Limb Muscle Formation Research Articles

Spatial Transcriptomics Analysis: Maternal Obesity Impairs Myogenic Cell Migration and Differentiation during Embryonic Limb Development.

Limb muscle is responsible for physical activities and myogenic cell migration during embryogenesis is indispensable for limb muscle formation. Maternal obesity (MO) impairs prenatal skeletal muscle development, but the effects of MO on myogenic cell migration remain to be examined. C57BL/6 mice embryos were collected at E13.5. The GeoMx DSP platform was used to customize five regions along myogenic cell migration routes (myotome, dorsal/ventral limb, limb stroma, limb tip), and data were analyzed by GeomxTools 3.6.0. A total of 2224 genes were down-regulated in the MO group. The GO enrichment analysis showed that MO inhibited migration-related biological processes. The signaling pathways guiding myogenic migration such as hepatocyte growth factor signaling, fibroblast growth factor signaling, Wnt signaling and GTPase signaling were down-regulated in the MO E13.5 limb tip. Correspondingly, the expression levels of genes involved in myogenic cell migration, such as Pax3, Gab1, Pxn, Tln2 and Arpc, were decreased in the MO group, especially in the dorsal and ventral sides of the limb. Additionally, myogenic differentiation-related genes were down-regulated in the MO limb. MO impedes myogenic cell migration and differentiation in the embryonic limb, providing an explanation for the impairment of fetal muscle development and offspring muscle function due to MO.

How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

Stepwise participation of HGF/MET signaling in the development of migratory muscle precursors during vertebrate evolution

BackgroundThe skeletal musculature of gnathostomes, which is derived from embryonic somites, consists of epaxial and hypaxial portions. Some hypaxial muscles, such as tongue and limb muscles, undergo de-epithelialization and migration during development. Delamination and migration of these myoblasts, or migratory muscle precursors (MMPs), is generally thought to be regulated by hepatocyte growth factor (HGF) and receptor tyrosine kinase (MET) signaling. However, the prevalence of this mechanism and the expression patterns of the genes involved in MMP development across different vertebrate species remain elusive.ResultsWe performed a comparative analysis of Hgf and Met gene expression in several vertebrates, including mouse, chicken, dogfish (Scyliorhinus torazame), and lamprey (Lethenteron camtschaticum). While both Hgf and Met were expressed during development in the mouse tongue muscle, and in limb muscle formation in the mouse and chicken, we found no clear evidence for the involvement of HGF/MET signaling in MMP development in shark or lamprey embryos.ConclusionsOur results indicate that the expressions and functions of both Hgf and Met genes do not represent shared features of vertebrate MMPs, suggesting a stepwise participation of HGF/MET signaling in MMP development during vertebrate evolution.

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.

Distinct modes of vertebrate hypaxial muscle formation contribute to the teleost body wall musculature

The formation of the body wall musculature in vertebrates is assumed to be initiated by direct ventral extension of the somites/myotomes. This contrasts to the formation of limb muscles and muscles involved in feeding or respiration/ventilation, which are founded by migratory muscle precursors (MMPs) distant to the somites. Here, we present evidence from morphology and expression of molecular markers proposing that the formation of the two muscle layers of the teleost body wall involves both of the above mechanisms: (1) MMPs from somites 5 and 6 found an independent muscle primordium-the so-called posterior hypaxial muscle (PHM)-which subsequently gives rise to the most anterior two segments of the medial obliquus inferioris (OI) muscle. (2) Direct epithelial extension of the hypaxial myotomes generates the OI segments from somite 7 caudalward and the entire lateral obliquus superioris (OS) muscle. The findings are discussed in relation to the evolution of hypaxial myogenic patterning including functional considerations. We hypothesise that the potential of the most anterior somites to generate migratory muscle precursors is a general vertebrate feature that has been differently utilised in the evolution in vertebrate groups.

Stable, conditional, and muscle‐fiber‐specific expression of electroporated transgenes in chick limb muscle cells

Limb muscle formation is spread out over time and, consequently, muscle cells are not easy to target at any particular stages. We aimed to design a technique to study gene function in the different steps of limb muscle formation. We have associated transposon-mediated gene transfer and a tetracycline-dependent activation method with forelimb somite electroporation. In addition, we have established a new vector combining a differentiated-muscle-cell-specific promoter and the transposon system, which allows stable gene expression in limb differentiated muscle cells and not in muscle progenitors. Using these methods, we observed that conditional Fgf4 expression in muscle cells at the onset of fetal muscle differentiation and specific Fgf4 expression in differentiated muscle cells alter muscle fiber formation in chick limbs. Limb somite electroporation with these set of vectors allowing stable, conditional, and differentiated-muscle-specific expression of transgenes opens new perspectives for investigating gene function at various steps of limb muscle formation.

