Dystrophic Chick Embryos Research Articles

Dystrophic chick brain lacks a particular neurotrophic factor

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.

Increased viability and differentiation of normal and dystrophic striated muscle in vitro.

Primary cultures of muscle from normal (line 412) and dystrophic (line 413) chick embryos were exposed to corticosterone-21-acetate (C-21-A) or sodium ibuprofen (Motrin) for 28 d after myotube formation. Ibuprofen (0.5 to 500 micrograms/ml) or C-21-A (0.4 to 40 micrograms/ml)-treated cultures were fixed and assessed semiquantitatively using phase microscopy. On this basis, ibuprofen (50 micrograms/ml) and C-21-A (40 micrograms/ml) seemed to be effective in maintaining both normal and dystrophic muscle cultures. Using ibuprofen and C-21-A at these concentrations, experiments were repeated and analyzed quantitatively. Ibuprofen maintained culture viability (up to 68% more myotubes than untreated controls) but had no significant effect on the number of striated cells. C-21-A effectively maintained culture viability (up to 73% increase) and strongly promoted the formation of striated cells in these cultures (up to a sixfold increase). Both normal and dystrophic cultures were affected similarly by these agents, but the dystrophic cultures showed more consistent if not more extensive improvements in the parameters examined here. Thus, it seems that ibuprofen and C-21-A may affect both normal and dystrophic muscle directly to maintain survival and even promote differentiation.

Connective tissue metabolism in muscular dystrophy. Early amino acid changes in collagen types isolated from the gastrocnemius muscle of developing dystrophic chicken embryos

1. 1. 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. 2. 2. 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. 3. 3. 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. 4. 4. 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). 5. 5. 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.

Developmental neuron cell death in dystrophic and normal chickens

The numbers of motoneurons in the brachial lateral motor columns were determined from the 6th embryonic day through the 5th week posthatching in age-matched dystrophic and normal chickens. The overall magnitude of cell loss was not significantly different between the dystrophic and normal strains. However there was a trend toward more motoneurons in the brachial lateral motor columns of dystrophic embryos and posthatch chickens which was found to be statistically significant at the 8th and 9th days of embryonic life. These observations are taken to suggest that, in addition to strain differences unrelated to the dystrophic gene, there may exist a temporal delay in developmental neuron cell death in dystrophic chick embryos that may be secondary to the primary defect within the dystrophic muscle.

Turnover of myofibrillar proteins in cultured muscle cells from normal and dystrophic chick embryos

Rates of protein degradation and synthesis were determined in myofibrillar and non-myofibrillar fractions and in myofibrillar subunits of cultured muscle cells from normal and dystrophic chick embryos of genetically closely related lines. Growth characteristics of normal and dystrophic cells were identical as measured by DNA, RNA and protein accumulation. Degradation rates of myofibrillar and non-myofibrillar protein determined from label-chase experiments were the same in normal and dystrophic cultures. In similar experiments in which degradation rates of eight different components of the myofibrillar fraction were measured, a spectrum of degradation rates was obtained indicating that myofibril components turn over individually or in groups, rather than as an intact unit. Myosin heavy chain was the slowest turning over component of those measured, while actin was among the most rapid. No differences were found between normals and dystrophics in the turnover of any of the components. Fractional rates of protein synthesis were also determined for the myofibrillar and non-myofibrillar fractions of cultured cells. The rates obtained with normal and dystrophic cells were indistinguishable and were the same for the myofibrillar and nonmyofibrillar fractions. Initial amino acid incorporation rates into 17 separate components of the myofibrillar fractions were compared in normal and dystrophic cells. No significant differences were found.

Similarities in protein synthesis and degradation in normal and dystrophic muscle cultures

Protein synthesis and degradation were compared in cultured muscle cells obtained from normal and dystrophic chick embryos under conditions where labeled amino acid reincorporation was not a complicating factor, where fibroblast contamination was minimized, and where the animals compared were as genetically similar as possible. Under these conditions both cell types exhibited a half-time of protein turnover of 34 h. Degradation in both was inhibited 21% by leupeptin (50 μg/ml), and both showed parallel increases in degradation rates under ‘step-down’ conditions.

Motoneuron Formation in the Brachial Spinal Cord of the Dystrophic Chick 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.

