Different subsets of axonal guidance cues are essential for sensory neurite outgrowth to cutaneous and muscle targets in the dorsal ramus of the embryonic chick.

The dorsal ramus nerve diverges dorsally from each spinal nerve to innervate the epaxial muscle and dermis that are derived in situ from each dermamyotome. The outgrowth of both the sensory and motor components of this nerve are sensitive to the proximity of the dermamyotome. Motoneurons display a direct target response that is not dependent upon the concurrent outgrowth of sensory neurites (Tosney: Dev. Biol. 122:540-588, 1987). Likewise, the outgrowth of sensory neurites could be directly dependent on the dermamyotome. Alternatively, sensory neurites could be dependent on motor axons that in turn require the dermamyotome for outgrowth. To distinguish between these possibilities, motor outgrowth was abolished by unilateral ventral neural tube deletion and the patterns of subsequent sensory neurite outgrowth were assessed. The cutaneous nerve branch formed in all cases. In contrast, neither of the epaxial muscle nerves formed in the absence of epaxial motoneuron outgrowth. Furthermore, sensory neurites could not be detected diverging into muscle from the cutaneous nerve or entering muscle via other novel routes. We conclude that motoneurons are essential for sensory outgrowth to epaxial muscle but not to cutaneous targets. It is clear that different subsets of navigational cues guide sensory afferents to muscle and to cutaneous destinations.

Expression of nerve-muscle topography during development.

Previous studies have indicated that in 2 muscles of the adult rat, the anterior serratus and the diaphragm, the rostrocaudal axis of the motoneuron pool projects topographically onto the rostrocaudal axis of the muscle. In the present work we have asked whether this orderly topography emerges as a function of postnatal synaptic rearrangement or whether this pattern is already established at birth. The anterior serratus muscle was studied over the period ranging from embryonic day 17 through postnatal day 30. Using 2 criteria of topography, average segmental innervation and average target field of cervical roots C6 and C7, we found that a topographic distribution of the motoneuron pool is already present prior to birth and maintained throughout the postnatal period. Moreover, both C6 and C7 form an orderly map over the surface of the serratus in the embryo, and the topography is sharpened during postnatal periods. The diaphragm also is topographically innervated at birth and undergoes a comparable sharpening of the projection map postnatally. We conclude that the topographic projection of motoneurons is established prior to birth in these muscles, and postnatal synaptic rearrangement serves to sharpen the topographic map toward the adult pattern. These results also suggest that the pursuit of basic mechanisms underlying topography should be directed toward initial embryonic nerve-muscle contacts.

Formation of the transverse nerve in moth embryos. I. A scaffold of nonneuronal cells prefigures the nerve.

We have studied the embryonic development of the transverse nerve (TN), an unpaired segmental nerve of the moth Manduca sexta. Two identified motor neurons and 16 identified neuroendocrine neurons project axons within the larval TN; therefore, the TN is both a peripheral nerve and a neurohaemal organ. At 33% of embryogenesis, and prior to the arrival of any neuronal growth cones, the position, shape, and trajectory of the TN are anticipated by two groups of nonneuronal cells that we call the strap and the bridge. At this time the strap and the bridge together consist of approximately 100 cells, all of which express a cell surface epitope recognized by the monoclonal antibody TN-1. As development proceeds, both the number of nonneuronal cells within the strap and the bridge and the fraction that expresses the TN-1 antigen(s) decrease. Moreover, individual cells within the strap become morphologically identifiable before the arrival of the neuronal growth cones. Most of the axons that project to the TN also express the TN-1 antigen(s) during their period of outgrowth. The two motor neuron growth cones are the first to reach the environment of the strap and the bridge, doing so at approximately 37%; having encountered these cellular structures, the growth cones restrict their navigation to this preexisting scaffolding, until they reach their muscle target. The neuroendocrine growth cones arrive later and also grow within the confines of the strap and the bridge (J.N. Carr and P.H. Taghert, 1988, Dev. Biol, 130, 500-512). In this first paper we describe the development of the strap and the bridge, and the interactions of the motor neuron growth cones with these structures. The observations are novel in documenting the extent and precision to which a peripheral nerve pathway is prefigured by a contiguous assemblage of nonneuronal cells.

The regulation of intramuscular nerve branching during normal development and following activity blockade.