Sim1 and Sim2 expression during chick and mouse limb development

The Drosophila Single minded (Sim) transcription factor is a master regulator of cell fate during midline development. The homolog mouse Sim1 and Sim2 genes are important for central nervous system development. Loss of mSim1 activity leads to an absence of specific neuroendocrine lineages within the hypothalamus, while overexpression of mSim2 leads to behavioural defects. We now provide evidence that vertebrate Sim genes might be important for limb muscle formation. We have examined by in situ hybridisation the expression of the Sim1 and Sim2 genes during limb development in chick and mouse embryos. The expression of both Sim genes is mainly associated with limb muscle formation. We found that each Sim gene has a similar temporal and spatial expression pattern in chick and mouse embryonic limbs, although with some differences for the Sim2 gene between species. In chick or mouse embryonic limbs, Sim1 and Sim2 display non-overlapping expression domains, suggesting an involvement for Sim1 and Sim2 proteins at different steps of limb muscle formation. Sim1 gene expression is associated with the early step of muscle progenitor cell migration in chick and mouse, while the Sim2 gene is expressed just after the migration process. In addition, chick and mouse Sim2 gene expression is enhanced in limb ventral muscle masses versus dorsal ventral muscle masses. Our results provide a basis for further functional analysis of the Sim genes in limb muscle formation.

Distinct transcriptional control and action of fibroblast growth factor receptor 4 in differentiating skeletal muscle cells

Although FGF signaling promotes myoblast proliferation and represses myogenic differentiation, one of the FGF receptors (FGFR), FGFR4, is expressed mainly in mature skeletal muscle. Disruption of FGFR4 signaling interrupts chick limb muscle formation. To determine the developmental regulation of FGFR4 expression, we compared the transcriptional control and action of FGFR4 in myoblasts and myotubes. We identified higher FGFR4 expression in differentiated myotubes than precursor myoblasts. FGFR4 promoter activity was localized within a region 115 bp upstream of the transcription start site. Overlapping fragments of this promoter displayed a distinct difference when compared by electromobility shift assay (EMSA) using nuclear extracts from myoblasts and myotubes. While fragments B (−95/−56) and C (−65/−26) formed specific complexes in both cell types, these complexes were consistently more intense in myotubes than myoblasts. These complexes were efficiently competed by an Sp-type oligonucleotide and were supershifted by Sp1 and by Sp3 antibodies. Deletions of the Sp-binding sites in fragment B (−95/−56) confirmed their critical contribution to promoter activity. Moreover, Sp1 expression correlated with FGFR4-expression in myotubes. To determine whether FGFR4 expression regulates myoblast differentiation, we infected a soluble dominant-negative FGFR4-containing adenovirus into these cells. This significantly impeded Erk1/2 phosphorylation and differentiation of myoblasts into MHC-expressing myotubes. Our findings point to distinct transcriptional regulation and action for FGFR4 in differentiating skeletal muscle cells.

Inhibition of myoblast migration via decorin expression is critical for normal skeletal muscle differentiation

During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglycans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-β activity. To evaluate decorin’s role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis.

Myogenic Regulatory Factors and the Specification of Muscle Progenitors in Vertebrate Embryos

Embryological and genetic studies of mouse, bird, zebrafish, and frog embryos are providing new insights into the regulatory functions of the myogenic regulatory factors, MyoD, Myf5, Myogenin, and MRF4, and the transcriptional and signaling mechanisms that control their expression during the specification and differentiation of muscle progenitors. Myf5 and MyoD genes have genetically redundant, but developmentally distinct regulatory functions in the specification and the differentiation of somite and head muscle progenitor lineages. Myogenin and MRF4 have later functions in muscle differentiation, and Pax and Hox genes coordinate the migration and specification of somite progenitors at sites of hypaxial and limb muscle formation in the embryo body. Transcription enhancers that control Myf5 and MyoD activation in muscle progenitors and maintain their expression during muscle differentiation have been identified by transgenic analysis. In epaxial, hypaxial, limb, and head muscle progenitors, Myf5 is controlled by lineage-specific transcription enhancers, providing evidence that multiple mechanisms control progenitor specification at different sites of myogenesis in the embryo. Developmental signaling ligands and their signal transduction effectors function both interactively and independently to control Myf5 and MyoD activation in muscle progenitor lineages, likely through direct regulation of their transcription enhancers. Future investigations of the signaling and transcriptional mechanisms that control Myf5 and MyoD in the muscle progenitor lineages of different vertebrate embryos can be expected to provide a detailed understanding of the developmental and evolutionary mechanisms for anatomical muscles formation in vertebrates. This knowledge will be a foundation for development of stem cell therapies to repair diseased and damaged muscles.

