Differentiation of muscle fiber types in the chicken hindlimb

The differentiation of myotubes into fiber types was studied by examining the ATPase staining characteristics of chicken embryo thigh muscles. Two distinct fiber types, designated type IEMB and IIEMB, could be distinguished as early as stage 29. Paralysis of the embryo with d-tubocurarine prevented the differentiation of type IEMB but not type IIEMB characteristics. The two embryonic fiber types differed from each other, and mature type I and II fibers, in the acid and alkali labilities of their ATPases. Myotubes which were type IEMB at stage 29 matured into type I fibers, whereas those which were type IIEMB predominantly but not exclusively developed into type II fibers. The process of maturation involved sequential changes in the staining characteristics of the myotubes. Thus, the ultimate fiber type of a myotube can be detected long before it expresses its mature characteristics.     

Distribution of fiber types in embryonic chick limb muscles innervated by foreign motoneurons

The differentiation of distinct myotube fiber types in chick limb muscle development is coincident with innervation. The role of motoneurons in influencing fiber type differentiation was analyzed by causing chick hind limb muscles to be innervated by inappropriate motoneurons and then examining experimental muscles for changes in the distribution of myosin ATPase fiber types. Motoneuron innervation of limb muscles was altered by performing either limb shifts, limb reversals, or large spinal cord reversals on early neural tube or limb bud stage chick embryos. The distribution of fiber types was then analyzed in muscles from stage 36 (E10) to stage 45 (E20) embryos after processing hind limb sections for myosin ATPase histochemistry. In the majority of experimental muscles examined (267/312), the distribution of myosin ATPase fiber types was unaltered. In the remaining experimental muscles (14%), alterations in the distribution of myosin ATPase fiber types occurred, indicating that in some cases, foreign innervation may alter the developmental program of differentiating myotubes. The results suggest that myotubes differentiate myosin ATPase staining characteristics according to an intrinsic program and that these differentiating myotubes are selectively innervated by motoneurons of the appropriate type under most conditions including normal development. Under exceptional circumstances of motoneuron-muscle fiber type mismatch, embryonic motoneurons can alter fiber type expression.     

Differentiation of the thyroid in the hypophysectomized chick embryo

The influence of hypophyseal hormones on the normal development of the thyroid gland was investigated by decapitating chick embryos at a time before the hypophysis has begun to form. Thyroids from decapitated embryos were compared with thyroids of normal embryos from shortly after decapitation to the time when the embryo should hatch. The thyroids were examined for ultrastructural differences and for accumulation of thyroglobulin. Thyroxine levels in the blood were also examined. It was concluded that the thyroids in decapitated embryos develop normally up to about 12 days of development, by which time morphogenesis and differentiation of the gland is essentially complete. The rates of thyroid growth and accumulation of thyroglobulin are diminished after 12 days of development. The amount of thyroxine in the blood is decreased by about one-half. Thyroxine was found in the yolk of unincubated eggs at levels sufficient to sustain blood thyroxine levels throughout embryonic development. We conclude that hormones that are synthesized by the embryonic hypophysis affect thyroid development only through their influence on generalized growth and metabolic activity.     

Distribution of muscle fiber types and EMG activity in cat intercostal muscles

The electromyogram (EMG) activity and histochemical properties of intercostal muscles in the anesthetized cat were studied. The parasternal muscles were consistently active during inspiration. The external intercostals in the rostral spaces and the ventral portions of the midthoracic spaces were also recruited during inspiration. The remaining external intercostals were typically silent, regardless of the level of respiratory drive. The internal intercostal muscles located in the caudal spaces were occasionally recruited during expiration. There was a clear correlation between recruitment patterns of the intercostals and the histochemically defined fiber type properties of the muscles. Intercostal muscles that were routinely recruited during inspiration had a significantly higher proportion of slow-oxidative muscle fibers.     

Calcium in the developing skeletal muscles of the chick embryo

The osmium-pyroantimonate technique was used for the ultrastructural study of Ca2+-localization in two types of chick embryo skeletal muscles: m. pectoralis and m. soleus. In 8- and 12-day old embryos the pyroantimonate precipitate was found on plasmalemma, condensed chromatine and ribosomes and in N-lines of I-band. During myogenesis (15-, 21-day old embryos) the calcium precipitate is redistributed from the above mentioned sites to terminal cisternae and N-line of I-band. It is proposed that calcium of N-lines may be involved in the glycogenolysis, its association with the muscle contraction occurring particularly at early developmental stages.     

