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Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a dramatic neuromuscular disease in which crippling muscle weakness is evident from birth. Here, we use the dyW mouse model for human MDC1A to trace the onset of the disease during development in utero. We find that myotomal and primary myogenesis proceed normally in homozygous dyW-/- embryos. Fetal dyW-/- muscles display the same number of myofibers as wildtype (WT) muscles, but by E18.5 dyW-/- muscles are significantly smaller and muscle size is not recovered post-natally. These results suggest that fetal dyW-/- myofibers fail to grow at the same rate as WT myofibers. Consistent with this hypothesis between E17.5 and E18.5 dyW-/- muscles display a dramatic drop in the number of Pax7- and myogenin-positive cells relative to WT muscles, suggesting that dyW-/- muscles fail to generate enough muscle cells to sustain fetal myofiber growth. Gene expression analysis of dyW-/- E17.5 muscles identified a significant increase in the expression of the JAK-STAT target gene Pim1 and muscles from 2-day and 3-week old dyW-/- mice demonstrate a dramatic increase in pSTAT3 relative to WT muscles. Interestingly, myotubes lacking integrin α7β1, a laminin-receptor, also show a significant increase in pSTAT3 levels compared with WT myotubes, indicating that α7β1 can act as a negative regulator of STAT3 activity. Our data reveal for the first time that dyW-/- mice exhibit a myogenesis defect already in utero. We propose that overactivation of JAK-STAT signaling is part of the mechanism underlying disease onset and progression in dyW-/- mice., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7β1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.

Neuromuscular disorders are a broad group of inherited conditions affecting the structure and function of the motor system with polymorphic clinical presentation and disease severity. Although individually rare, collectively neuromuscular diseases have an incidence of 1 in 3,000 and represent a significant cause of disability of the motor system. The past decade has witnessed the identification of a large number of human genes causing muscular disorders, yet the underlying pathogenetic mechanisms remain largely unclear, limiting the developing of targeted therapeutic strategies. To overcome this barrier, model systems that replicate the different steps of human disorders are increasingly being developed. Among these, the zebrafish (Danio rerio) has emerged as an excellent organism for studying genetic disorders of the central and peripheral motor systems. In this review, we will encounter most of the available zebrafish models for childhood neuromuscular disorders, providing a brief overview of results and the techniques, mainly transgenesis and chemical biology, used for genetic manipulation. The amount of data collected in the past few years will lead zebrafish to became a common functional tool for assessing rapidly drug efficacy and off-target effects in neuromuscular diseases and, furthermore, to shed light on new etiologies emerging from large-scale massive sequencing studies.

The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin beta2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.

Duchenne muscular dystrophy (DMD) is a common X-linked disease resulting from the absence of dystrophin in muscle. Affected boys suffer from incurable progressive muscle weakness, leading to premature death. Stem cell transplantation may be curative, but is hampered by the need for systemic delivery and immune rejection. To address these barriers to stem cell therapy in DMD, we investigated a fetal-to-fetal transplantation strategy. We investigated intramuscular, intravascular, and intraperitoneal delivery of human fetal mesenchymal stem cells (hfMSCs) into embryonic day (E) 14-16 MF1 mice to determine the most appropriate route for systemic delivery. Intramuscular injections resulted in local engraftment, whereas both intraperitoneal and intravascular delivery led to systemic spread. However, intravascular delivery led to unexpected demise of transplanted mice. Transplantation of hfMSCs into E14-16 mdx mice resulted in widespread long-term engraftment (19 weeks) in multiple organs, with a predilection for muscle compared with nonmuscle tissues (0.71% vs. 0.15%, p < .01), and evidence of myogenic differentiation of hfMSCs in skeletal and myocardial muscle. This is the first report of intrauterine transplantation of ontologically relevant hfMSCs into fully immunocompetent dystrophic fetal mice, with systemic spread across endothelial barriers leading to widespread long-term engraftment in multiple organ compartments. Although the low-level of chimerism achieved is not curative for DMD, this approach may be useful in other severe mesenchymal or enzyme deficiency syndromes, where low-level protein expression may ameliorate disease pathology.

