Philip H. Bonner

Clonal analysis of vertebrate myogenesis. I. Early developmental events in the chick limb.

Abstract Early developmental events occurring in the prospective muscle tissue region of chick embryo leg buds have been subjected to an in vitro clonal analysis. Colony-forming cells are present at stage 20 (72 hr incubation), but none of the colonies exhibit morphological signs of muscle differentiation. After an additional 8 hr of incubation (stage 21), approximately 10% of the colony-forming cells have acquired the capacity to form multinucleated cells in vitro , and the percentage of clonable myoblasts increases to a level of approximately 60% during the next 3 days of incubation. Clonal analysis of myoblast populations within regions of the developing limb have indicated that, between stages 21 and 27, the dorsal and ventral segments of the myogenic region contain appreciably more clonable muscle cells than the anterior and posterior segments. In addition, during stages 21 and 22 there is a 3-fold difference in muscle-colony-forming cells between the proximal and distal halves of the dorsal-ventral segments, as well as between the proximal and distal halves of the anterior-posterior segments. Thus at least two temporal and regional gradients—proximal to distal and medial to lateral—of clonable myoblast content can be delineated within the developing chick limb. In addition to changes in the proportions of muscle-colony-forming cells, the extent of multinuclearity within individual muscle colonies increases with the developmental age of the embryo from which the clonable myoblasts are derived. The progressive changes in the relative proportions of muscle-colony-forming cells and in clonal morphology are discussed in terms of their possible cell lineage implications.

Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

Abstract The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium. The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.

Nerve-dependent changes in clonable myoblast populations.

Abstract Between the 3rd and 12th days of development at least four distinct classes of clonable myoblast are present in chick embryo leg skeletal muscle (N. K. White, P. H. Bonner, D. R. Nelson, and S. D. Hauschka, 1975, Develop. Biol. 44, 346–361) . In the present study, the behavior of each class has been examined quantitatively by clonal growth and differentiation of cells derived from denervated and normal embryos. Embryos functionally denervated by cauterization of the posterior spinal cord or by injection of the neuromuscular blocking agent d-tubocurarine exhibit changes in the two broad categories of clonable myoblast—fresh medium-sufficient (FMS) and conditioned medium-requiring (CMR) muscle cells. Clonal analysis of cells derived from leg muscle tissue of 10- to 12-day-old embryos denervated early in development (Days 3 to 6) has shown that the proportion of FMS clonable myoblasts is reduced to about 60% of the level found in normally innervated leg muscle. The CMR class is composed of three subclasses: CMR-I, CMR-II, and CMR-III (White et al., 1975) . Clonal analysis of cells from denervated muscle has shown that, while the total CMR clone proportion is unchanged, the three subclasses are rearranged. The proportions of CMR-I and CMR-II muscle clones are greatly increased and the CMR-III subclass is severely reduced or absent when compared to clones derived from normally innervated leg muscle tissue.

Differentiation of chick embryo myoblasts is transiently sensitive to functional denervation

Abstract Clonal analysis of myoblast differentiation has been used to assess effects of denervation on developing skeletal muscle: chick embryo legs denervated by spinal cord cautery yield reduced proportions of clonable myoblasts (P. H. Bonner, 1978, Develop. Biol. , 66, 207–219) . The present work examines the effects on clonable myoblasts of functional denervation by d -tubocurarine. Curare treatment during the third or fourth days of embryonic development had no effect on clonable myoblasts later in development, treatment during the fifth or sixth days resulted in reduced proportions of clonable myoblasts, and treatment during the eighth or ninth days again had no effect. Clonal analysis of treated and control embryo leg muscle cells was performed between Days 10 and 18. Embryos were also permanently denervated by spinal cord cautery late in the sixth day. These embryos showed no effect of denervation on clonable myoblast proportion. It is concluded that the differentiation of skeletal muscle myoblasts is affected by interference with normal nerve-muscle relationships only during a “window” of sensitivity and that this “window” extends approximately from Hamburger and Hamilton stage 27 to stage 30.