Signals regulating muscle formation in the limb during embryonic development.

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.

Morphogenesis and evolution of vertebrate appendicular muscle

Two different modes are utilised by vertebrate species to generate the appendicular muscle present within fins and limbs. Primitive Chondricthyan or cartilaginous fishes use a primitive mode of muscle formation to generate the muscle of the fins. Direct epithelial myotomal extensions invade the fin and generate the fin muscles while remaining in contact with the myotome. Embryos of amniotes such as chick and mouse use a similar mechanism to that deployed in the bony teleost species, zebrafish. Migratory mesenchymal myoblasts delaminate from fin/limb level somites, migrate to the fin/limb field and differentiate entirely within the context of the fin/limb bud. Migratory fin and limb myoblasts express identical genes suggesting that they possess both morphogenetic and molecular identity. We conclude that the mechanisms controlling tetrapod limb muscle formation arose prior to the Sarcopterygian or tetrapod radiation.

Morphogenesis and evolution of vertebrate appendicular muscle.

Two different modes are utilised by vertebrate species to generate the appendicular muscle present within fins and limbs. Primitive Chondricthyan or cartilaginous fishes use a primitive mode of muscle formation to generate the muscle of the fins. Direct epithelial myotomal extensions invade the fin and generate the fin muscles while remaining in contact with the myotome. Embryos of amniotes such as chick and mouse use a similar mechanism to that deployed in the bony teleost species, zebrafish. Migratory mesenchymal myoblasts delaminate from fin/limb level somites, migrate to the fin/limb field and differentiate entirely within the context of the fin/limb bud. Migratory fin and limb myoblasts express identical genes suggesting that they possess both morphogenetic and molecular identity. We conclude that the mechanisms controlling tetrapod limb muscle formation arose prior to the Sarcopterygian or tetrapod radiation.

Expression and localization of proteoglycans during limb myogenic activation.

After arriving at the limb bud, migrating myogenic precursor cells express transcription factors responsible for the induction of terminal skeletal muscle differentiation. One such factor is myogenin, a member of the basic helix-loop-helix family, known to activate the expression of muscle-specific genes. The extracellular signals involved in activating the myogenic program in the muscle precursor cells that reach the limb in vivo are not known. However, in vitro, it has been shown that proteoglycans, macromolecules composed of a core protein and glycosaminoglycan chains, modulate the triggering of myogenin activity. To understand the role of proteoglycans during limb muscle development, we assessed the synthesis of proteoglycans in limb bud explants at 10.5 days post coitum, when migrating cells arrive, evaluated the expression and nature of these macromolecules during in vivo early limb bud formation, and determined the colocalization of myoblasts expressing myogenin with specific proteoglycans. We found that the expression of myogenin was temporally and spatially coincident with the expression of syndecan-3 and decorin, two essential proteoglycans in the modulation of skeletal muscle differentiation. This article is the first report of myogenic activation and proteoglycan expression during limb muscle formation.

Sonic hedgehog is a survival factor for hypaxial muscles during mouse development.

Sonic hedgehog (Shh) has been proposed to function as an inductive and trophic signal that controls development of epaxial musculature in vertebrate embryos. In contrast, development of hypaxial muscles was assumed to occur independently of Shh. We here show that formation of limb muscles was severely affected in two different mouse strains with inactivating mutations of the Shh gene. The limb muscle defect became apparent relatively late and initial stages of hypaxial muscle development were unaffected or only slightly delayed. Micromass cultures and cultures of tissue fragments derived from limbs under different conditions with or without the overlaying ectoderm indicated that Shh is required for the maintenance of the expression of myogenic regulatory factors (MRFs) and, consecutively, for the formation of differentiated limb muscle myotubes. We propose that Shh acts as a survival and proliferation factor for myogenic precursor cells during hypaxial muscle development. Detection of a reduced but significant level of Myf5 expression in the epaxial compartment of somites of Shh homozygous mutant embryos at E9.5 indicated that Shh might be dispensable for the initiation of myogenesis both in hypaxial and epaxial muscles. Our data suggest that Shh acts similarly in both somitic compartments as a survival and proliferation factor and not as a primary inducer of myogenesis.