The dystrophin gene, which is defective in Duchenne muscular dystrophy (DMD), also encodes a number of smaller products controlled by internal promoters. Dp71, which consists of the two C-terminal domains of dystrophin, is the most abundant product of the gene in non-muscle tissues and is the major product in adult brain. To study the possible function of Dp71 and its expression during development, we specifically inactivated the expression of Dp71 by replacing its first and unique exon and a part of the concomitant intron with a beta-galactosidase reporter gene. X-Gal staining of Dp71-null mouse embryos and tissues revealed a very stage- and cell type-specific activity of the Dp71 promoter during development and during differentiation of various tissues, including the nervous system, eyes, limb buds, lungs, blood vessels, vibrissae and hair follicles. High activity of the Dp71 promoter often seemed to be associated with morphogenic events and terminal differentiation. In some tissues the activity greatly increased towards birth.

During a gene trap screen, an insertion of the gene trap vector into the dystrophin gene, creating a new allele for the Dmd gene, has been discovered. Because the ROSA beta geo vector was used, the new allele is called Dmd(mdx-beta geo). The insertion occurred 3' of exon 63 of the dystrophin gene, resulting in a mutation that affects all presently known dystrophin isoforms. In contrast to spontaneous or ENU-induced alleles, Dmd(mdx-beta geo) can be used to follow dystrophin expression by staining for beta-galactosidase activity. The high sensitivity of this method revealed additional and earlier expression of dystrophin during embryogenesis than that seen previously with other methods. Dystrophin promoters are active predominantly in the dermamyotome, limb buds, telencephalon, floor plate, eye, liver, pancreas anlagen, and cardiovascular system. Adult Dmd(mdx-beta geo) mice show reporter gene expression in brain, eye, liver, pancreas, and lung. In skeletal and heart muscle, beta-galactosidase activity is not detectable, confirming Western blot data that indicate the absence of the mutant full-length protein in these tissues. Hemizygous Dmd(mdx-beta geo) mice show muscular dystrophy with degenerating muscle fibers, cellular infiltration, and regenerated muscle fibers that have centrally located nuclei. Some mutant animals develop a dilated esophagus, probably due to constriction by the hypertrophic crura of the diaphragm.

Alpha dystroglycan (156 kDa DAG) and beta dystroglycan (43 kDa DAG) are encoded by the same gene and are components of the dystrophin-associated membrane glycoprotein complex. The dystroglycans together with dystrophin form a link between the extracellular matrix and the intracellular cytoskeleton of the muscle fibre. Using in situ hybridisation to mRNA in embryo sections we have examined the expression of the mouse dystroglycan gene. Dystroglycan transcripts are ubiquitously expressed throughout development but are most abundant in cardiac, skeletal and smooth muscle and in ependymal cells lining the developing neural tube and brain. The expression patterns of dystroglycan and dystrophin overlap in major muscle systems during development, suggesting that the dystrophin-dystroglycan complex plays an important role during myogenesis. In contrast, the major sites of utrophin expression do not co-localize with those of dystroglycan suggesting that utrophin may interact with a distinct membrane-associated complex in these non-muscle sites. In mdx embryos the pattern of distribution of dystroglycan mRNA remains unchanged, as do those of utrophin and apo-dystrophin mRNAs. This observation implies that the observed changes in the relative abundance of DAGs and utrophin in dystrophin-deficient muscle occur post-transcriptionally.

Dissociated interosseus muscle fibres of wildtype and mdx mice were investigated to characterize acetylcholine (ACh) receptors with the single channel recording patch-clamp technique. On the muscle fibres of mdx mutants, two types of nicotinic acetylcholine receptor (nAChR) channels were found. One channel (29 pS, mean open time 2.1 ms) resembles channels on denervated and embryonic muscle and was not found on wildtype muscle where exclusively a 48 pS channel (mean open time 1.3 ms) was seen.

