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K W Tosney

Publications and source records attributed to K W Tosney.

34 records · Page 2Linked to original sources
PubMed1989

A hierarchy of determining factors controls motoneuron innervation. Experimental studies on the development of the plantaris muscle (PL) in avian chimeras.

Quail leg buds were grafted in place of chick leg buds or chick wing buds and vice versa at stages 18 to 21 after colonization by muscle precursor cells had been completed. Motor endplate pattern in the plantaris muscle of the grafts was analyzed before hatching by means of esterase and acetylcholinesterase staining techniques. Muscle fibre types were made visual using the myosin ATPase reaction. Investigations are based on the species-specific endplate pattern of the plantaris muscle: multiply innervated fibres in the chick and focally innervated fibres in the quail. Muscle pieces isolated from the adjacent medial gastrocnemius muscle of the grafted legs were histologically examined to judge their species-specific composition. Horseradish peroxidase was injected into the plantaris muscles of both the grafted and the opposite leg as well as in the plantaris muscle of normal quail embryos, in order to be sure that the plantaris muscle of the grafts is innervated by appropriate motoneurons. This procedural design offers for the first time a possibility to test experimentally the influences of motoneurons on endplate pattern formation under conditions corresponding to those in normal ontogenesis. It is shown that such appropriate motoneurons of one species which project to the plantaris muscle of the other species dictate the endplate pattern. When the plantaris muscle is innervated by inappropriate motoneurons, the endplate pattern inherent in the muscle primordium itself becomes realized. A sequence of hierarchically acting factors is proposed to bring different results in line. According to this, the neuronally set programme has priority compared with that set in the muscle. This is true for the normal development and might generate the high neuro-muscular specificity. If under experimental conditions the neuronal programme and the peripheral programme differ, the axons and muscle fibres selectively interact with respect to their inherent characteristics and the muscle-specific programme becomes expressed. If there is a lack of a certain axon type, muscle fibres might become innervated by non-corresponding motoneurons which alter the muscle fibre type.

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PubMed1988

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.

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PubMed1988

Cell death delineates axon pathways in the hindlimb and does so independently of neurite outgrowth.

We wished to know whether the cell death and phagocytosis seen near the outgrowing nerve front in the hindlimb delineate axon pathways and, if so, whether the cells died only in the presence of growth cones. We unilaterally deleted the lumbosacral neural tube and reconstructed the patterns of neurite outgrowth and phagocytes during the stage when neurites first begin to colonize the thigh. In the control limbs, sensory and motor nerve pathways coincided with sites of phagocytosis, including those pathways that had yet to be colonized by growth cones. For instance, phagocytes were clustered at foci within the muscle masses where muscle nerves form a day later. However, they were not seen in adjacent, nonpathway regions such as posterior sclerotome or dorsal and ventral to the region of the plexus in which axons extend only posteriorly. Phagocytes were also seen in defined regions that are probably inaccessible to growth cones because they are too distant from pathways (i.e., subjacent to the apical ectodermal ridge) or express substances that are typical of precartilagenous tissues which may prohibit axon advance. In the experimental limbs, we conservatively estimated that neurite outgrowth was reduced to less than one-tenth (neurites were visible only with electron microscopy) or less than one-third of normal. Outgrowth extended less far distally and, in half the cases, motor innervation was completely abolished. Despite the extensive reduction in neurite outgrowth, the distribution of phagocytes was indistinguishable from that of the control side. Furthermore, the number of phagocytes did not differ significantly. We conclude that cell death delineates axon pathways remarkably well and does so without an interaction with growth cones; it is an independent characteristic of the axonal pathways and may be directly or indirectly important to axonal pathfinding. This is the first identification of a feature that characterizes prospective nerve pathways in the hindlimb.

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PubMed1988

Somites and axon guidance.

