Insulin-like growth factors,hepatocyte growth factor and transforming growth factor-alpha in mouse tongue myogenesis

Many reports have shown that tongue striated muscles have several unique characteristics not found in other skeletal muscles such as limb and trunk. Several peptide growth factors are reported to play important roles in skeletal myogenesis. In this article, the roles of insulin-like growth factors (IGF), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-alpha in mouse tongue myogenesis were studied using an organ culture system of the mandible or tongue obtained from mouse embryos. It was found that IGF-I promotes the differentiation of tongue myoblasts. HGF plays an essential role in the migration and proliferation of tongue myogenic cells, and inhibits the differentiation of tongue myoblasts. TGF-alpha does not play an essential role in the proliferation of tongue myogenic cells, but does promote the early differentiation of tongue myoblasts. The role of IGF-I in the differentiation of tongue myoblasts, and that of HGF in the migration, proliferation and differentiation of tongue myogenic cells appear to be almost identical to their roles in the myogenesis of limb and cultured myogenic cell lines. However, the role of TGF-alpha in the proliferation and differentiation of tongue myogenic cells appears to be different from its role in the myogenesis of limb and cultured myogenic cell lines such as C2 and L6.     

Overexpression of nPKC theta is permissive for myogenic differentiation

Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.     

Possible application of muscle specific conditional mouse-derived induced pluripotent stem cells for muscle research

The Cre-driver mouse line, which allows for in vivo regulation of target gene(s) in specific cells, is an indispensable tool for recent muscle research. In this study, I aimed to explore new applications of muscle specific Cre-driver mouse line in muscle research. For this purpose, I generated an iPS cells from a myofiber specific conditional mouse with tamoxifen inducible GFP expression, and then I checked whether homologous recombination was induced in the iPS-derived myogenic cells by tamoxifen administration. Fibroblasts were isolated from the tails of Myf6^CE/wt::CAG-EGFP mice, which expressed GFP specifically in Myf6 lineages by tamoxifen injection, and then iPS cells was generated by transfection with a vector based on sendai-virus and containing OSKM genes. Muscle specific conditional mouse-derived iPS cells (mCM-iPSCs) were successfully differentiated to myogenic cells, such as Pax7⁺ muscle progenitors, MyoD⁺ myoblasts, and MHC⁺ myotubes, under myogenic differentiation conditions. Using this model, I examined whether homologous recombination was induced in mCM-iPSC-derived myotubes by 4-hydroxytamoxifen (4OH-TAM) administration. As a result, multinucleated myotubes showed GFP expression, while no GFP signals were detected in both Pax7⁺ muscle progenitor and non-myogenic cells. These results indicated that homologous recombination could be induced in mCM-iPSC–derived myotubes by tamoxifen administration, and that this system operated normally even in reprogrammed cells. Also, I evidenced that GFP reporter was expressed in myoblasts in addition to multinucleated myotubes when tamoxifen-pulse was applied at an early phase of myogenesis. Taken together, Myf6^CE/wt::CAG-EGFP mouse-derived iPS cells reproduced at least in part Myf6 expression during mouse myogenesis. This study demonstrated a novel application of muscle specific conditional mouse in addition to in vivo application, and mCM-iPSCs could also be used in in vitro investigations with muscle specific conditional knock-out mouse.     

The expression of slow myosin during mammalian somitogenesis and limb bud differentiation

