Embryo Muscle Research Articles

Loss of Smyhc1 or Hsp90α1 Function Results in Different Effects on Myofibril Organization in Skeletal Muscles of Zebrafish Embryos

BackgroundMyofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90α1 (Hsp90α1) has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90α1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90α1 function or indirectly through the disorganization of myosin thick filaments.Methodology/Principal FindingsIn this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide) in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1) resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90α1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines.Conclusion/SignificanceTogether, these studies indicate that the hsp90α1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90α1 may play a role in the assembly and organization of other sarcomeric structures.

Stable‐isotope comparisons between embryos and mothers of a placentatrophic shark species

Stable nitrogen (delta(15)N) and carbon (delta(13)C) isotopes of Atlantic sharpnose shark Rhizoprionodon terraenovae embryos and mothers were analysed. Embryos were generally enriched in (15)N in all studied tissue relative to their mothers' tissue, with mean differences between mother and embryo delta(15)N (i.e. Deltadelta(15)N) being 1.4 per thousand for muscle, 1.7 per thousand for liver and 1.1 per thousand for cartilage. Embryo muscle and liver were enriched in (13)C (both Deltadelta(13)C means = 1.5 per thousand) and embryo cartilage was depleted (Deltadelta(13)C mean = -1.01 per thousand) relative to corresponding maternal tissues. While differences in delta(15)N and delta(13)C between mothers and their embryos were significant, muscle delta(15)N values indicated embryos to be within the range of values expected if they occupied a similar trophic position as their respective mothers. Positive linear relationships existed between embryo total length (L(T)) and Deltadelta(15)N for muscle and liver and embryo L(T) and Deltadelta(13)C for muscle, with those associations possibly resulting from physiological differences between smaller and larger embryos or differences associated with the known embryonic nutrition shift (yolk feeding to placental feeding) that occurs during the gestation of this placentatrophic species. Together these results suggest that at birth, the delta(15)N and delta(13)C values of R. terraenovae are likely higher than somewhat older neonates whose postpartum feeding habits have restructured their isotope profiles to reflect their postembryonic diet.

LRT, a tendon-specific leucine-rich repeat protein, promotes muscle-tendon targeting through its interaction with Robo

Correct muscle migration towards tendon cells, and the adhesion of these two cell types, form the basis for contractile tissue assembly in the Drosophila embryo. While molecules promoting the attraction of muscles towards tendon cells have been described, signals involved in the arrest of muscle migration following the arrival of myotubes at their corresponding tendon cells have yet to be elucidated. Here, we describe a novel tendon-specific transmembrane protein, which we named LRT due to the presence of a leucine-rich repeat domain (LRR) in its extracellular region. Our analysis suggests that LRT acts non-autonomously to better target the muscle and/or arrest its migration upon arrival at its corresponding tendon cell. Muscles in embryos lacking LRT exhibited continuous formation of membrane extensions despite arrival at their corresponding tendon cells, and a partial failure of muscles to target their correct tendon cells. In addition, overexpression of LRT in tendon cells often stalled muscles located close to the tendon cells. LRT formed a protein complex with Robo, and we detected a functional genetic interaction between Robo and LRT at the level of muscle migration behavior. Taken together, our data suggest a novel mechanism by which muscles are targeted towards tendon cells as a result of LRT-Robo interactions. This mechanism may apply to the Robo-dependent migration of a wide variety of cell types.

Biphasic Myopathic Phenotype of Mouse DUX, an ORF within Conserved FSHD-Related Repeats

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX), embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models highlight the myopathic nature of sequences within the FSHD-related repeat array.

Activity of matrix metalloproteinases in skeletal muscle of chick embryos of different ages

Activity of matrix metalloproteinases in skeletal muscle of chick embryos of different ages

Distinct Origins and Genetic Programs of Head Muscle Satellite Cells

Adult skeletal muscle possesses a remarkable regenerative capacity, due to the presence of satellite cells, adult muscle stem cells. We used fate-mapping techniques in avian and mouse models to show that trunk (Pax3(+)) and cranial (MesP1(+)) skeletal muscle and satellite cells derive from separate genetic lineages. Similar lineage heterogeneity is seen within the head musculature and satellite cells, due to their shared, heterogenic embryonic origins. Lineage tracing experiments with Isl1Cre mice demonstrated the robust contribution of Isl1(+) cells to distinct jaw muscle-derived satellite cells. Transplantation of myofiber-associated, Isl1-derived satellite cells into damaged limb muscle contributed to muscle regeneration. In vitro experiments demonstrated the cardiogenic nature of cranial- but not trunk-derived satellite cells. Finally, overexpression of Isl1 in the branchiomeric muscles of chick embryos inhibited skeletal muscle differentiation in vitro and in vivo, suggesting that this gene plays a role in the specification of cardiovascular and skeletal muscle stem cell progenitors.

