Muscle Pattern Formation Research Articles

Characterizing the functional impact of embryonic myosin mutations in stem cell-derived skeletal muscle.

Divergent contractile properties of different myosins suggest that each isoform performs distinct functions in embryonic and adult skeletal muscles. To better understand the role of embryonic myosin heavy chain 3 (MYH3) in regulating skeletal muscle pattern formation, growth and regeneration, our group investigated the functional defects caused by multiple mutations. Our team generated human induced pluripotent stem cell (iPSC) lines bearing T178I or R672C mutations in MYH3. Human iPSCs bearing these mutations were differentiated into skeletal muscle and evaluated for differences in the structure, maturation and functional performance of the sarcomere. Isolated myofibril mechanics showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to controls. Myofibril preparations were also used to compare ATP binding rates and showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. When incorporated into 3D engineered tissues, these changes in MYH3 mechanics resulted in slower relaxation kinetics and an incomplete relaxation of the tissue in response to repetitive tetanic stimulation, which may explain the emergence of developmental contractures in patients possessing MYH3 mutations. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the β-sheet and SH-Helix than in controls. Mutations also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

Cryptorchidism in the orl rat is associated with muscle patterning defects in the fetal gubernaculum and altered hormonal signaling.

Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.

Reception of Slit requires only the chondroitin–sulphate-modified extracellular domain of Syndecan at the target cell surface

Syndecan (Sdc) is a conserved transmembrane heparan sulfate proteoglycan (HSPG) bearing additional chondroitin sulfate (CS) modifications on its extracellular domain. In vertebrates, this extracellular domain of Sdc is shed and acts as a soluble effector of cellular communication events, and its cytoplasmic domain participates in intracellular signaling needed to maintain epithelial integrity. In Drosophila, Sdc has been shown to be necessary for Slit signaling-dependent axon and myotube guidance during CNS development and muscle pattern formation. We report that Sdc acts in a cell-autonomous manner in Slit-receiving cells and that its membrane-anchored extracellular domain is sufficient to mediate Slit signaling. Sdc activity can be replaced by the human homolog hsdc2. However, the HSPG Dally-like protein (Dlp), which lacks CS modifications at its extracellular domain, can only partially substitute for Sdc function, and its activity is not restricted to the Slit target cells. Our results suggest that Sdc and Dlp act in a cooperative but nonredundant fashion in axon and myotube guidance. We propose that Dlp, which lacks CS modifications, participates in the transfer of Slit from its site of expression to the target cells, where CS-modified Sdc concentrates and presents the ligand.

α PS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin

During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (α- and β-subunits). The α-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the β-integrin subunit is expressed in both tissues. Mutations of β-integrin cause a severe muscle detachment phenotype, whereas α-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. Here, we report that mutations in the Drosophila gene thrombospondin ( tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern formation as well as the initial contact between muscle and tendon cells nor muscle-to-muscle attachments are affected. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the α PS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells.

Gain-of-Function Screen for Genes That Affect Drosophila Muscle Pattern Formation

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.

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.

Transient expression of collagen type XIV during muscle development and its reappearance after denervation and degeneration.

In the formation of muscle pattern, the architectural arrangement is believed to be controlled by the local connective tissue cells. In this study we examined the immunohistological localization of Type XIV collagen recognized by a monoclonal antibody, MAb DBM, in embryonic chick hind limbs from stage (St.) 27 to 2 weeks post hatching. DBM staining was transiently observed in the epimysium from St. 30, in the perimysium of the dorsal region from St. 37, and in the entire perimysium from St. 39. After hatching, DBM staining was notably diminished in both epimysium and perimysium. In contrast, DBM staining and in situ hybridization signals for Type XIV collagen mRNA increased in the muscle connective tissues after denervation and around the regenerating muscle fibers. Therefore, Type XIV collagen expression appears to coincide with muscle activity and muscle regenerating conditions, and Type XIV collagen is considered to play roles in muscle development and regeneration.