We previously reported that differentiating G0 myotube cells cultured from normal chicken embryos exhibit a histone synthesis pattern that is highlighted by transitions in the expression of the minor replacement variants H3.3 and perhaps H2A.Z (Wunsch and Lough, Dev. Biol. 119 (1987) 94-99). Because these proteins may be synthesized to maintain chromatin structure during the differentiation and maturation of the skeletal muscle fiber, it was of interest to determine whether they are made at normal levels during the differentiation of dystrophic muscle. To this end, the synthesis of histone proteins in cultured myoblasts and myotubes from normal and dystrophic avian embryos has been characterized by two-dimensional polyacrylamide gel electrophoresis and fluorography. Proliferating myoblasts (day 1) as well as two stages of differentiating myotubes (days 3, 4) exhibited histone synthesis patterns that were indistinguishable when comparing normal and dystrophic cells. It is noteworthy that this study also revealed that, in both cell types, the change in H2A.Z synthesis during the myoblast/myotube transition was remarkable, increasing from approximately 20% of the non-ubiquitinated H2As in myoblasts to 80% in myotubes. Also, gel staining patterns and immunoblotting detected no differences in the degree of histone ubiquitination between normal and dystrophic cells. These findings indicate that, up to this point in dystrophic differentiation, neither the synthesis nor ubiquitination of histones are perturbed.

Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is encoded by a 14 kb transcript of over 65 exons. A point mutation in the homologous mouse gene causes muscular dystrophy in mdx mice. We have examined the developmental regulation of transcription of this gene in skeletal mouse muscle and also the tissue specificity of the transcript in muscle and brain, by using the polymerase chain reaction to amplify overlapping segments of dystrophin mRNA spanning the entire coding sequence and 5'-untranslated region. We have characterised a specific embryonic transcript that would encode dystrophin with a different C-terminus and have shown that this persists from the earliest stages to the adult in mdx skeletal muscle. The brain transcript shows striking sequence homology to rat and human, being highly conserved at the 5'-untranslated region and is present in both wild-type and mdx mice.

Intraspecific chick/chick chimaeras were prepared by transplanting thoracic somitic mesoderm from donor chick embryos with hereditary muscular dystrophy to replace extirpated brachial somites of normal host embryos at stage 13 (48-52 h in ovo). Since the wings of unoperated dystrophic embryos exhibit significantly reduced motility between day-10 in ovo (day-10E) to day-15E, this parameter was used as a marker both to verify the viability of the transplant and to assess if the dystrophic phenotype of impaired functional activity is preserved in the mutant wing muscles innervated by brachial nerves of normal embryos. Our motility analyses of the chimaeras confirmed that transplanted thoracic somitic mesoderm gives rise to brachial musculature and that the experimental muscles maintained the inherent dystrophic phenotype.

Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.

Myelin-forming Schwann cells in the peripheral nervous system characteristically surround and myelinate only single axons. Polyaxonal myelination is an anomaly of this one-to-one relationship whereby one normal-appearing Schwann cell myelinates multiple axons. We examined the ventral roots and the proximal sciatic and posterior tibial nerves of developing normal mice and of dy2J/dy2J dystrophic mice with proximal failure of myelination. Polyaxonal myelination was a rare feature in normal nerves. Examples of polyaxonal myelination were observed six times more often in dystrophic than in normal mice and were most abundant in proximal sciatic nerves. Polyaxonal myelination could result from either an axonal or a Schwann-cell abnormality, or it may be the nonspecific response of uncommitted Schwann cells to an early failure of myelination.

During development ex ovo, the avian mutant with an hereditary form of muscular dystrophy demonstrates biochemical, histochemical, and physiological (functional) abnormalities which may result from impaired muscle-nerve interaction. To investigate if impaired functional activity also characterizes the dystrophic process during development in ovo, limb motility, an index of embryonic functional muscle-nerve interaction, was compared between normal and dystrophic embryos from day 6E through day 16E. A highly significant reduction in this parameter was exhibited by dystrophic wings from day 11E to day 14E inclusive. In contrast, genotypically dystrophic hind limbs demonstrated values equivalent to normal legs. Thus, in the dystrophic embryo, impaired muscle-nerve interaction characterized the brachial region exclusively during a specific period of embryogenesis.