Trophic activity of sheep sciatic nerve extracts in skeletal muscle cultures from normal and dystrophic chick embryos: Failure of dystrophic muscle to respond

Chickens with hereditary muscular dystrophy have been reported to have abnormalities in acetylcholinesterase (AChE) activity and therefore muscles of this genotype may not be able to respond to neural influences. We compared the trophic control of AChE activity in muscle cultured from dystrophic embryos with that from normal embryos. With standard culture media, no differences were noted in the development of AChE activity in muscle cultured from normal and dystrophic embryos. Addition of culture media containing a neural extract prepared by ammonium sulfate precipitation of sheep sciatic nerve consistently produced a significant increase in AChE activity in normal but not in dystrophic muscle. The data, in addition to showing that trophic activity is present in ammonium sulfate-precipitated fractions of sheep sciatic nerve, demonstrate an inability of muscle of the dystrophic genotype to respond to a neurotrophic influence. Under standard culture conditions, skeletal muscle of the dystrophic genotype has the capability to synthesize AChE, and therefore the failure to respond to a trophic influence may indicate the absence of a receptive mechanism for detecting the mediator(s) of the neurotrophic effect.

An analysis of the integrity of the brachial motor unit in the dystrophic chick embryo.

In New Hampshire chickens, the primary clinical symptom of dystrophy is limitation of wing motility. Examination of the brachial-level motor unit in chick embryos homozygous for dystrophy reveals abnormalities in both muscular and neural components. Wing motility in these embryos is abnormal as early as six days, and there is a corresponding lack of differentiation of the pectoralis major muscle. The findings suggest that delayed development of brachial-level neuronal pathways is responsible for the decreased wing motility and early degeneration of the pectoral muscle.

Effect of leupeptin of protein turnover in normal and dystrophic chicken skeletal muscle cells in culture

The effect of leupeptin on turnover of the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain was evaluated in muscle cell cultures. Cultures were prepared from the breast muscle of 12-day normal (white leghorn) and dystrophic (line 307) chick embryos. After 7 days in culture, cells were labeled for 16 hr with 1 μCi/ml of [ 3H]Leu and then “chased” for an additional 24 hr in culture medium containing no [ 3H]Leu and 0–75 μg/ml leupeptin. Cultures were analyzed for radioactivity in the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Leupeptin (75 μg/ml) virtually eliminated loss of radioactivity from the soluble protein fraction, but only minimally affected loss of radioactivity from the myofibrillar fraction or myosin heavy chain. Normal and dystrophic muscle cells responded identically to leipeptin treatment. Thus, the muscle proteases that are specifically inhibited by leupeptin seem to have no major role in initiating myofibrillar protein turnover.

Altered protein synthesis and creatine kinase in breast muscle cell cultures from dystrophic chick embryos: Corrective influence of leupeptin, pepstatin and diphenylhydantoin

The total protein synthesis (TPS), myosin synthesis (MS) and creatine kinase (CK) levels in muscle cell cultures obtained from 400 normal (strain 454) and 400 dystrophic chick embryos (strain 455) were investigated. The cultures were obtained from breast muscles of 12 day chick embryos by dissociation in 0.25% trypsin, preplating and plating of 5 × 10 5 floating cells on gelatin coated dishes in Minimal Essential Medium, 10% horse serum and 2% chick embryo extract. After 6 days, when electron-microscopic studies demonstrated good muscle differentiation, cell cultures were labeled with [ 3H]leucine. TPS and MS, respectively, showed 85% and 65% increases in breast muscle cell cultures from dystrophic chick embryos. The half-life times for total protein and myosin from dystrophics were 19 and 32 hr, respectively, as compared with 36 and 48 hr from controls. Noncollagen protein content (NCP) showed 27% decrease in postfusion stage (12 days) of cell cultures from dystrophics. The CK level showed 30% lower values in the cells from dystrophics but 50% higher values in their culture medium. The addition of leupeptin plus pepstatin (50 μg/ml) to these cultures restored NCP content, total protein and myosin turnover to normal values and significantly increased TPS and MS. The addition of diphenylhydantoin (DPH) (20 μg/ml) to cell cultures from dystrophics did not change the NCP content nor the turnover for total protein and myosin but significantly increased TPS, MS and CK while medium CK significantly decreased. The addition of leupeptin plus pepstatin or DPH to muscle cell cultures from normal chick embryos also significantly stimulated TPS and MS.