In vertebrates, approximately 50% of the lumbosacral motoneurons die during a short period of development that coincides with synaptogenesis in the limb. Although it has been postulated that these motoneurons die because they fail to obtain adequate trophic support from the muscles, it is not clear how this factor is supplied. The mechanism by which activity blockade prevents motoneurons cell death is also unknown. In order to begin to understand the nature of these proposed trophic interactions, we have examined the temporal sequence of axonal invasion and ramification within two muscles of the chick hindlimb, the predominantly slow iliofibularis and the fast posterior iliotibialis, during the cell death period. We found striking differences in intramuscular nerve ingrowth and branching between fast and slow muscle. We also observed differences in the molecular composition of fast and slow myotubes that may contribute to the nerve pattern differences. In addition, we observed a progressive increase in the degree of intramuscular nerve fasciculation as well as a precise temporal sequence of nerve branching. The earliest detectable response to chronic curarization was a dramatic decrease in the degree of intramuscular nerve fasciculation. Activity blockade also greatly enhanced nerve branching within the muscles from the time that nerve branches normally formed, and, additionally, interfered with the normal cessation of axon growth. Our results support the idea that nerve endings are the sites of trophic uptake. Furthermore, although our results do not allow us to exclude other activity-dependent influences on motoneuron survival, they suggest the following testable hypotheses: (1) the normal regulation of motoneuron survival may result from the precise control of intramuscular nerve branching, (2) activity blockade may increase motoneuron survival by enhancing intramuscular nerve branching, and (3) anything which affects this complex process of nerve branching may also alter motoneuron survival.

Distinct roles for adhesion molecules during innervation of embryonic chick muscle.

In vitro studies have suggested that the cell adhesion molecules NCAM and G4/L1 contribute to a variety of events during neural development. We have directly tested the role played by these molecules in the process of initial nerve ingrowth and ramification in the embryonic chick iliofibularis muscle by in ovo injections of specific adhesion-blocking antibodies and analysis of the resultant nerve branching pattern in muscle whole mounts. Antibodies against both molecules produced axonal defasciculation, which resulted in an enhanced transverse projection to the fast region of the muscle. In the case of anti-G4/L1, we also observed a large increase in the number of side branches that form from nerve trunks in the slow region and an enhancement of nerve branching in the fast region. Conversely, anti-NCAM produced a striking decrease in both the number and length of side branches in the slow region, and a reduction in nerve branching in the fast region. A similar reduction of nerve branching was obtained following injection of an endosialidase, which removes sialic acid from NCAM, and which was observed to enhance fiber-fiber apposition, presumably by increasing cell adhesion. Based on their biochemical properties in vitro and their in vivo distribution, both NCAM and G4/L1 are in a position to contribute to axon-axon adhesive interactions, whereas NCAM would be expected to also promote axon-myotube interactions. Our observations in fact indicate that these two adhesion molecules play different but complementary roles during muscle innervation and, specifically, that axon-axon fasciculation is influenced by both NCAM and G4/L1 in an anatomically distinct manner to regulate the overall pattern of nerve branching and that NCAM-mediated axon-myotube interactions are necessary for the attainment of the normal stereotyped pattern of nerve branching in both fast and slow regions of this muscle.

Changes in myosin and C-protein isoforms proceed independently of the conversion to singly innervated neuromuscular junctions in developing pectoral muscle.

Changes in contractile protein expression during myogenesis are usually categorized as developmentally programmed or neuronally dependent. Studies on aneurogenic chick embryos indicated that the neuronally dependent phase begins at about Embryonic Day 15, immediately prior to the fetal transition in myosin and C-protein expression. The prime candidate for the neuronal event that induces the fetal transition is the conversion to the adult form of singly innervated neuromuscular junctions (NMJs), which occurs contemporaneously with the fetal transition. Using curare to inhibit the conversion to focal innervation, we find that the fetal transition proceeds unimpaired, demonstrating that there is no causal link between the fetal transition and the conversion to focal innervation. Furthermore, because the doses of curare used inhibit motor activity by more than 80%, the fetal transition can occur in the absence of normal levels of motor activity. These observations show that the fetal transition in ovo is not induced by either a specific change in innervation or use. Rather, the dependence on innervation seems to be a consequence of the need for muscle activity to prevent atrophy, and the fetal transition appears to have characteristics more like the preprogrammed contractile protein transitions that precede it.

Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: partial and complete deletion of the somite.