Muscle morphogenesis in the absence of myogenic cells

Experimental evidence indicates, that the myogenic cells themselves are not responsible for the muscle pattern formation. We report on a chance observation that reveals that muscle pattern formation can occur even in the absence of myogenic cells. Epiblastic cells from a quail embryo in the primitive streak stage were implanted into the wing bud of a chick embryo. The grafted quail cells developed into mononucleate, fibroblast-like cells that formed the muscle belly of the extensor medius longus muscle. This showed essentially normal form and topography as revealed by computer-aided 3D-reconstruction. This finding shows, that the formation of muscles does not depend on the presence of myogenic cells.

Ontogeny of architectural complexity in embryonic quail visceral arch muscles.

Understanding the mechanisms of muscle pattern formation requires that the complete sequence of ontogenetic events be defined, particularly in the emergence of architectural complexity and in the spatial relations between muscles and skeletal elements. This analysis of visceral arch myogenesis in quail (Coturnix coturnix japonica) embryos identifies the location of premuscle condensations and subsequent segregation of individual muscles, documents the initial orientation of myofibers and changes in alignment associated with maturation, and describes the spatial and temporal relations between muscle development and the formation of connective tissues. Premuscle condensations form within the visceral arches on embryonic days 2-4, before skeletal elements make their appearance. Discrete muscles may form from the subdivision of a muscle mass after fiber orientations have been established (e.g., jaw adductor and hyobranchial muscles) or by the segregation of a mesenchymal cluster from the condensation prior to the appearance of oriented fibers (e.g., protractor, muscle of the columella). The rate and pattern of subsequent muscle maturation are closely associated with the development of the hard tissues. Myogenesis in 4-9-day embryos centers around the quadrate cartilage, the retroarticular process of the mandibular (Meckel's) cartilage, and the epibranchial cartilage. Muscles form attachments on these elements and remain without additional attachments until the appropriate elements (e.g., otic capsule, pterygoid bone) develop. No single description of myogenic events applies to all visceral arch muscles, nor is there an arch-specific pattern of ontogeny. Rather, each muscle has distinctive characteristics based on its spatial relations within the developing head.

Positional signals: evidence for a possible role in muscle fibre-type patterning of the embryonic avian limb

Abstract Current evidence favours the hypothesis that avian muscle fibre-type differentiation is intrinsically programmed during early embryogenesis and proceeds independent of neuronal influences (for review, Miller & Stockdale, 1987; Sanes, 1987). Previous interspecific (chick/quail) and intraspecific (chick/chick) chimaeric studies, involving the heterotopic transplantation of unsegmented somitic mesoderm, indicate that the ability of premyogenic cells to form specific limb muscles is determined by positional cues operative in the host embryo. To determine if similar influences are associated with embryonic fibre-type differentiation, the limb musculature of intraspecific (chick/chick) chimaeras was assessed using differential myosin-ATPase activity. The limb musculature analysed histochemically was derived from donor unsegmented thoracic somitic mesoderm transplanted to replace extirpated brachial somites of a host embryo at 48 –52 h in ovo. Our results demonstrated that (1) the donor tissue formed, on schedule, an appropriate brachial musculature and (2) the experimental muscles duplicated the fibre-type patterning characteristic of control muscles. Thus, our data suggest that an extramyogenic, non-neural factor(s) operative in the host limb-forming region is associated with both muscle pattern formation and muscle fibre-type pattern formation. Whether or not the same putative factor(s) influences both processes remains to be determined.

Development of wing-bud-derived muscles in normal and wingless chick embryos: a computer-assisted three-dimensional reconstruction study of muscle pattern formation in the absence of skeletal elements.

