Myotomal Cells Research Articles

Development of the vertebra and fin skeleton in the lamprey and its implications for the homology of vertebrate vertebrae.

Although vertebrae are the defining character of vertebrates, they are found only in rudimentary form in extant agnathans. In addition, the vertebrae of agnathans possess several unique features, such as elastin-like molecules as the main matrix component and late (post-metamorphosis) differentiation of lamprey vertebrae. In this study, by tracing the developmental process of vertebrae in lamprey, we examined the homology of vertebrae between lampreys and gnathostomes. We found that the lamprey somite is first subdivided mediolaterally, with myotome cells differentiating medially and non-myotome cells emerging laterally. Subsequently, collagen-positive non-myotome cells surround the myotome. This pattern of somitogenesis is rather similar to that in amphioxi and sheds doubt on the presence of a sclerotome, in terms of mesenchyme cells induced by a signal from the notochord, in lamprey. Further tracing of non-myotome cell development revealed that fin cartilage develops in ammocoete larvae approximately 35 mm in body length. The development of the fin cartilage occurs much earlier than that of the vertebra whose development proceeds during metamorphosis. We propose that the homology of vertebrae between agnathans and gnathostomes should be discussed carefully, because the developmental process of the lamprey vertebra is different from that of gnathostomes.

Fetal development and growth of the human erector spinae with special reference to attachments on the surface aponeurosis.

The longissimus (LO) and iliocostalis (IC) of adults consist of myofibers extending from the superolateral to the inferomedial side of the back and, because of the same course, they are fused in the thoracolumbar region. The LO also has a medial attachment to the long myofibers of the transversospinalis (TS) showing a course from the superomedial to the inferolateral side. However, there is apparently no information regarding when and how these similar longitudinal muscles differentiate from a cluster of dorsomedial myotome cells. We examined sagittal and horizontal sections of the trunks of 39 human embryos and fetuses (18-330mm crown-rump length). At 6-7weeks gestational age (GA), the surface aponeurosis appeared prior to and independent of the thoracolumbar fascia. At 6-9weeks GA, the LO myofibers had a postero-inferior course, from the transverse process to the initial aponeurosis, whereas the TS myofibers had a postero-superior course, from a lateral extension of the intertransverse ligament to the aponeurosis. However, the IC consisted of supracostal longitudinal myofibers and was distant from the LO until 12weeks GA. Because of the lack of ligamentous attachments and ribs, myofibers of the TS, LO, and IC took a similar inferior course in the lumbar region. When the early TS was represented by the transverso-aponeurotic muscle, consequently, the LO corresponded to the aponeuro-transversal muscle and was independent from the IC. The classical model of TS and LO development does not recognize the essential role of the aponeurosis identified here.

Development of the shoulder girdle musculature.

The muscles of the shoulder region are important for movements of the upper limbs and for stabilizing the girdle elements by connecting them to the trunk. They have a triple embryonic origin. First, the branchiomeric shoulder girdle muscles (sternocleidomastoideus and trapezius muscles) develop from the occipital lateral plate mesoderm using Tbx1 over the course of this development. The second population of cells constitutes the superficial shoulder girdle muscles (pectoral and latissimus dorsi muscles), which are derived from the wing premuscle mass. This muscle group undergoes a two-step development, referred to as the "in-out" mechanism. Myogenic precursor cells first migrate anterogradely into the wing bud. Subsequently, they migrate in a retrograde manner from the wing premuscle mass to the trunk. SDF-1/CXCR4 signaling is involved in this outward migration. A third group of shoulder muscles are the rhomboidei and serratus anterior muscles, which are referred to as deep shoulder girdle muscles; they are thought to be derived from the myotomes. It is, however, not clear how myotome cells make contact to the scapula to form these two muscles. In this review, we discuss the development of the shoulder girdle muscle in relation to the different muscle groups.

SDF-1 controls the muscle and blood vessel formation of the somite.