Analysis of expression of heavy myosin chains during in vitro differentiation of satellite cells and myoblasts derived from rat skeletal muscles

Differentiation of cultured myogenic progenitor cells (satellite cells and mononucleated myoblasts) derived from hindlimb muscles of rat embryos and newborn animals was studied. Immunocytochemical methods and PCR analysis revealed expression of heavy myosin chains at the earliest stages of myogenesis (in mononucleated myoblasts). Expression of the gene encoding the embryonic form of myosin and a low level of expression of the gene encoding perinatal myosin in cultured progenitor cells derived from embryonic muscles was detected by PCR. Cells derived from muscles of newborn animals also expressed these two myosin forms, though at a lower level. The progenitor cells derived from muscles of rat embryos and newborn animals were found to express myosin 2a, which is characteristic of fast-twitch definitive muscle fibers.

The extracellular matrix protein TGFBI promotes myofibril bundling and muscle fibre growth in the zebrafish embryo

The extracellular matrix protein Tgfbi has been shown to localise to myotendinous junctions in mouse and postulated to interact with the transmembrane protein Integrin alpha7beta1, which, in parallel with the Dystrophin-associated protein complex, is critical for linkage between the extracellular matrix and the cytoskeleton of muscle fibres. Here we use a GFP-tagged form of Tgfbi to analyse its distribution in the developing skeletal muscle of the zebrafish embryos and antisense morpholino oligonucleotides to investigate the function of the endogenous protein. We find that although tagged Tgfbi accumulates at the myosepta, the attachment of muscle fibres to the myosepta is established and maintained normally in morphant embryos; however, the fibres show a marked reduction in their growth and a disruption of their myofibril bundles.

Heat-shock protein 90α1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos

Heat-shock protein 90alpha (Hsp90alpha) is a member of the molecular chaperone family involved in protein folding and assembly. The role of Hsp90alpha in the developmental process, however, remains unclear. Here we report that zebrafish contains two Hsp90alpha genes, Hsp90alpha1, and Hsp90alpha2. Hsp90alpha1 is specifically expressed in developing somites and skeletal muscles of zebrafish embryos. We have demonstrated that Hsp90alpha1 is essential for myofibril organization in skeletal muscles of zebrafish embryos. Knockdown of Hsp90alpha1 resulted in paralyzed zebrafish embryos with poorly organized myofibrils in skeletal muscles. In contrast, knockdown of Hsp90alpha2 had no effect on muscle contraction and myofibril organization. The filament defects could be rescued in a cell autonomous manner by an ectopic expression of Hsp90alpha1. Biochemical analyses revealed that knockdown of Hsp90alpha1 resulted in significant myosin degradation and up-regulation of unc-45b gene expression. These results indicate that Hsp90alpha1 plays an important role in muscle development, likely through facilitating myosin folding and assembly into organized myofibril filaments.

Excretion Products of Shigella dysenteriae and Apoptotic Cell Death on Chick Embryo Muscle Tissue

El objetivo del presente trabajo ha sido evaluar el dano celular agudo generado por el producto de excrecion de Shigella dysenteriae sobre el tejido muscular del miembro inferior de embrion de pollo, asi como sobre los mioblastos obtenidos a partir del cultivo de explantes de tejido muscular desarrollado en gota pendiente. Tres controles fueron definidos: a) solucion de tiroide b) caldo de infusion cerebro-corazon (CCC), c) producto de excrecion bacteriano no toxigenico de E. coli O157:H7. El producto de excrecion fue obtenido a partir de la centrifugacion y filtrado del medio de cultivo de Shigella dysenteriae con 24 horas de crecimiento. Luego de una hora de tratamiento, los cambios morfologicos del tejido muscular fueron evaluados a traves del examen histopatologico, con tecnicas de analisis de imagenes sobre cortes tenidos con hematoxilina-eosina (H&E) y Tricromico de Gomori. El ensayo enzimatico TUNEL fue utilizado para evaluar el indice de apoptosis. Senales morfologicas de muerte celular fueron evaluadas en mioblastos en cultivo En contraste con los controles, el tejido muscular presento signos de atrofia, con fibras desconectadas y perdida del 57,14% del numero total de celulas asi como perdida de la matriz de colageno. Un incremento en el indice apoptotico y una reduccion de indice mitotico fueron registrados. Esto ultimo fue no significativo. Los mioblastos en cultivo presentaron signos morfologicos de apoptosis tales como protuberancias en la membrana, vacuolizacion, agregacion de cromatina alrededor del nucleo y perdida de la adhesion celular. Se concluye que el producto de excrecion afecta al tejido muscular esqueletico del miembro inferior de embrion de pollo e induce lesiones de toxicidad relacionadas con la muerte celular por apoptosis