Creatine phosphokinase in cultured control and dystrophic chick embryo muscle

Myogenesis and development of control and dystrophic chick embryo muscle cells in tissue culture were followed for periods up to 57 days. Control myofibers generally showed earlier and more vigorous contractile activity than dystrophics, and in controls detachment of the center of the cell mat occurred at about 3–4 weeks. CPK activity of control cultures increased to maximum values by 3–4 weeks and then declined. In dystrophic cultures, CPK activities increased to levels somewhat greater than controls before declining, and maximum levels occurred over a wider time range. In the medium from dystrophic cultures, CPK activity increased markedly, when cell CPK activity was rapidly declining. Maximum medium CPK activities in dystrophics were, on the average, two and one-half times greater than any control medium value. In controls there was no consistent correlation between maximum medium CPK activity and any phase of cell CPK activity. There were no marked differences, in DNA and protein content, between control and dystrophic cultures, and these parameters primarily reflected the fibroblast component of the culture. None of the differences between control and dystrophic cultures was markedly affected by the proportion of myoblasts to fibroblasts in the original tissue culture inoculum. These results are discussed in reference to the validity of determining CPK activity as a quantitative approach to investigating dystrophic muscle in tissue culture.

Multiunit activity patterns in the brachial spinal cord of dystrophic chick embryos

The frequency of polyneuronal burst discharges was examined in different regions of the cervical spinal cord (segments 14 and 15) of normal and dystrophic chick embryos from days 18 to 20 of incubation. On the dorsoventral axis in a midlateral position, the pattern of change in burst frequency was quite different in normal and dystrophic embryos. Although bursts were encountered in each, in approximately the same region and at about the same frequency, significant declines in frequency were encountered only in deeper regions of the spinal cord of dystrophic embryos. In normal embryos, bursts maintained their frequency independently of the position of the sampling electrode. If these frequency declines in dystrophic cord are associated with changes in the pattern of motor outflow from this region, they may have consequences in subsequent muscle differentiation.

Myogenic defect in muscular dystrophy of the chicken

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at 3 1 2 days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.

Myogenic defect in acetylcholinesterase regulation in muscular dystrophy of the chicken.

To determine whether inherited muscular dystrophy of the chicken is neurogenic or myogenic in origin, limb buds from homozygous normal and dystrophic chick embryos were exchanged prior to muscle differentiation and innervation. Biceps muscles of hatched chicks, in which muscle of the donor was innervated by nerves of the host, were analyzed for embryonic properties of muscle acetylcholinesterase and for fiber diameter, two distinctive markers for expression of the dystrophic gene. The results indicate that muscular dystrophy of the chicken is caused by an initial biochemical lesion in the limb and its muscle rather than in its innervating nerve.

"Trophic" influences of nerve on muscle.

"Trophic" influences of nerve on muscle.

Growth and metabolism of normal and genetically dystrophic chick embryo cells on a phosphate buffered medium.

SummaryA phosphate buffered physiological saline useful in formulation of tissue culture media and solutions is described. Data concerning the growth of cells from the pectoral region of 14-day-old normal and genetically dystrophic chick embryos on medium using the phosphate buffered saline are presented. The rate of protein synthesis of the cultures was log-linear but slower than the rate of cell division. Cells tended to form lipid globules during growth without losing their capacity to divide. No differences were found between the free amino acids, the division rates, the peak populations and the protein synthesis rates of cells from normal and dystrophic lines.

ENZYME ACTIVITIES ON THE DEVELOPING MUSCLE AND LIVER OF NORMAL AND GENETICALLY DYSTROPHIC CHICK EMBRYOS.

SummaryActivities of succinic dehydroge-nase, cytochrome c oxidase and glutamic oxaloacetic transaminase (GOT) have been assayed in embryos and day-old chicks from a genetically dystrophic and normal strain of New Hampshire chicks. Results point out possible defects in the cytochrome c oxidase and succinic dehydrogenase systems as the site for the metabolic disturbance in the hereditary muscular dystrophy in chicks.