Stromal-cell-derived factor-1 (SDF-1), the only ligand of the chemokine receptor CXCR4, is involved in skeletal muscle development. However, its role in the proliferation, differentiation and migration of somite cells is not well understood. Here, we investigated its function during somite development in chicken embryos by using gain-of-function and loss-of-function experiments. Overexpression of SDF-1 was performed by electroporating SDF-1 constructs into the ventrolateral part of the somite, or by injecting SDF-1-expressing cells into the somites of stages HH14-16 chicken embryos. We found that enhanced SDF-1 signaling induced cell proliferation in the somite. This resulted in an increase in number of both myotomal and endothelial cells. In contrast, inhibition of SDF-1/CXCR4 signaling led to a reduction of myotomal cells. Injection of SDF-1 producing cells into the somite induced ectopic localization of myotomal cells in the sclerotome. Although many SDF-1-expressing somite cells colonized the limb, only a few of them developed into muscle cells. This resulted in a reduction of the limb muscle mass. This means that most myogenic progenitors were stopped on their migration towards the limb due to the high concentration of the SDF-1 signal in the somite. Most of the SDF-1-expressing somite cells found in the limb were of endothelial cell fate and they contributed to the increase in limb blood vessels. These results reveal that SDF-1 promotes the proliferation of both myogenic and angiogenic progenitor cells of the somite and controls myotome formation. Furthermore, SDF-1 controls muscle and blood vessel formation in the limb in different ways.

Developmental Expression of Calcium Activated Chloride Ion Channels Anoctamin 5 in Mouse Skeletal Muscle

Anoctamin 5 (Ano5), also named TMEM16E, belongs to the Anoctamin gene family. The mutations in the Ano5 gene cause Limb-girdle muscular dystrophy (LGMD) 2L type and Miyoshi muscular dystrophy (MMD3). Both patients showed sarcolemmal lesions. The studies showed that TMEM16E mRNA expressed in the somites during embryogenesis, particularly in the myotomal cells, and also in the muscle myotome-derived progenitor cells. However, no report has been done to examine Ano5 expression during mouse skeletal muscle development. In the present study, we investigated the distribution and quantification of Ano5 in the skeletal muscles of mice during their development, with the methods of immunofluorescence, Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. The results indicated that Ano5 mRNA and protein are expressed in skeletal muscle of the mouse from 1 day to 6 months, but with development and aging, the expression of Ano5 reduced gradually. Taken together, our results demonstrate that Ano5 expression level decreased throughout development and aging, and this may explain why the musculardystrophy syndrome of Ano5 mutant patients only starts during the later stage of their lives.

A dynamic analysis of muscle fusion in the chick embryo.

Skeletal muscle development, growth and regeneration depend upon the ability of muscle cells to fuse into multinucleated fibers. Surprisingly little is known about the cellular events that underlie fusion during amniote development. Here, we have developed novel molecular tools to characterize muscle cell fusion during chick embryo development. We show that all cell populations arising from somites fuse, but each with unique characteristics. Fusion in the trunk is slow and independent of fiber length. By contrast, the addition of nuclei in limb muscles is three times more rapid than in trunk and is tightly associated with fiber growth. A complex interaction takes place in the trunk, where primary myotome cells from the medial somite border rarely fuse to one another, but readily do so with anterior and posterior border cells. Conversely, resident muscle progenitors actively fuse with one another, but poorly with the primary myotome. In summary, this study unveils an unexpected variety of fusion behaviors in distinct embryonic domains that is likely to reflect a tight molecular control of muscle fusion in vertebrates.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

MUSCLE DEVELOPMENT IN AMPHIOXUS: MORPHOLOGY, BIOCHEMISTRY, AND MOLECULAR BIOLOGY

ABSTRACT This review concerns the structure and biochemistry of muscle in amphioxus. Most work has focused on the segmented swimming (axial) muscles. These muscles derive from the medial wall of the somites, which arise as evaginations from the gut wall. The myotomal muscle cells of amphioxus, unlike those of vertebrates, never fuse, but remain mononucleate, contain only one myofibril, and span the entire length of the myotome. The muscle cells are very thin and lack a T-tubule system. There are two, maybe three, types of fibers. Innervation is via muscle tails, which contact the basal lamina of the nerve cord. The notochord is also composed of striated muscle cells, which similarly send muscle tails to the nerve cord. Less is known about the biochemistry of muscle. The notochord, like molluskan catch muscle, contains paramyosin. Among the muscle-specific proteins sequenced are alkali myosin light chain, troponin C and sarcoplasmic calcium-binding proteins, calcium-vector protein, and its target protein c...

How is Myogenesis Initiated in Chordates?