Ubiquitous expression of myostatin in chicken embryonic tissues: Its high expression in testis and ovary

The skeletal muscle of mammals is known to express myostatin (GDF-8) that acts as a potent negative regulator of skeletal muscle growth. However, the function of GDF-8 is not limited to skeletal muscle, because of its ubiquitous expression in fish. Here we investigated whether GDF-8 is expressed in various tissues including gonads during chicken embryogenesis. As revealed by RT-PCR and Western blotting, the transcript and protein for GDF-8 were detected in brain, eye, gizzard, muscle, heart, small gut, large gut, mesonephroi, testis and ovary of chicken embryos at E12, but not in liver. GDF-8 was constitutively expressed in testis and ovary as well as muscle at E6-E21, as demonstrated by in situ hybridization on section and whole-mount. Some cell population in testis, but not identified, highly expressed GDF-8. On the other hand, the medulla and germinal epithelium of ovary highly expressed it. Collectively, these results indicate that GDF-8 is ubiquitously expressed in various tissues of chicken embryos including testis and ovary through the stage of embryogenesis.

Effect of Incubator Temperature and Oxygen Concentration at the Plateau Stage in Oxygen Consumption on Turkey Embryo Muscle Growth and Development

Effect of Incubator Temperature and Oxygen Concentration at the Plateau Stage in Oxygen Consumption on Turkey Embryo Muscle Growth and Development

Muscle‐specific expression of the smyd1 gene is controlled by its 5.3‐kb promoter and 5′‐flanking sequence in zebrafish embryos

Zebrafish SmyD1 is a SET and MYND domain-containing protein that plays an important role in myofiber maturation and muscle contraction. SmyD1 is required for myofibril organization and sarcomere assembly during myofiber maturation. Whole-mount in situ hybridization revealed that smyd1 mRNAs are specifically expressed in skeletal and cardiac muscles in zebrafish embryos. However, it is unknown if smyd1 is expressed in other striated muscles, such as cranial and fin muscles, and moreover, the regulatory elements required for its muscle-specific expression. We report here the analyses of smyd1 expression using smyd1-gfp transgenic zebrafish. smyd1-gfp transgenic zebrafish were generated using the 5.3-kb smyd1 promoter and its 5'-flanking sequence. GFP expression was found in the skeletal and cardiac muscles of smyd1-gfp transgenic embryos. GFP expression appeared stronger in slow muscles than fast muscles in transgenic zebrafish larvae. In addition, GFP expression was also detected in cranial and fin muscles of smyd1-gfp transgenic zebrafish larvae. In situ hybridization confirmed smyd1 mRNA expression in these tissues, suggesting that the expression of the smyd1-gfp transgene recapitulated that of the endogenous smyd1 gene. Deletion analysis revealed that the 0.5-kb sequence in the proximal promoter of smyd1 was essential for its muscle specificity. Together, these data indicate that smyd1 is specifically expressed in most, if not all, striated muscles, and the muscle specificity is controlled by the 5.3-kb promoter and flanking sequences.

Neuromuscular stimulation causes muscle phenotype‐dependent changes in the expression of the IGFs and their binding proteins in developing slow and fast muscle of chick embryos

Insulin-like growth factor (IGF) -1 and -2 and binding protein (IGFBP-2, -4, and -5) expression can be affected by several environmental factors, but the impact of movement on the IGF axis during late embryogenesis has yet to be fully characterized. Movement was promoted in chick embryos during mid-embryogenesis using 4-aminopyridine (4-AP). The results indicate an increase in IGF-1 (P < 0.01) and a decrease in IGFBP-2 (P < 0.05) mRNA expression in slow muscle of the stimulated group compared with the control group. In fast muscle, there was a decrease in IGF-2 (P < 0.01) on embryonic day (ED) 16 and an increase in IGFBP-2 (P < 0.01) and IGFBP-4 (P < 0.05) and in IGFBP-5 (P < 0.05) expression on ED18 in the stimulated group compared with the control group. These results indicate that neuromuscular stimulation with 4-AP influences IGF axis gene expression in a muscle fiber type-dependent manner. Consequences of the changes in the IGF system for each muscle are discussed.