Histochemical determination of muscle fiber types in locomotor muscles of anuran amphibians.

1. A histochemical study using myosin ATPase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase reactions and a morphometric analysis with image analyser, was carried out in sartorius and gastrocnemius muscles of two anuran species, Rana perezi and Bufo calamita, that show different locomotor activities. 2. Four types of muscle fiber were found. There were interspecific variations in their proportions, with a predominance of oxidative muscle fibers in Bufo calamita. 3. These results agree with those obtained previously for the metabolic profile of several tissues from both species and point to a clear metabolic basis for the differences in locomotor activities between these two species.     

Kinetics of chick embryo cell types in culture

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.     

Origin of the iris sphincter muscle in chick embryo

The origin of the iridial sphincter muscle in chick embryo was investigated by means of immunohistochemistry. Desmin immunoreactive cells are shown in the mesenchymal stroma overlying the anterior epithelial layer of the iris in 4 1/2-day chick embryos. In 9-11-day chick embryos also some cells of the posterior epithelium near the pupillary margin, and of the iridial lamella show a slighter desmin-immunoreactivity. This finding agrees with a double origin of the iridial sphincter muscle: an early mesenchymal one and a later epithelial other.     

Glycogen metabolic enzymes in the skeletal muscles of the developing chick embryo

In the skeletal muscles of the chick embryo from the 10th till the 15th day of embryogenesis, phosphorylase (EC. 2.4.1.1) is represented by two isozymes one of which corresponds, by electrophoretic mobility, to the liver phosphorylase and another to phosphorylase of the skeletal muscles of the adult rat. From the 17th day of embryogenesis on only one isozyme of phosphorylase is found in the skeletal muscles which is identical with that of the skeletal muscles of the adult bird. The isozyme spectrum of phosphorylase of the whole 4 days old embryo contains, besides phosphorylase L, a special "embryonic" isozyme which differs from that of the skeletal muscles by immunochemical characteristics and electrophoretic mobility. From the 10th day of embryogenesis till hatching, the activity of phosphorylase of the skeletal muscles increases more than 50 times and that of glycogen synthetase (EC. 2.4.1.11) only 4 times.     

Differentiation of fiber types in wing muscles during embryonic development: effect of neural tube removal

The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.     

Pyruvate dehydrogenase activation in the skeletal muscles of the chick embryo during electrostimulation

The pyruvate dehydrogenase activity in the skeletal muscle during chicken embryo-genesis and early postnatal life amounts to 550 +/- 50 mU per g tissue, on the average; electrostimulation (1 Hz) induced a two-fold increase in its activity starting from the 15th day of incubation.     

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

Summary 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.Supported in part from institutional funds from the College of Osteopathic Medicine and Surgery and the National Osteopathic Foundation     

Differentiation in the early chick embryo: effects of bromodeoxyuridine on erythropoiesis

Organ cultures of chick embryos at the definitive streak stage will normally show the first appearance of hemoglobin in erythroid cells after 20 hr of culture. Addition of 5-bromodeoxyuridine (BrdU) will prevent hemoglobin formation when added at the beginning of culture, but the tissue becomes resistant to the inhibitor when addition is delayed for approximately 10 hr. The inhibitory effect of BrdU is canceled or reversed if thymidine replaces BrdU at the beginning of culture or later. Transfer to thymidine containing medium even after 20 hr permits hemoglobin formation to occur at almost the normal time, thus making it unlikely that a complete cell cycle or DNA replication during a complete S period is required for the reversal of inhibition.Even when the appearance of cytologically detectable hemoglobin is inhibited by BrdU, some globin is synthesized and RNA sequences specific for globin are present, but in decreased amount, unless the inhibitor is given very early. BrdU does not affect the synthesis of any particular RNA species or of polyadenylic acid, but it does lower the rate of uptake of adenosine into the ATP pool. While not affecting cell cycle times in cell cultures, BrdU greatly reduces cell numbers.