The development of patterned axon outgrowth and dorsal root ganglion (DRG) formation was examined after partially or totally removing chick somitic mesoderm. Since the dermamyotome is not essential and a full complement of limb muscles developed, alterations in neural patterns could be ascribed to deletion of sclerotome. When somitic tissue was completely removed, axons extended and DRG formed, but in an unsegmented pattern. Therefore the somite does not elicit outgrowth of axons or migration of DRG precursors, it is not a manditory substratum and it is not required for DRG condensation. These results suggest that posterior sclerotome is relatively inhibitory to invasion, an inhibition that is released when sclerotome is absent. When somites were partially deleted, axonal segmentation was not lost proportionally with the amount of sclerotome removed, suggesting that properties that may vary with sclerotome volume (such as diffusible cues) do not play a primary role. Instead, spinal nerves lost segmentation only when ventral sclerotome was deleted, regardless of whether dorsal sclerotome was or was not removed. This strongly suggests that axonal segmentation is imposed by direct interactions between growth cones and extracellular matrices or surfaces sclerotome cells. While DRG tended to be normally segmented when ventral sclerotome was deleted and to lose segmentation when dorsomedial sclerotome was absent, a coordinate loss of DRG segmentation with sclerotome volume could not be ruled out. However it is clear that axonal and DRG segmentation are independent. Observations on a subset of embryos in which the notochord was displaced relative to the spinal cord suggest that the ventromedial sclerotome surrounding the notochord inhibits axon advance. Posterior and ventromedial sclerotome are hypothesized to act as barriers to axon outgrowth due to some feature of their common cartilaginous development. Specific innervation patterns were also examined. When the notochord was displaced toward the control limb, axons on this side made and corrected projection errors, suggesting that the notochord can influence the precision of axonal pathway selection. In contrast, motor axons that entered the limb on all operated sides innervated muscle with their normal precision despite the absence of the somite and axonal segmentation. Therefore, the somite and the process of spinal nerve segmentation are largely irrelevant to the specificity of motoneuron projection.

Development of postsynaptic-like specializations of the neuromuscular synapse in the absence of motor nerve.

It was previously reported that the acetylcholine receptor clusters and acetylcholinesterase appear on embryonic superior oblique muscle cells developing in vivo without motor nerve contacts. The objective of this study was to examine whether some other components of neuromuscular junction also form on muscle cells developing in vivo in the absence of motor neurons. In the present study, postsynaptic specializations such as junctional folds, postsynaptic density and basal lamina were studied in normal and aneural muscles. The superior oblique muscle of duck embryos was made aneural by permanent destruction of trochlear motor neurons by cauterizing midbrain on embryonic day 7; 3 days before the motor neurons normally project their axons into the muscle. Normal and aneural muscles from embryonic days 10 to 25 were processed for electron microscopy. The results indicate that morphological specializations such as junction-like folds, postsynaptic-like density, and basal lamina also develop in the absence of motor neuron contacts. Whether the differentiation of specialized synaptic basal lamina is dependent on the presence of motor neurons was examined by utilizing a monoclonal antibody against heparan sulfate proteoglycan. Immunohistochemical studies indicate that specialized synaptic basal lamina differentiates in the absence of motor neurons. Thus, the mechanism of development of postsynaptic components of neuromuscular junction in this muscle is not dependent on motor neuron contacts. These results also suggest that the postsynaptic cell plays a more active role in synapse formation than previously realized. The results are discussed in relation to the control of synapse numbers by the postsynaptic cell.

Developmental approaches to the analysis of vertebrate central pattern generators.

The isolated spinal cord of the chick embryo is a new preparation for analyzing the neural mechanisms and development of vertebrate motor activity. The embryonic cord can be isolated in vitro during the period of development when antagonist alternation of hindlimb motoneurons matures. The preparation is spontaneously active in vitro generating episodes of motor activity that can be recorded from muscle nerves and the ventral roots. The neural mechanisms responsible for the development and genesis of motor activity are being investigated using intra- and extracellular recording from motoneurons and electrotonic recordings of motoneuron synaptic activity from muscle nerves. The results suggest that alternating motor activity in the isolated chick cord may be generated by a mechanism in which a synaptically induced motoneuronal shunt conductance regulates the time of discharge of flexor and extensor motoneurons.

Formation of primary and secondary myotubes in rat lumbrical muscles.