SmyD1, a histone methyltransferase, is required for myofibril organization and muscle contraction in zebrafish embryos

Histone modification has emerged as a fundamental mechanism for control of gene expression and cell differentiation. Recent studies suggest that SmyD1, a novo SET domain-containing protein, may play a critical role in cardiac muscle differentiation. However, its role in skeletal muscle development and its mechanism of actions remains elusive. Here we report that SmyD1a and SmyD1b, generated by alternative splicing of SmyD1 gene, are histone methyltransferases that play a key role in skeletal and cardiac muscle contraction. SmyD1a and SmyD1b are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. Knockdown of SmyD1a and SmyD1b expression by morpholino antisense oligos resulted in malfunction of skeletal and cardiac muscles. The SmyD1 morphant embryos (embryos injected with morpholino oligos) could not swim and had no heartbeat. Myofibril organization in the morphant embryos was severely disrupted. The affected myofibers appeared as immature fibers with centrally located nuclei. Together, these data indicate that SmyD1a and SmyD1b are histone methyltransferases and play a critical role in myofibril organization during myofiber maturation.

Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

Non-sense mutations in the dihydropyridine receptor β1 gene, CACNB1, paralyze zebrafish relaxed mutants

Contractions by skeletal muscle require proper excitation–contraction (EC) coupling, whereby depolarization of the muscle membrane leads to an increase in cytosolic Ca 2+ and contraction. Changes in membrane voltage are detected by dihydropyridine receptors (DHPR) that directly interact with and activate ryanodine receptors to release Ca 2+ from the sarcoplasmic reticulum into the cytosol. A genetic screen for motility mutations isolated a new allele of the immotile zebrafish mutant relaxed. Muscles in relaxed embryos do not contract in response to potassium chloride (KCl) thus appear unresponsive to membrane depolarization, but do contract when stimulated by caffeine, an agonist of ryanodine receptors. This suggests that relaxed mutant muscles are defective in EC coupling. Indeed, immunohistochemical analysis demonstrated that mutants lack DHPRs in skeletal muscles. The mutant phenotype results from non-sense mutations in the zebrafish CACNB1 gene that encodes the DHPR β1 subunit. The zebrafish CACNB1 gene is expressed in skeletal muscles, PNS and CNS. Electrophysiological recordings showed no obvious abnormalities in the motor output of relaxed mutants, presumably due to redundancy provided by other β subunits. The structural and functional homology of CACNB1 in zebrafish and mammals, suggests that zebrafish can be useful for studying EC coupling and potential neuronal function of CACNB1.

A Hox Regulatory Network Establishes Motor Neuron Pool Identity and Target-Muscle Connectivity

Spinal motor neurons acquire specialized "pool" identities that determine their ability to form selective connections with target muscles in the limb, but the molecular basis of this striking example of neuronal specificity has remained unclear. We show here that a Hox transcriptional regulatory network specifies motor neuron pool identity and connectivity. Two interdependent sets of Hox regulatory interactions operate within motor neurons, one assigning rostrocaudal motor pool position and a second directing motor pool diversity at a single segmental level. This Hox regulatory network directs the downstream transcriptional identity of motor neuron pools and defines the pattern of target-muscle connectivity.

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.

Endoplasmic reticulum stress signaling transmitted by ATF6 mediates apoptosis during muscle development

Although apoptosis occurs during myogenesis, its mechanism of initiation remains unknown. In a culture model, we demonstrate activation of caspase-12, the initiator of the endoplasmic reticulum (ER) stress-specific caspase cascade, during apoptosis associated with myoblast differentiation. Induction of ER stress-responsive proteins (BiP and CHOP) was also observed in both apoptotic and differentiating cells. ATF6, but not other ER stress sensors, was specifically activated during apoptosis in myoblasts, suggesting that partial but selective activation of ER stress signaling was sufficient for induction of apoptosis. Activation of caspase-12 was also detected in developing muscle of mouse embryos and gradually disappeared later. CHOP was also transiently induced. These results suggest that specific ER stress signaling transmitted by ATF6 leads to naturally occurring apoptosis during muscle development.