The somites are arrayed in a repeating pattern along the longitudinal axis of the embryo, as are the developing sensory and sympathetic ganglia and the spinal nerves. This pattern is not a coincidence: the somite imposes a segmental pattern on the cells and axons that invade it. Both neural crest cells and axons prefer the anterior portion of the sclerotome (the ventral part of the somite) for outgrowth. What differences in anterior and posterior sclerotome are responsible? I used scanning electron microscopy to ask whether these populations differed on the tissue level in chick embryos. This study shows that differences in tissue organization are of insufficient magnitude or develop too late to explain the preference of neural crest cells and axons for the anterior half of each sclerotome. For instance, the extracellular matrix does not differ dramatically in density at the dorsal sclerotome boundary and yet neural crest cells promptly enter the anterior sclerotome when they reach this boundary. These cells have access to the cell processes of somitic cells that extend through the matrix. This suggests that neural crest cells could detect important differences in anterior and posterior populations by direct cell contact. Likewise, barriers and consistent differences in cell density, shape or orientation were not obvious before or during initial axon outgrowth. The absence of significant differences in tissue organization suggests that axons and neural crest cells become segmented by responding to diffusible cues, to differences in extracellular material or to the cell surfaces of individual anterior and posterior sclerotome cells.

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PubMed1987

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

The role of the dermamyotome (the dorsal portion of the somite which gives rise to muscles and dermis) in the development of patterned axon outgrowth was examined under conditions where limb development was substantially undisturbed. One or more chick dermamyotomes were removed before or during early neurite outgrowth and subsequent development was examined. Several developmental processes suspected to depend on the dermamyotome were not altered by its removal: (1) Neural crest cells that form sensory ganglia migrated and condensed in their normal segmental pattern. (2) The distal progression, dorsal-ventral organization, and segmentation of spinal nerves were unaltered. (3) Motoneuron pathway selection and projection patterns in the limb were normal in all respects. The most interesting finding was that the formation of the dorsal ramus is dependent on the nearby dermamyotome which provides the targets for this nerve. When a single or two adjacent dermamyotomes were removed, the metameric epaxial muscles derived from each dermamyotome were absent and the dorsal ramus extended into epaxial muscle in the closest adjacent segment. However, when dermamyotomes in both adjacent segments had also been removed or substantially reduced, the dorsal ramus did not form. These results strongly suggest that the target provides a chemotactic signal for proper outgrowth of dorsal ramus axons.

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PubMed1986

The distribution of NCAM in the chick hindlimb during axon outgrowth and synaptogenesis.

We have determined the distribution and form of the neural cell adhesion molecule (NCAM) in the chick hindlimb from initial axon outgrowth (stage 17 1/2) until 3 days posthatching by immunohistological staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots. Axons stained intensely for NCAM at all ages, whereas nonneuronal limb components exhibited dynamic changes in staining. Mesenchymal cells in the sclerotome adjacent to the neural tube developed NCAM immunoreactivity in an anterior-posterior sequence which correlated with the sequence of axonal outgrowth. Low to moderate amounts of NCAM were detected within and surrounding presumptive nerve pathways, consistent with a permissive role for NCAM in axon extension, but not with a precise delineation of pathway boundaries. On myotubes immunoreactivity for NCAM remained low from stage 26 to 30 when it increased dramatically in both aneural and control limbs, indicating that its appearance is not triggered by nerve-dependent activity or trophic interactions. The increase was temporally associated with muscle cleavage and may encourage subsequent axon ramification as well as synaptogenesis. Staining remained high on muscle fibers during secondary myotube formation and only declined during the week before hatching when polyneuronal innervation is withdrawn and the mature synaptic pattern becomes stabilized. This loss of muscle NCAM occurred first on fast and then on slow muscle fibers. Together these results suggest that the timing of innervation may be controlled by the muscle, through NCAM expression, but that the subsequent suppression of muscle NCAM may occur as a result of nerve-mediated activity.

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PubMed1986

Neurites and growth cones in the chick embryo. Enhanced tissue preservation and visualization of HRP-labeled subpopulations in serial 25-microns plastic sections cut on a rotary microtome.

Study of axonal guidance in developing vertebrates has been hindered by an inability to readily visualize individual growth cones, determine the neuronal population from which they originate, trace their trajectories, and discern their interactions with their embryonic environment. We report a method that combines plastic embedding and serial sectioning with horseradish peroxidase labeling of subpopulations of neurons in the chick embryo. This method labels individual neurites from the soma to the tip of the growth cones, allowing their trajectory to be inferred and their identity to be determined by the position of the somata. As sections are up to 25 micron thick, entire growth cones can often be visualized without laborious reconstruction. Tissue preservation is much better than with similar material embedded in paraffin. Sections are cut relatively quickly using a steel knife on a standard rotary microtome and are suitable for subsequent electron microscopy.