Numbers of myoblasts, primary myotubes and secondary myotubes in developing rat embryo hindlimb IVth lumbrical muscles were counted at daily intervals up until the time of birth, using electron microscopy. Motoneurone death at the spinal cord level supplying the lumbricals was assessed by counting axons in the 4th lumbar ventral root. Death of the motoneurones that supply the intrinsic muscles of the hindfoot was monitored by comparing the timecourse of development of total muscle choline acetyltransferase activity in control embryos with that in embryos where motoneurone death was inhibited by chronic paralysis with TTX, and by counting axons in the mixed nerve trunks at the level of the ankle at daily intervals. Condensations of undifferentiated cells marking the site of formation of the muscle were seen on embryonic day 15 (E15). Primary myotubes began to appear on E16 and reached a stable number (102 +/- 4) by E17. Secondary myotubes first appeared two days later, on E19, and numbered 280 at the time of birth (E22). The adult total of about 1000 muscle fibres, derived from both primary and secondary myotubes, was reached at postnatal day 7 (PN7) so considerable generation of secondary myotubes occurred after birth. There was a linear correlation between the number of undifferentiated mononucleate cells in a muscle and the rate of formation of secondary myotubes. The major period of motoneurone death in lumbar spinal cord was during E16-E17, when axon numbers in the L4 ventral root fell from 12,000 to 4000, but a discontinuity in the curve of muscle ChAT activity versus time indicated that death in the lumbrical motor pool occurred during E17-E19, after all primary myotubes had formed and before generation of secondary myotubes began. We suggest that motoneurone death, by regulating the final size of the motoneurone pool, regulates the ratio of secondary to primary myotube numbers in a muscle.

Motoneuron projection patterns in chick embryonic limbs with a double complement of dorsal thigh musculature.

During the normal development of the chick, lateral motoneurons within the lumbosacral motor column of the spinal cord consistently project to muscles of dorsal origin within the limb while medial motoneurons project to muscles of ventral origin. To determine if specific cues arising from each type of target are the dominant guidance cues used by lateral and medial motoneurons to create this pattern, I examined motoneuron projections in embryonic chick limbs with a double complement of dorsal thigh musculature and no ventral musculature. Results indicate that cues associated with muscles of a specific developmental origin do not invariably dominate. Before and after the major period of motoneuron death, all muscles in dorsal limb regions (host) were innervated by lateral or dorsal pool neurons. Most ventrally positioned (donor) muscles were innervated by medial or ventral pool neurons. Only the donor iliofibularis, a muscle located very near to its original source of innervation, received projections from some lateral neurons. Within the limb proper, medial or ventral pool neurons projected to donor muscles in a patterned manner suggesting that they were following nonspecific regional cues and perhaps also responding to the availability of uninnervated target tissue. I conclude that axon sorting into distinct lateral and medial classes is independent of limb target complement and that subsequent pathway choice is a separate event governed by both specific target cues and other guidance mechanisms.

Increased motor neuron projection during development does not increase the number of neuromuscular synapses.

We investigated in developing embryos whether the total number of neuromuscular synapses is determined by the muscle or by the number of innervating motor neurons. The superior oblique muscle of duck embryos was hyperinnervated by preventing the naturally occurring death of trochlear motor neurons using immunoglobulin G from patients with acquired myasthenia gravis. In spite of a significant increase in the number of motor neurons innervating the muscle, a corresponding increase in the number of neuromuscular synapses did not occur. These results suggest that the total number of synapses in a muscle is independent of the number of innervating motor neurons and that it is determined intrinsically by the muscle itself.

Nerve-induced remodeling of muscle basal lamina during synaptogenesis.

To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis.

Spontaneous release of transmitter from the growth cones of Xenopus neurons in vitro: the influence of Ca2+ and Mg2+ ions.

To determine whether spontaneous release of transmitter from the growth cones of neurons exhibits properties similar to the spontaneous release which occurs from the neurons at the neuromuscular junction, release of transmitter from the growth cones of Xenopus neurons in culture was monitored in salines containing varying calcium and magnesium concentrations. Release was monitored by use of an outside-out piece of muscle membrane attached to a patch clamp electrode. Spontaneous release of transmitter from the growth cones in standard saline (2 mM CaCl2, 1 mM MgCl2) produces clusters of single-channel openings in the muscle membrane. Clusters are seen to consist of two types: a series of high-frequency channel openings, called "bursts," and clusters of low-frequency channel openings called "singles." The bursts were identified and examined for their possible relationship to MEPP-producing release, and the singles were identified and examined for their possible relationship to "leak" release of the neuromuscular junction. When the external saline contains high calcium (10 mM CaCl2, 1 mM MgCl2) or high magnesium (2 mM CaCl2, 9 mM MgCl2), the frequencies of both "bursts" and "singles" was greatly reduced. This reduction in release persists if the neurons are grown in the high-calcium or high-magnesium solutions. When the saline is a low-calcium solution (0 mM CaCl2, 3 mM MgCl2) the growth cones release transmitter at rates similar to those from standard saline. These results indicate that although the spontaneous release from the growth cone shares one characteristic with the leak release, neither the burst nor the singles release from the growth cones share exact relationship with either the MEPP producing release or the leak release. This suggests that further development of the mechanisms for spontaneous release of neurotransmitter occurs after nerve-muscle contact.