Expression of transcriptor factors MyoD, Myf5, myogenin and MRF4 forms the basis of myogenesis. In Acrania, Pisces and Amphibia, as in Aves, myogenesis is initiated by MyoD. In Mammalia expression of Myf5 initiates myogenesis. Signal proteins Wnt and Shh induce the expression of genes encoding for MyoD or Myf5. In fishes and amphibians expression of MyoD starts in non-segmented mesoderm and then in myotomal cells. In birds and mammals expression of MyoD or Myf5 is initiated in the cells of the dermatomyotome. Embryonic myotomes are post-mitotic. Proliferating cells Pax3 and Pax7-positive and mesenchymal cells take part in the growth of myotomal muscles. Cells migrating to the limb bud contain regulatory proteins Six4/Six1, Pax3, Lbx1 and c-met. Rectus abdominis develops from cells that contain Pax3 and Lbx1.

The translational repressor 4E-BP mediates the hypoxia-induced defects in myotome cells

Cell growth, proliferation, differentiation and survival are influenced by the availability of oxygen. The effect of hypoxia on embryonic cells and the underlying molecular mechanisms to maintain cellular viability are still poorly understood. In this study, we show that hypoxia during Xenopus embryogenesis rapidly leads to a significant developmental delay and to cell apoptosis after prolonged exposure. We provide strong evidence that hypoxia does not affect somitogenesis but affects the number of mitotic cells and muscle-specific protein accumulation in somites, without interfering with the expression of MyoD and MRF4 transcription factors. We also demonstrate that hypoxia reversibly decreases Akt phosphorylation and increases the total amount of the translational repressor 4E-BP, in combination with an increase of the 4E-BP associated with eIF4E. Interestingly, the inhibition of PI3-kinase or mTOR, with LY29002 or rapamycin, respectively, triggers the 4E-BP accumulation in Xenopus embryos. Finally, the overexpression of the non-phosphorylatable 4E-BP protein induces, similar to hypoxia, a decrease in mitotic cells and a decrease in muscle-specific protein accumulation in somites. Taken together, our studies suggest that 4E-BP plays a central role under hypoxia in promoting the cap-independent translation at the expense of cap-dependent translation and triggers specific defects in muscle development.

Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse

Skeletal myogenesis is extensively influenced by the surrounding environment. However, how the extracellular matrix (ECM) affects morphogenesis of muscles is not well understood. We mapped the three-dimensional (3D) organization of fibronectin, tenascin, and laminin by immunofluorescence during early epaxial myogenesis in mouse embryos. We define four stages of dermomyotome/myotome development and reveal the 3D organization of myogenic cells within their ECM during those stages. Fibronectin is abundant in all interstitial tissues, while tenascin is restricted to intersegmental borders. Bundles of fibronectin and tenascin also penetrate into the myotome, possibly promoting myocyte alignment. A laminin matrix delineates the dermomyotome and myotome and undergoes dynamic changes, correlating with key developmental events. Our observations cast new light on how myotomal cells interact with their environment and suggest that, as the segmented myotomes transform into the epaxial muscle masses, the laminin matrix disassembles and myocytes use the abundant fibronectin matrix to reach their final organization.

Myogenesis – Possibilities of its Stimulation in Chickens

Due to selection for increased body weight modern broilers are 3-4 times heavier as compared with chickens of the laying type. The muscle mass is mainly determined by the total number of muscle fibres (hyperplasia), their thickness (hypertrophy) and different fibre types. Hyperplasia occurs during either embryogenesis or the early posthatching period. Skeletal muscles originate from the dermatomyotome, which differentiates into four myogenic cell populations: myotomal cells, embryonic myoblasts, fetal myoblasts and satellite cells; the latter are the adult myoblasts, present within adult skeletal muscles to serve as a cell source for both muscle regeneration and self-renewal. Pax3 keeps migrated precursor cells non-differentiated, thereby controlling transcription of the MyoD gene, whereas Pax7 is a significant regulator of the satellite cell population. Manipulation of temperature and light quality and quantity have been proposed as methods of both pre- and postnatal myogenesis stimulation. Being thermogenic stimulants, both thyroid and adrenal hormones substantially stimulate metabolism. Short-term exposure of embryos to increased temperature between days 16 and 18 of incubation directly influences the proliferation and differentiation of muscle fibres, which manifest themselves in increased hyperplasia. Ultraviolet radiation is an effective means for disinfection of hatching eggs, resulting in a change of embryonic mortality rate during breeding. Especially, green light influences both body weight and the satellite cell number in the first days posthatch, thereby enhancing the growth of embryos, and causing a significant increase in both muscle and body weight. In ovo green stimulation probably enhances the proliferation and differentiation of myoblasts, subsequently causing an increase in muscle weight. The present paper highlights the possibilities of enhancing growth and development of skeletal muscles in birds by manipulation of many aspects of their regulation, thereby contributing to a further increase in production efficiency.