Evolutionary origins of vertebrate appendicular muscle.

The evolution of terrestrial tetrapod species heralded a transition in locomotor strategies. While most fish species use the undulating contractions of the axial musculature to generate propulsive force, tetrapods also rely on the appendicular muscles of the limbs to generate movement. Despite the fossil record generating an understanding of the way in which the appendicular skeleton has evolved to provide the scaffold for tetrapod limb musculature, there is, by contrast, almost no information as to how this musculature arose. Here we examine fin muscle formation within two extant classes of fish. We find that in the teleost, zebrafish, fin muscles arise from migratory mesenchymal precursor cells that possess molecular and morphogenetic identity with the limb muscle precursors of tetrapod species. Chondrichthyan dogfish embryos, however, use the primitive mechanism of direct epithelial somitic extensions to derive the muscles of the fin. We conclude that the genetic mechanism controlling formation of tetrapod limb muscles evolved before the Sarcopterygian radiation.

Early specification of limb muscle precursor cells by the homeobox gene Lbx1h.

During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs. Regulatory molecules such as receptor tyrosine kinases like c-Met ( ref. 6) and those encoded by homeobox-containing genes, including c-Met (ref. 6), Tbx1 (ref. 7), Mox2 (ref. 8), Six1 and Six2 (ref. 9), Pitx2, Pax3 (refs 10,11) and Lbx1h (refs 12,13), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevented an analysis of later functions of these genes during limb muscle development. Based on quail-chick chimaera studies, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Here we show that Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells, led to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect was caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. Our results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles.

Fibroblast-growth-factor-induced additional limbs in the study of initiation of limb formation, limb identity, myogenesis, and innervation.

In this review, we focus on the additional limb induced by members of the fibroblast growth factor (FGF) family in the flank of chick embryos. The "additional limb" was first reported 73 years ago by Balinsky in 1925. He grafted otic vesicle to the flank of newt embryos and observed the formation of the "additional limb." In 1995, formation of an additional limb was found to be induced by FGF in the chick embryo. This finding subsequently led to the recent understanding of how the limb bud is initially formed, how the limb position is determined, and how the limb identity is determined. Thus, the additional limb has been recognized as a useful experimental system for the study of limb development and its relation to the regionalization of the body. Furthermore, since limb muscles are formed from cells which have migrated from somites and innervation to them takes place from the spinal cord, the additional limb would also be a powerful tool with which to study the relation of limb morphogenesis to developmental processes of the spinal cord and somites. This review consists of five sections: (1) "Introduction," (2) "How to make additional limbs," (3) "Characteristics of the additional limb," (4) "Studies with the additional limb," and (5) "Concluding remarks." In the second section, techniques to make additional limbs are reviewed, showing that additional limbs can be made by fairly easy manipulation of the chick embryo. In the third section, the characteristics analyzed so far of the additional limb are summarized, focusing on its morphology. In the fourth section, recent studies on the use of the additional limb are reviewed: experiments on the additional limb have been performed to elucidate the mechanisms governing determination of limb identity by Hox codes and the Tbx family and initiation of limb formation by FGF10. In addition, the roles of SF/HGF in the formation of limb muscles have also been investigated using the additional limb. In the near future, the additional limb will be also used in the study of innervation from the spinal cord, and probably migration of neural crest cells.

The four populations of myoblasts involved in human limb muscle formation are present from the onset of primary myotube formation.

To understand how and when myogenic precursor cells become committed to their particular developmental programs, we have analysed the different populations of myoblasts which grow out from explants of muscle tissue isolated from human limb buds from the beginning of primary fibre formation throughout subsequent development and post-natal growth. Four phenotypically distinct types of myoblasts were identified on the basis of their expression of desmin, myogenin and myosin heavy chain isoforms (MyHC), and after 5 and 20 divisions, cells were cloned. All four types of myoblasts were present at the beginning of primary myogenesis. Each respective phenotype was stably heritable through cloning and subsequent proliferation. The type 1 clones correspond to a novel class of myoblasts never described during human development, that biochemically differentiates, but does not fuse. Type 2 clones are composed of small myotubes expressing only embryonic MyHC. Type 3 clones are composed of thin and long myotubes expressing both embryonic and fetal MyHCs. The type 4 clones are composed of myotubes that have a phenotype very similar to human satellite cells. Contrasting with others species, no other population of myoblasts appear during fetal development and only the relative number of these four types changes.