⁺ sparks is >10-fold higher after 5–7 days in culture than in days 1–3. With the use of fibers cultured for 7 days, application of the Ca²⁺ channel blockers Co²⁺ or nifedipine almost completely suppressed the occurrence of Ca²⁺ sparks, as previously shown in embryonic fibers, suggesting that Ca²⁺ sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca²⁺ sparks, suggesting that Ca²⁺ sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling. calcium ion signaling; muscle remodeling; fluo 4; calcium ion imaging     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle

Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of mysoin light chains in addition to α and β forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, α-fast, α-slow, and β-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, β-tropomyosin and the α-fast form of tropomyosin, are found. In addition, β-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

ULTRASTRUCTURAL ORGANIZATION OF OBLIQUELY STRIATED MUSCLE FIBERS IN ASCARIS LUMBRICOIDES

The somatic musculature of the nematode, Ascaris, is currently thought to consist of smooth muscle fibers, which contain intracellular supporting fibrils arranged in a regular pattern. Electron microscopic examination shows that the muscle fibers are, in fact, comparable to the striated muscles of vertebrates in that they contain interdigitating arrays of thick and thin myofilaments which form H, A, and I bands. In the A bands each thick filament is surrounded by about 10 to 12 thin filaments. The earlier confusion about the classification of this muscle probably arose from the fact that in one longitudinal plane the myofilaments are markedly staggered and, as a result, the striations in that plane of section are not transverse but oblique, forming an angle of only about 6° with the filament axis. The apparent direction of the striations changes with the plane of the section and may vary all the way from radial to longitudinal. A three-dimensional model is proposed which accounts for the appearance of this muscle in various planes. Z lines as such are absent but are replaced by smaller, less orderly, counterpart "Z bundles" to which thin filaments attach. These bundles are closely associated with fibrillar dense bodies and with deep infoldings of the plasma membrane. The invaginations of the plasma membrane together with intracellular, flattened, membranous cisternae form dyads and triads. It is suggested that these complexes, which also occur at the cell surface, may constitute strategically located, low-impedance patches through which local currents are channeled selectively.     

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.     

Identification and Profiling of MicroRNAs in the Embryonic Breast Muscle of Pekin Duck

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in embryonic breast muscle of duck are unclear. In this study, we analyzed the miRNAs profiling in embryonic breast muscle of Pekin duck at E13 (the 13^th day of hatching), E19, and E27 by high-throughput sequencing. A total of 382 miRNAs including 359 preciously identified miRNAs 23 novel miRNA candidates were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in Pekin duck was high conserved. The expression of identified miRNAs were significantly different between E13 and E19 as well as between E27 and E19. Fifteen identified miRNAs validated using stem-loop qRT-PCR can be divided into three groups: those with peak expression at E19, those with minimal expression at E19, and those with continuous increase from E11 to E27. Considering that E19 is the fastest growth stage of embryonic Pekin duck breast muscle, these three groups of miRNAs might be the potential promoters, the potential inhibitors, and the potential sustainer for breast muscle growth. Among the 23 novel miRNAs, novel-miRNA-8 and novel-miRNA-14 had maximal expression at some stages. The stem-loop qRT-PCR analysis of the two novel miRNAs and their two targets (MAP2K1 and PPARα) showed that the expression of novel-mir-8 and PPARα reached the lowest points at E19, while that of novel-mir-14 and MAP2K1 peaked at E19, suggesting novel-miRNA-8 and novel-miRNA-14 may be a potential inhibitor and a potential promoter for embryonic breast muscle development of duck. In summary, these results not only provided an overall insight into the miRNAs landscape in embryonic breast muscle of duck, but also a basis for the further investigation of the miRNAs roles in duck skeletal muscle development.     

Monoclonal antibodies distinguish titins from heart and skeletal muscle

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

M line appearance in developing striated muscle

Summary Myofilament assembly occurs in a definite sequence. Myofibrils first appear within embryonic myotomes as non-striated, linear arrangements of parallel thick and thin myofilaments (crude sarcomeres) with periodic dense cross bands (Z lines). In the center of sarcomeres within these early myofibrils, faint M lines are often detected. In older embryos, after typical cross striations became apparent, the M lines can be detected bisecting each A band.Research supported by The Muscular Dystrophy Association, U.S.A.     

Accumulation of tropomyosin in developing chicken gizzard smooth muscle

Smooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to alpha and beta isoforms (Hosoya et al. (1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of alpha and beta isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.