Development of semitendinosus muscle in pig fetuses with spinal cord lesions.

The effect of upper motor neuron regulation on the development of the semitendinosus muscle was studied in the fetus. A region of the fetal spinal cord at the level of the upper cervical vertebrae was destroyed by cauterization at 45 days of gestation. Fetuses with intact spinal cords served as controls. One cauterized fetus and one control fetus were obtained from each of six crossbred sows at 110 days of gestation. From each fetus one semitendinosus muscle was removed for histochemistry and the contralateral muscle was removed, weighed and utilized for biochemical analyses. Body weights and muscle weights were not significantly different (p greater than 0.05) between the two groups. Transverse sections (cryostat) of muscle were stained for lipid and the following enzymes: acid ATPase, NADH-TR, and esterase. Lipid and enzyme cytochemistry showed that sections from cauterized and control fetuses had identical fiber type patterns. Motor endplates, as studied with esterase reactions, were not affected by spinal cord cauterization. Mean values for percentage of muscle dry weight, DNA, RNA, protein, glycogen content and minimum fiber diameters were similar for cauterized and control fetuses (p greater than 0.05). These data illustrate that in the porcine fetus the central nervous system proximal to the alpha-motoneuron exerts little control over muscle cell development.

Myotube clusters do not bear any quantitative relation to the extent of motoneuron survival.

We conducted a quantitative study of the trochlear motoneurons and the myotube clusters in the corresponding superior oblique muscle of the Japanese quail, before, during, and after the period of normally occurring motoneuron degeneration. A ratio of approximately 1:1 between myotube clusters and neurons was observed at the onset of motoneuron degeneration. The number of myotube clusters prior to neuron death was 37% greater than the number of neurons surviving after cell death. These results are in contradiction with the hypothesis that the number of myotube clusters is the deciding factor for the survival of motoneurons during the critical stages of development.

Development of neuromuscular specificity in the grasshopper embryo: guidance of motoneuron growth cones by muscle pioneers.

In the grasshopper embryo, neuromuscular specificity develops between individual identified motoneurons whose cell bodies are located in the central nervous system, and specific skeletal muscles in the periphery. We previously reported on a class of large mesodermal cells, called muscle pioneers (MPs), that arise early in development (Ho, R. K., E. E. Ball, and C. S. Goodman (1983) Nature 301: 66-69). We suggested that the MPs might be involved in orchestrating the coordinated development of nerve and muscle. In this paper, we describe the development of the MP for coxal muscle 133a in the metathoracic limb bud, and its innervation by two excitatory motoneurons (fast, Df, and slow, Ds). Although many motoneuron growth cones extend out of nerve 5 and quite likely come in contact with the 133a MP between 35% and 45% of development, only Df and Ds display a high affinity for its surface; the other motoneurons innervate more distal leg muscles. When the 133a MP is ablated before arrival of motoneurons in the limb bud, the Df growth cone extends past the location where it normally gets off nerve 5 and continues to extend distally along the same pathway taken by its sibling motoneuron. Although there is a mass of small mesodermal cells in the area where the differentiated coxal muscle 133a normally forms, evidently it does not provide the necessary guidance cue for the Df growth cone. These results indicate the important role played by MPs in the specific guidance of motoneuron growth cones in the grasshopper embryo.

Ultrastructural study of motoneurons in Werdnig-Hoffmann disease.

Ultrastructural studies of the lumbar spinal cord in three children with Werdnig-Hoffmann (W-H) disease type Ia revealed numerous small neurons which appeared both atrophic and immature. We compared these motoneurons with anterior horn cells of a 3-month-old child, a 27-week and a 16-week human fetus, and found (1) that the motoneurons were much smaller in W-H disease, and (2) the Nissl substance was peripherally located and less developed. Signs of motoneuron immaturity as well as secondary degenerative changes suggest that in W-H disease neurons die either because they fail to make adequate peripheral contact or because the neurons are genetically intrinsically defective.