Neural induction of chick myoblast differentiation in culture

Abstract Developing chick embryo leg muscle contains a series of clonally distinct myoblasts. One of these myoblast classes, CMR-III, appears during the eighth day of development and, by Day 10, comprises up to 50% of all clonable cells in the leg muscle tissue. Denervation earlier in development prevents the appearance of CMR-III. The nature of neural influence on CMR-III production has been studied with a completely in vitro system which allows functional interaction between cultured neurons and myogenic cells. Spinal cord cells from 4- to 6-day-old embryos are grown on gelatin-coated dishes and, after neuronal differentiation, myogenic cells from the legs of 10- to 12-day-old denervated embryos are added to the cultures. After 48 hr of coculture the cells are removed from the plates and myoblast populations assayed by clonal analysis. Cocultures produce CMR-III myoblasts as efficiently as do normal embryos—up to 50% of all clonable cells—while control cultures remain nearly free of CMR-III. The induction of CMR-III from precursor is inhibited by curare and by physical separation of neurons and myogenic cells. Induction is not mediated by soluble or insoluble, stable or unstable components released by spinal cord cells. Nonneuronal cells from skeletal muscle, liver, adrenal, and heart muscle do not induce CMR-III. It is suggested that contact between neurons and sensitive precursor myogenic cells is required for conversion of precursor into CMR-III.

Muscle type–specific myosin isoforms in crustacean muscles

Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased toward the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.

Botulinum A toxin stimulates neurite branching in nerve-muscle cocultures

In addition to skeletal muscle paralysis, type A botulinum toxin commonly causes sprouting of motor axons in various experimental whole-animal systems. The use of type A botulinum toxin in clinical treatment of muscle spasm disorders is becoming increasingly popular. The eventual, unwanted return of involuntary activity in the treated muscles may be a consequence of such axon sprouting. We have developed a coculture model allowing the quantification of botulinum toxin-induced sprouting that shows promise for future studies on its mechanism and control. Chick embryo ciliary ganglion motor neurons were cocultured with chick leg muscle cells. The presence of type A botulinum toxin in the coculture medium was correlated with significantly increased branching frequency of neurites. Toxin-increased branching frequency occurred even when the neurons and muscle cells were separated from each other on the culture dishes, suggesting a presynaptic effect of toxin. Cocultures incubated in the presence of curare, a post-synaptic blocker, had control levels of neurite branching, ruling out the possibility that simple synaptic blockade causes sprouting but again supporting the hypothesis of a pre-synaptic activity of botulinum toxin.

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae.

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneiders and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.

The oculomotor periphery: the clinician's focus is no longer a basic science stepchild

The study of the oculomotor periphery, the extraocular muscles and their orbital attachments, is undergoing a rapid expansion. This is an important progression for both basic and clinical communities as, for too long, the ophthalmologist has worked primarily in the periphery and the basic researcher has been occupied with study of the central components of the oculomotor system. From recent studies, it is clear that the morphology, cell and molecular biology, and genetics of the eye muscles and their corresponding motoneuron pools, and muscle attachments within the orbit are more complex than has heretofore been appreciated.

Clonal analysis of vertebrate myogenesis: V. Nerve-muscle interaction in chick limb bud chorio-allantoic membrane grafts

Abstract The clonable cell populations of innervated and noninnervated chorio-allantoic membrane (CAM) grafts of early chick embryo leg buds have been compared. Co-grafts of stage 21 or 22 leg buds with stage 22–26 spinal cord segments exhibit a near twofold greater proportion of clonable muscle cells than noninnervated control grafts (leg bud alone). A statistically significant increase is apparent by the 8th graft day. Grafts constructed of stage 17–20 leg buds plus spinal cord and grown for up to 11 days do not exhibit a proportion of myoblasts greater than noninnervated controls. This stimulation of relative muscle cell content suggests a role of motor nerves in regulation of the single-cell population of developing skeletal muscle.