Effects of hypoxia on myogenesis and muscular differentiation

Oxygen (O2) is an environmental and developmental regulator during several events such as energy homeostasis, development and process of progenitor cells differentiation. Hypoxia corresponds to a decrease in oxygen partial pressure that leads to adaptive responses of skeletal muscle. Activation, proliferation and differentiation of myogenic precursor cells required for muscle growth seem to be mediated by recruitment of several intracellular pathways including calcineurin, p38–MAPK and Akt/mTOR. To examine the hypoxia-induced alterations in these main intracellular signalling pathways during myogenesis two models were used, myoblasts from the L6 line which differentiate in vitro and the embryonic myogenesis using Xenopus laevis embryos. In culture cells, hypoxia delays myoblast differentiation, affecting both calcineurin and Akt signalling pathways, as well as the mTOR downstream, the translation factor S6. In the embryo, hypoxia exposure causes growth retardation and affects differentiation of the myotome cells. These cell events were correlated to an activation of p38-MAPK and a cytoplasmic accumulation of the 4E-BP1 protein specifically in the axial tissues such as the notochord, somites and floor plate. The data show that the 4E-BP1 is a target of hypoxia that reversibly inhibits the cap-dependent translation of mRNA.

Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome

Different levels and timing of Hedgehog (Hh) signalling activity have been proposed to specify three distinct cell types in the zebrafish myotome. Two of these, the medial fast-twitch fibres (MFFs) and the slow-twitch muscle pioneers (MPs) are characterised by expression of eng1a, -1b and -2a and require the highest levels of Hh for their specification. We have defined a minimal eng2a element sufficient to drive reporter expression specifically in MPs and MFFs. This element binds both Gli2a, a mediator of Hh signalling, and activated Smads (pSmads), mediators of bone morphogenic protein (BMP) signalling, in vivo. We found a strict negative correlation between nuclear accumulation of pSmad, and eng2a expression in myotomal cells and show that abrogation of pSmad accumulation results in activation of eng2a, even when Hh signalling is attenuated. Conversely, driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. Nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. We show that a synthetic form of the Gli2 repressor interacts with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signalling activity. Our results demonstrate that the eng2a promoter integrates repressive and activating signals from the BMP and Hh pathways, respectively, to limit its expression to MPs and MFFs. We suggest a novel basis for crosstalk between the Hh and BMP pathways, whereby BMP-mediated repression of Hh target genes is promoted by a direct interaction between Smads and truncated Glis, an interaction that is abrogated by Hh induced depletion of the latter.

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3′-Terminal Exon

Alternative splicing of 3'-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3'-untranslated regions that regulate their fate and their expression. The Xenopus alpha-tropomyosin pre-mRNA possesses a composite internal/3'-terminal exon (exon 9A9') that is differentially processed depending on the embryonic tissue. Exon 9A9' is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3'-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9' as a 3'-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9' in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9' as a 3'-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3'-end processing of the alpha-tropomyosin pre-mRNA.

Somite cell cycle analysis using somite‐staging to measure intrinsic developmental time

Somite stages were employed as units of intrinsic developmental time to measure cell doubling rate and other cell cycle parameters of chick forelimb level somites. Somite cell nuclei doubled over an interval corresponding to approximately 7+ somite stages (7+ ss; approximately 11 hr) and approximately 24 new primary myotome cells are born per somite stage ( approximately 16/hr). FACS analysis of DNA content in dissociated paraxial mesoderm cells indicated that slightly more than half are in G1/G0 phase of the cell cycle and that the average combined length of the S phase and G2 phase intervals is approximately 3 ss ( approximately 4.5 hr). A wavefront of increased mitotic nuclei per segment coincident with somite budding potentially reflects a surge in the number of cells entering S phase 3 ss earlier as each PSM segment becomes unresponsive to FGF signaling as it passes through the determination front.