The developmental pattern of slow myosin expression has been studied in mouse embryos from the somitic stage to the period of secondary fiber formation and in myogenic cells, cultured from the same developmental stages. The results obtained, using a combination of different polyclonal and monoclonal antibodies, indicate that slow myosin is coexpressed in virtually all the cells that express embryonic (fast) myosin in somites and limb buds in vivo as well as in culture. On the contrary fetal or late myoblasts (from 15-d-old embryos) express in culture only embryonic (fast) myosin. At this stage, muscle cells in vivo, as already shown (Crow, M.T., and F.A. Stockdale. 1986. Dev. Biol. 113:238-254; Dhoot, G.K. 1986. Muscle & Nerve. 9:155-164; Draeger, A., A.G. Weeds, and R.B. Fitzsimons. 1987. J. Neurol. Sci. 81:19-43; Miller, J.B., and F.A. Stockdale. 1986. J. Cell Biol. 103:2197-2208), consist of primary myotubes, which express both myosins, and secondary myotubes, which express preferentially embryonic (fast) myosin. Under no circumstance neonatal or adult fast myosins were detected. Western blot analysis confirmed the immunocytochemical data. These results suggest that embryonic myoblasts in mammals are all committed to the mixed embryonic-(fast) slow lineage and, accordingly, all primary fibers express both myosins, whereas fetal myoblasts mostly belong to the embryonic (fast) lineage and likely generate fibers containing only embryonic (fast) myosin. The relationship with current models of avian myogenesis are discussed.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

Myogenin is required for late but not early aspects of myogenesis during mouse development

Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD- lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts.     

Real-time functional imaging for monitoring miR-133 during myogenic differentiation

MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events.     

Desmin expression during early mouse tongue morphogenesis.

Occipital somites provide progenitor cells for craniofacial muscle development including the tongue musculature. Serum-derived factors are assumed to be pre-requisite for myogenesis in vitro. To test these assertions, we designed experiments to determine whether early mouse tongue development in general, and desmin localization in particular, were expressed during the development of embryonic mouse first branchial arch explants cultured in serumless, chemically-defined medium. Immunohistochemical techniques determined the chronology and positions of desmin expression during early craniofacial development. Occipital somites expressed desmin at E9 (9 days +/- 2 h post-fertilization, 18-20 somites). A discrete cell migration pathway initiating in the somites and terminating in the lateral lingual processes of the tongue primordium was defined based upon desmin expression patterns in E9-E11 embryos and computer-assisted three dimensional reconstructions. The in vitro model system was permissive for tongue morphogenesis, allowing development and fusion of the lateral lingual processes with the tuberculum impar. During culture myoblasts were not observed to fuse into myotubes with sarcomeric assembly, even though explant myoblasts produced muscle-specific protein. E10 explants cultured for 9 days demonstrated a five-fold increase in cell number that expressed desmin (P less than 0.05) when compared to the E10 starting material. We interpret these results to indicate that the tongue myogenic cell lineage was determined between E8 and E11, and that this resident population expanded within explants cultured in serumless medium by several explanations: (i) cells other than progenitor myoblasts (e.g., satellite cells) were induced to become myoblasts, and/or (ii) progenitor myoblasts within the original explants expanded by cell division in the absence of serum factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attempt to produce a chick/mouse heteroclass musculature

Summary Heteroclass chick/mouse chimaeras were prepared by transplanting somitic presumptive myogenic cells or limb bud myoblasts from donor mouse embryos into chick hosts, to replace (1) previously extirpated brachial somitic mesoderm or (2) experimentally deleted limb premuscular masses. Since mouse and chick cells can be distinguished by differential staining affinities, this parameter was used to verify the viability of the implant and to assess its fate. Our analyses showed that transplanted mouse somitic myogenic stem cells or limb bud myoblasts did not participate in the host brachial musculature, whatever the experimental conditions.     

Rat embryonic myoblasts are restricted to forming primary fibres while later myogenic populations are pluripotent.

Three populations of myoblasts, embryonic, foetal and adult, appear sequentially during myogenesis. The present study uses retroviruses to mark myoblasts clones in vivo from these populations. Myoblasts labelled at E15 (embryonic) contributed to primary fibres only. The majority of marked primary fibres were slow but a small number of clones contained marked primaries which were no longer slow at E19. Myoblasts labelled at E17 (foetal) fused with both primary and secondary fibres and most clones contained both fast and slow fibres. Similarly, adult myoblasts marked at P0 fused with all fibre types. These results indicate that embryonic myoblasts are restricted to producing only primary fibres which are initially slow but which can convert to being fast. Clones of foetal and adult myoblasts fuse with both primary and secondary fibres which may be either fast or slow.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

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.