The analysis of hereditary neuro-muscular diseases in the mouse and in other vertebrates may contribute to our understanding of the developmental interactions between spinal cord and sceletal muscle. Meaningful biochemical analysis must be preceded by "biological mixing experiments" to demonstrate whether a given mutation is cell-autonomous, and, if so, which cell type caries the primary defect. Techniques are available to carry out the critical experiment in vivo (artificial chimaeras) or in culture.

The amino acid composition of all collagen types present in the gastrocnemius muscle of dystrophic chick embryos showed an altered profile at both day 14 and day 20 in ovo when compared with the controls. The changes observed at both day 14 and day 20 in ovo suggests that there is a removal of polar side-chains in dystrophic collagen and substitution with non-polar amino acids. The amino acid composition data between day 14 and day 20 indicated: (a) a decrease in hydroxylation (hydroxyproline and hydroxylysine) with a concurrent increase in proline and lysine and a decrease in the levels of arginine; (b) the levels of glycine and alanine did not change with age; and (c) the ratios of glycine to hydroxyproline and proline to hydroxyproline changed significantly in all dystrophic collagen types between day 14 and day 20. Contrast analysis results clearly showed that the changes in amino acid composition observed in each dystrophic type of collagen between day 14 and day 20 were not due to the effect of aging but to some other factor(s). This study provides more evidence that a problem lies in the biosynthesis of collagen present in developing muscles of dystrophic chick embryos, particularly with respect to the transcription or translation of procollagen genes and/or a failure in the processing and differentiation of collagen types.

Abnormalities have previously been reported in the pectoral muscle of embryos and young chicks from a pure strain of New Hampshire Red chickens homozygous for inherited muscular dystrophy. Fine structural studies of the musculus complexus in normal and dystrophic embryos were undertaken because of a sharp decrease in hatching by the diseased birds. Ultrastructural differences found between the normal and dystrophic embryos included a leached sarcoplasm, swollen and distorted mitochondria and tubular components, a lack of polyribosomes (myosin synthesis), and the formation of pseudostraps during differentiation of the myopathic hatching muscle. These differences may curtail differentiation until a point after the critical hatching time.

Creatine kinase (CK) and acetylcholinesterase (AChE) were followed during the course of development of embryonic muscle in tissue culture for periods of up to 8 weeks. Control muscle cultures released CK and AChE into the medium for the first 2-3 weeks, after which time measurable enzyme release ended. In dystrophic muscle cultures release of CK and AChE continued over most of the 8-week culture period. Cumulative measurable activity of CK was significantly greater than that of controls by the 3rd week and thereafter and cumulative AChE release was greater than that of controls by the 5th week and averaged about twice that of controls by the end of the culture period. Total cell CK activity of the dystrophic muscle cultures was greater than control values but results of cellulose acetate electrophoretic analysis of CK isozyme composition indicated that control muscle cultures attained higher percentage levels of MM isozyme type and lower levels of MB isozyme. Breast muscle extracts from adult dystrophic chickens had an approximately 10--13% content of MB isozyme while in similar extracts of control chicken muscle only the MM isozyme was detectable and total activity was greater than in dystrophics. Bound AChE activity of dystrophic muscle was significantly greater than control levels subsequent to the 2nd week in culture. Soluble AChE activity of dystrophic muscle was somewhat greater than comparable control activity in 2 of 3 tissue culture series at approximately the same period. In 7--10 week-old dystrophic chickens, both soluble and bound AChE activities of dystrophic breast muscle extracts were markedly increased over control values.

Early aspects of in vivo sarcomere organization were studied using normal and dystrophic New Hampshire chick embryos. Brachial somites 17-22 were removed from stages 16 through 28 embryos, processed for electron microscopy, and analyzed. Comparisons of normal and dystrophic material disclosed that thick filaments appeared later in dystrophic myotomal cells. This was correlated with a similar delay in the appearance of long polyribosomes. By stage 28, normal myotomal cells contained well-defined sarcomeres, whereas dystrophic sarcomeres were frequently unorganized, with myofilaments in poor longitudinal alignment. Periodic tubules were poorly developed or lacking.