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PubMed1985

Development of the major pathways for neurite outgrowth in the chick hindlimb.

To elucidate mechanisms that may control development of the gross anatomical nerve pattern, motoneuron outgrowth into the chick hindlimb was examined using orthograde labeling, scanning and transmission electron microscopy, and Alcian blue staining. Results show that growth cones are not guided by contact with oriented extracellular fibrils, aligned mesenchyme cells, the myotome, or the vasculature. Pathways are not delineated by cell-free space or channels of lower cell density; however, densely packed mesenchyme may form barriers that channel outgrowth. In addition, abundant mesenchymal cell death was seen at the nerve front. This cell death may provide space that encourages growth cone advancement. Pathways often lie along interfaces between areas that stain darkly and lightly with Alcian blue, which specifically stains glycosaminoglycans, and growth cones never penetrate areas that stain intensely, such as the pelvic girdle, which is known to be a barrier to outgrowth. Leading growth cones form specialized contacts with mesenchyme cells, but the predominant contacts are interneuronal. It is proposed that the anatomical pattern of outgrowth is determined by the distribution of preferred substrata, the most preferred substratum being other neurites. Further, neurites tend to prefer loose mesenchyme to dense mesenchyme or areas rich in glycosaminoglycans.

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PubMed1985

Specificity of early motoneuron growth cone outgrowth in the chick embryo.

During development, chick lumbosacral motoneurons have been reported to form precise topographic projections within the limb from the time of initial outgrowth. This observation implies, first, that motoneurons select the appropriate muscle nerve pathway and, second, that they restrict their ramification within the primary uncleaved muscle masses to appropriate regions. Several reports based on electrophysiology and orthograde horseradish peroxidase (HRP) labeling have shown muscle nerve pathway selection to be fairly precise. However, studies based on retrograde labeling with HRP have produced conflicting reports on the extent to which vertebrate motoneurons make projection errors. Since it is difficult to distinguish between true projection errors and HRP leakage when using retrograde labeling, we decided to assess the distribution of labeled growth cones in 25-micron serial plastic sections, following orthograde labeling of identifiable subpopulations of motoneurons during the period of initial axon outgrowth. Examination of a large number of muscle nerves revealed no segmentally inappropriate axons, confirming earlier reports that muscle nerve pathway selection is very accurate. In addition, we observed that growth cones take widely divergent trajectories into the same muscle nerve, suggesting that growth cones are responding independently to some specific environmental cue rather than being passively channeled at this point. The distribution of labeled growth cones within the muscle masses provided direct evidence that motoneurons did not at any time project to obviously inappropriate muscle regions.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Growth cone morphology and trajectory in the lumbosacral region of the chick embryo.

We quantitatively analyzed several features of orthogradely labeled peripheral growth cones in the lumbosacral region of the chick embryo. We compared motoneuron growth cones in regions where they appear to express specific directional preferences (the plexus region and regions where muscle nerves diverge from main nerve trunks), which we operationally defined as "decision regions," to motoneuron growth cones in other pathway regions (the spinal nerve, nerve trunk, and muscle nerve pathways) which we termed, for contrast, "non-decision region." We found that motoneuron growth cones are larger, more lamellepodial, and have more complex trajectories in decision regions. Sensory growth cone populations, which are thought to be dependent upon motoneurons for outgrowth (Landmesser, L., and M. Honig (1982) Soc. Neurosci. Abstr. 8: 929), do not enlarge or become more lamellepodial in motoneuron decision regions, suggesting that this local environment does not affect all species of growth cones equally and that the alterations in motoneuron growth cones in these regions may be relevant to their specific guidance. In addition, the resemblance between the sensory population and other closely fasciculating growth cones lends support to the suggestion that sensory neurons utilize motoneuron neurites as a substratum. We suggest that the convoluted trajectories, enlarged size, and more lamellepodial morphology of motoneuron growth cones in decision regions is either related directly to the presence of specific cues that guide motoneurons or to some aspect of this environment that allows them to respond to specific cues.