A Rare Variation in the Pattern of Flexor Muscles of the Foot: Absence of Flexor Digitorum Brevis is compensated by Two Accessory Flexors to Digiti Minimi

The tendon of the Flexor Digitorum Brevis (FDB) to the 5th toe was observed to be absent in the right foot of one 76 year old female cadaver. In addition to the missing muscle, two accessory muscles, which we named Accessory Flexor Digitorum Brevis (AFDB) pars cuneus and pars calcaneus according to their boney attachments, appear to compensate morphologically and biomechanically for the missing portion of FDB. The AFDB pars cuneus was found to attach to the inferolateral aspect of the 1st cuneiform, had a muscular belly which crossed superficial to the tendons of flexor digitorum longus (FDL) for the 2nd, 3th and 4th toes and had a slender tendon which accompanied the medial aspect of the FDL tendon into the fibrous digital sheath of the 5th toe. The AFDB pars calcaneus attached to the medial aspect of the calcaneus in common with the medial head of the Quadratus Plantae (QP). The AFDB pars calcaneus tendon crossed superficial to the medial and lateral heads of the QP and accompanied the lateral aspect of the FDL tendon into the fibrous digital sheath of the 5th toe. The tendons of the two accessory flexor muscles fused into a common tendon just proximal to the 5th metatarsophalangeal joint. This common tendon accurately replaced the missing FDB tendon, splitting and attaching to the sides of the middle phalanx of the 5th toe with the FDL tendon passing through the splitting terminal tendons. In this case two accessory flexor muscles appeared to compensate for the missing portion of the FDB muscle. This finding supports the hypothesis that biomechanical compensation may play a role in guiding myotomal cell migration during embryogenesis and may help to explain the occurrence of muscular variants.

Histopathological effects induced by paraquat during Xenopus laevis primary myogenesis

The oxidative agent paraquat induced tail abnormalities during Xenopus laevis development. Specimens exposed from blastula to the tadpole stage revealed pear-shaped myocytes and irregular intersomitic boundaries. The histological feature of the axial musculature was evaluated in embryos sampled at significant stages of the primary myogenesis. During the somitogenesis PQ-treated embryos showed normal appearing myotomes, but reduced PAS activity in the post-rotating myotomal cells, and myoblasts with slight vacuolations. Once etched from the vitelline envelope, embryos showed severely altered myoblasts with irregular cellular apexes, heavy sarcoplasmic vacuolations, pyknotic nuclei and disorganizing intersomitic boundaries. Myotomes with many necrotic myocytes containing disorganized contractile material and heavily malformed intersomitic boundaries characterized the late myogenic stages. Our results evidence the heaviest PQ histopathological effects to affect myogenesis of post-etched embryos, suggesting a possible linkage between the swimming activity and the oxidative damage to muscle tissue.

The zic1 gene is expressed in chick somites but not in migratory neural crest

Zic transcription factors regulate the expression of neural and neural crest-specific genes and are expressed in the cells of the dorsal neural tube and the premigratory neural crest. Here we characterize zic1 expression in the chick embryo during somite formation and neural crest migration. zic1 is expressed in the dorsomedial portion of epithelial somites and subsequently in the dorsomedial lip of the dermomyotome. Although zic1 is expressed in cells of the nascent myotome, it is absent from differentiated myotome cells that express myosin. As the dorsal root ganglia form, zic1 is expressed at high levels in the dorsal sclerotome and zic1 expression is more pronounced in the caudal regions of the somites. Double-label experiments showed that cells expressing zic1 are not labeled by the HNK-1 antibody specific for migratory neural crest cells. Thus, migrating neural crest cells do not express zic1.

Asymmetric localization of numb in the chick somite and the influence of myogenic signals

Whereas Notch signaling is known to play an essential role in the formation of somites, its role during later stages of somite maturation is less well understood. Here, we examine the signals and transcription factors that control the expression of the Notch antagonist, Numb, during somite maturation in the chick embryo. Numb mRNA is present in the epithelial somite and is increased in expression in the forming myotome. Numb protein displays a very specific subcellular localization and dynamic expression during somite maturation. Numb protein is asymmetrically localized in a cortical crescent on the basal side of dividing cells in the dorsomedial lip of the dermomyotome and is subsequently uniformly distributed throughout differentiated myotomal cells. Treatment of somites with either the combination of Wnt-3a and Shh, or ectodermal signals plus noggin, both of which induce somitic myogenesis, did not significantly affect Numb transcript levels but did lead to a dramatic increase in the levels of Numb protein, which was uniformly distributed throughout the cytoplasm of the resultant myotubes. Forced expression of MyoD in somites similarly induced high levels of Numb protein throughout the cytoplasm, without affecting Numb mRNA levels. We also found that signals that promote somitic myogenesis or forced MyoD expression induced expression of the Notch ligand, Serrate-2. Our findings suggest that Notch signals are specifically repressed in the myotome and that asymmetric expression of Numb in dividing cells of the dorsomedial lip of the dermomyotome may modulate whether these cells continue to divide or differentiate into myotomal cells.