Growth patterns during myogenesis were observed in vitro using normal and dystrophic New Hampshire Red chick embryos. The pectoral muscles from 6 d old embryos were removed, dissociated and grown in culture. Smaller muscle straps, both in size and number, as well as pseudostraps were seen in cultures from the myopathic embryos. There was a 1 1/2 d delay in the appearance of thick myosin-like myofilaments as well as large polyribosomes actively synthesizing myosin in the dystrophic muscle cells. Myosin may be a necessary constituent during myogenesis for the occurrence of normal fusion and thus myotubes.

Extracts from normal embryonic chick brain act as "trophic" agents in the newt blastemata assay where they stimulate the incorporation of amino acids into protein. Equivalent extracts prepared from the brains of dystrophic (am/am) chick embryos are apparently devoid of any equivalent activity.

A unique lipid, ethanolamine plasmalogen, is not only present abnormally in the plasmalemmae of erythrocytes from ex ovo dystrophic chickens but is also present abnormally in the membranes from embryonic myoblasts of in ovo dystrophic chickens.

This paper presents an ultrastructural study of the changes occurring within the tendon and at the insertion of the developing gastrocnemius of White Leghorn chickens and the dystrophic lines 413 and 423 from Davis, CA. The study revealed that the Davis lines contained abnormalities in these areas as early as 13 d in ovo with progressive deterioration to 19 d in ovo. The fibroblasts, collagen, and myofibers showed significant alterations as early as 13 in ovo. Fibroblasts contained abnormal mitochondria and altered Golgi bodies. At day 19, many were ruptured. The average diameter of collagen fibrils was smaller in the dystrophic chickens and myofibers showed a variety of alterations, some of which were severe. The details of these alterations are described and their possible relationship with the etiology of genetic muscular dystrophy is discussed.

Previous analyses of experimental chick embryos of normal lineage demonstrate the inability of brachial muscles to sustain a successful union with foreign nerves derived from a thoracic neural tube segment transplanted to the brachial region at day 2 in ovo (day 2E). The present experiments were performed to determine if mutant chick embryos afflicted with hereditary muscular dystrophy would respond similarly to this experimental manipulation. Using the same criteria applied to our analysis of experimental normal embryos, our results demonstrated that dystrophic brachial muscles were capable of maintaining a compatible union with foreign thoracic nerves throughout the experimental period analysed. Significant muscle growth occurred, intramuscular nerve branches were maintained, motor endplates formed and wing motility was equivalent to that of unoperated dystrophic embryos. Thus, foreign nerves rejected by normal brachial muscles were accepted by brachial muscles of the mutant dystrophic embryo.

Motor neuroblasts comprising the brachial segments of the lateral motor column in the dystrophic chicken were counted at 6, 7, 9, 12, and 18 days of incubation and at 5 days' post-hatching. Comparisons with similar counts from normal chick embryos disclosed that there was a significantly greater depletion of motor neurons in normal embryos between days 9 and 12. These findings suggest that nerve-muscle interaction may be altered early in the development of the motor unit in the dystrophic chick embryo.

We have examined the development of the brachial lateral motor column in White Leghorn chickens that are homozygous for muscular dystrophy. We found an accelerated loss of motor neurons between days 6 and 11 in ovo in the dystrophic embryos such that at the end of this time they had only 80% of the population in age-matched controls. After day 11 in ovo the rate of motor neuron loss was the same in both normal and dystrophic birds. To determine whether the accelerated motor neuron loss was due to expression of the dystrophic gene within the spinal cord itself or whether it was secondary to abnormalities in some other tissue, we exchanged the brachial region of the spinal cord between normal and dystrophic embryos at 2 days in ovo, allowed the resulting chimeras to develop until 11 days in ovo and estimated the motor neuron population in the transplanted segment of cord. We found that spinal cords transplanted into normal hosts had significantly higher populations of motor neurons than spinal cords transplanted into dystrophic hosts. We concluded that the accelerated motor neuron loss seen in dystrophic birds is not intrinsic to the cord but is influenced by other tissues in the embryo.