The mechanisms whereby the normal pattern of muscles within the developing chick limb bud is generated are largely unexplored. It has been proposed that the muscle pattern is established independently of the pattern for the limb skeletal elements to which the muscles normally attach (Shellswell and Wolpert: "The Pattern of Muscle and Tendon Development in the Chick Wing."In: Vertebrate Limb and Somite Morphogenesis. Cambridge University Press, Cambridge, pp. 71-86, 1977). To further examine this possibility we studied the formation of the proximal wing muscles in normal and wingless chick embryos. The muscles of the shoulder region (including the pectoralis) arise as part of the dorsal and ventral premuscle masses of the developing limb bud. These secondarily migrate out of the limb to take origin from the pectoral girdle while inserting onto the humerus (Sullivan: Aust. J. Zool., 10:458-516, 1962). With rare exceptions, wingless embryos have complete absence of wing skeletal elements, but they may possess more than 40% of the normal volume of wing-bud-derived muscles. The muscles that remain in wingless embryos are primarily shoulder muscles, and to a varying extent, the pectoralis. The question we sought to answer was whether in wingless embryos the proximal wing muscles could form a normal pattern in the absence of the humerus and distal wing skeletal elements. By examining three-dimensional reconstructions of the proximal wing region in normal and wingless embryos, we found that the initial subdivision of the dorsal and ventral premuscle masses proceeded normally in the absence of the wing skeleton. This resulted in a grossly normal pattern of proximal wing muscles despite the absence of wing skeletal elements. However, some subsequent cleavages of individual muscles within premuscle mass divisions did not occur in wingless embryos. This suggests that the skeleton may be required for this step in muscle morphogenesis to occur. We also observed that the wing-bud-derived muscles in wingless embryos were nearly always anchored to the pectoral girdle at both ends. Sometimes this resulted in muscles making abnormal tendonous fusions with other muscles derived from the opposite (i.e., dorsal or ventral) premuscle mass. Therefore, attachment to the skeleton may be important for some facet of muscle development. Finally, the supracoracoideus muscle was absent in all but one wingless embryo we examined in the present study. In that one, it was substantially reduced in volume compared to normal. absence of this muscle, the space normally occupied by the supracoracoideus was maintained beneath the pectoralis.(ABSTRACT TRUNCATED AT 400 WORDS)

The Role of Collagen in the Development of Muscle and its Relationship to Eating Quality

Our identification of a family of genetically distinct collagens specifically distributed in the connective tissue of muscle now poses questions on their possible role in myoblast differentiation, muscle development, turnover and function in the living muscle, and post-rigor in the tenderness of meat. Using collagen-type specific antibodies we have shown temporal transitions in the type of collagen synthesized during myogenesis. Similar temporal transitions are currently being observed during muscle development. Immunofluorescence techniques also indicated that collagen did not play a role in muscle pattern formation. Once the muscle pattern has been established, the specific distribution of the collagen types in the endo-mysium (IV and V), perimysium (III and I) and epimysium (I) is maintained during growth. Using an arrested growth model we demonstrated that there was no significant loss of the pre-existing collagen, indicating that the mature collagen fibers do not restrict growth. As the growth rate decreased and the animal matures, the collagen fibers, initially stabilized by intermolecular head-to-tail cross-links, form a stable network via multivalent cross-links. These cross-links determine the extent of the tension generated during heating and the residual adhesion between muscle fibers and hence, the greater the proportion present the tougher the meat. It is therefore the quality rather than the quantitiy of the collagen that determines its contribution to the texture of meat.

Tissue interactions in the organization and maintenance of the muscle pattern in the chick limb

Recent investigations on a hereditary muscular dysgenesis (cn/cn) in the chicken (Kieny, Mauger, Hedayat & Goetinck, 1983) have suggested that limb muscle pattern development and subsequent maintenance are two independent steps in the formation of the musculature. The respective activities or muscle cells and connective tissue cells in the ontogeny of the musculature have been investigated in avian embryos 1) by in ovo administration of drugs interfering with collagen biosynthesis, and 2) by heterogenetic somite-exchange experiments between normal and mutant embryos. None of the drugs administered to the chick embryo caused any disturbance of muscle pattern formation or maintenance whether treatment occurred before (5 days) or after (7.5 days) the muscle splitting period. Heterogenetic implantations were performed at 2 days of incubation either at the leg or at the wing level. Somitic mesoderm from non-mutant quail embryo was grafted to replace a piece of somitic mesoderm in putative mutant (cn/cn) chick embryos. The introduction of normal myogenic cells into a mutant leg or wing led to a normally patterned musculature, which demonstrates that the muscular dysgenesis cn/cn results from a defect of the somitic myogenic cell line.