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PubMed1984

Pattern and specificity of axonal outgrowth following varying degrees of chick limb bud ablation.

Motoneurons grow into the chick hindlimb via consistent pathways, within which they make specific decisions leading to their correct targets. To determine which axonal guidance features dictate the position of the pathways and to examine the distribution of specific cues, we totally or partially ablated the early hindlimb bud and determined how the subsequent pattern of nerve outgrowth related to the distribution of tissue remnants. Our results suggest that local elements determine the gross anatomical pattern of outgrowth. First, determinants of individual pathways could be selectively removed without altering the pattern in other regions. Second, neurites were restricted to the plexus region at the base of the leg (within which, for unknown reasons, they proceeded posteriorly) unless distal permissive pathways or nearby target remnants were present. Finally, we found that the central region of the pelvic girdle, adjacent to the plexus region, determines the position where the major nerve trunks enter the leg. When gaps were introduced in this region of the girdle, nerves traversed the gaps and directly innervated adjacent muscle. The developing girdle is probably a nonpermissive environment for axon elongation, and axons enter the leg only where it is locally absent. Our results also support the concept that the specific cues that neurites use to reach their appropriate muscles are local. We find that neurites could make correct and specific decisions in the plexus region in the absence of all tissues distal to the pelvic girdle. This shows that the cues for these decisions are independent of the target and must reside in the local mesenchyme. In addition, when muscle remnants were present they were correctly innervated.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1983

Neuronal motility: the ultrastructure of veils and microspikes correlates with their motile activities.

We have documented the ultrastructural characteristics that correlate with protrusion, adhesion and retraction of neuronal veils and microspikes, by filming individual neurons of the chick ciliary ganglion and examining the same cells with high-voltage electron microscopy. We find that new veils invariably contain only a cortical meshwork of filaments and are devoid of microtubules, groups of vesicles and other organelles. At sites of recent veil retraction a cortical meshwork on the substratum side underlies a filament-free space containing vesicle clusters and a complexly folded upper membrane. Areas without veil activity are smooth-surfaced and contain a three-dimensional lattice of filaments. We discuss the implications of these observations for the mechanisms of surface recruitment and retrieval during motile activity. We also find that the ultrastructure of moving and attached extensions of the cell surface differs dramatically. Unattached microspikes and actively extending veils have an open, criss-cross array of filaments, whereas attached microspikes contain more aligned filaments, which extend as a small bundle into the growth cone. These results suggest that cell surface protrusion is mediated by meshworks of loosely packed filaments. More compact bundles of filaments are probably generated only at adhesion points.

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PubMedDate not supplied

Projection-neurons that send axons through the lumbar spinal cord of the chick embryo are not obviously distributed in a segmentally repetitive pattern.

Earlier studies have shown that the formation of the segmentally repetitive pattern of spinal nerves in avian embryos depends upon the segmental arrangement of the somites and does not reflect an intrinsic pattern of the spinal cord. These studies could not, however, rule out the possibility that some elements of the central nervous system are intrinsically segmented. The question remained, for instance, whether projection-neurons are distributed in a segmentally repetitive pattern within the spinal cord. To address this question, horseradish peroxidase was injected into one or two segments of the lumbar spinal cord of chick embryos, thereby labeling projection-neurons that had ascending or descending axons passing through the injection site. In all stages examined, the number of labeled projection-neurons in the anterior and posterior halves of each segment did not differ significantly. In addition, segmentally repetitive peaks or troughs in the numbers of labeled projection-neurons were not detected in the center of each segment. Three subpopulations of projection-neurons, defined by their position along the dorsal-ventral axis of the spinal cord, were also not segmentally distributed by these criteria. While these results do not rule out the possibility that subpopulations of projection-neurons defined by dendritic morphology, functional class, or some other parameter are segmentally arranged or that there is a basic modular repetition of neural populations from segment to segment, they do show that projection-neurons as a class and subpopulations of these neurons defined by their dorsal-ventral position are not obviously distributed in a repetitive segmental pattern.

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