On the origin and development of the ventrolateral abdominal muscles in the avian embryo. An experimental and ultrastructural study.

In avian embryos the formation of ventrolateral abdominal muscles was studied by (1) heterospecific grafting experiments between chick and quail embryos and (2) ultrastructural examinations of cells having part in this process. The results demonstrate that the muscle cells are of somitic origin while the connective tissue derives from the somatopleure. Somatopleural cells do not differentiate into myocytes, and somite cells which have entered the ventrolateral abdominal wall, do not contribute to the connective tissue. It is concluded that both dermatome and myotome cells undergo muscular differentiation. The formation of muscles is found to take place in four characteristic steps. During the 4th day of development, epithelially structured ventral somite buds enter the somatopleure. The light cells of the inner myotome layer are elongated in a cranio-caudal direction and contain randomly distributed microfilaments. On the 5th day, the buds lose their epithelial arrangement and change into compact processes in which cells intermingle. The myotome cells show short bundles of thin and thick microfilaments. The third step can be characterized by the appearance of intercellular spaces and the disaggregation of processes becoming invaded by somatopleural cells. Thus, subdivision in single muscle blastemata begins to occur. In 7-day embryos, the muscle anlagen are distinctly separated and the first myotubes containing regularly arranged myofibrils are found. Coincidentally, signs of cell death are observed. Up to the 10th day, the tendons being of somatopleural origin become plainly outlined and the muscle anlagen move to their definitive positions. It is assumed that the formation of muscle pattern is controlled by the somatopleure.

Has collagen a role in muscle pattern formation in the developing chick wing? 1. An immunofluorescence study

We have used antibodies to three of the isomorphic forms of collagen, types I, III and V, in an immunofluorescence microscopy study of myogenesis in the embryonic chick wing, concentrating on the period between stages 27 and 30 (5 to 7.5 days incubation) which is when the dorsal and ventral muscle masses separate into discrete muscle. We have demonstrated the presence of all three collagen types at the ectoderm-mesenchyme junction from stage 27 onwards. Type I collagen and then type III collagen are found in progressively deeper layers of the dermis at the later stages. Both types I and V collagen are initially present in the cartilage elements, but type I collagen becomes restricted to the periphery of these structure at later stages. The developing muscle areas show a lack of staining at all stages and it is only at the latest stages that types I and III collagen first appear in the surrounding epimysium. We discuss possible mechanisms for the division of the muscle masses in the light of this information on the distribution of collagen types.

The formation of muscles in regenerating limbs of the newt after denervation of the blastema

The purpose of this experiment was to examine the relationship, if any, between nerve fibers and the formation of muscle pattern in the regenerating amphibian limb. During embryogenesis, nerve fibers grow into the limb bud at the time when the common muscle blastemas subdivide into individual muscle primordia, whereas in regeneration nerve fibers are always present. In order to learn whether or not the muscle pattern could be laid down in the absence of nerves we amputated 58 limbs of newts (Notophthalmus viridescens) at the mid humeral level and allowed them to regenerate to the medium-bud or late-bud stage. The limbs were then denervated. The majority of limbs denervated at the medium-bud stage either regressed or failed to regenerate further. Regeneration after denervation failed in 9 of 25 limbs denervated at the late-bud stages. In those limbs that continued to regenerate after denervation, the formation of individual muscle primordia did occur, following the same sequence with respect to the gross stage of regeneration as innervated regenerates. In comparing these results with our previous results on the development of muscular pattern in aneurogenic limbs of the axolotl, we conclude that in neither the embryonic nor the regenerating amphibian limb are nerve fibers directly involved in the subdivision of common muscle blastemas into the primordia of individual muscles.