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K W Tosney

Publications and source records attributed to K W Tosney.

At least 19 recordsLinked to original sources
PubMed2001

Filopodial initiation and a novel filament-organizing center, the focal ring.

This study examines filopodial initiation and implicates a putative actin filament organizer, the focal ring. Filopodia were optically recorded as they emerged from veils, the active lamellar extensions of growth cones. Motile histories revealed three events that consistently preceded filopodial emergence: an influx of cytoplasm into adjacent filopodia, a focal increase in phase density at veil margins, and protrusion of nubs that transform into filopodia. The cytoplasmic influx probably supplies materials needed for initiation. In correlated time lapse-immunocytochemistry, these focal phase densities corresponded to adhesions. These adhesions persisted at filopodial bases, regardless of subsequent movements. In correlated time lapse-electron microscopy, these adhesion sites contained a focal ring (an oblate, donut-shaped structure approximately 120 nm in diameter) with radiating actin filaments. Filament geometry may explain filopodial emergence at 30 degree angles relative to adjacent filopodia. A model is proposed in which focal rings play a vital role in initiating and stabilizing filopodia: 1) they anchor actin filaments at adhesions, thereby facilitating tension development and filopodial emergence; 2) "axial" filaments connect focal rings to nub tips, thereby organizing filament bundling and ensuring the bundle intersects an adhesion; and 3) "lateral" filaments interconnect focal rings and filament bundles, thereby helping stabilize lamellar margins and filopodia.

Actins↗
PubMed2000

Identification of an invariant response: stable contact with schwann cells induces veil extension in sensory growth cones.

Growth cones sense cues by filopodial contact, but how their motility is altered by contact remains unclear. Although contact could alter motile dynamics in complex ways, our analysis shows that stable contact with Schwann cells induces motility changes that are remarkably discrete and invariant. Filopodial contact invariably induces local veil extension. Even when contacts are brief, veils always extend before the filopodia retract. Contact at filopodial tips suffices for induction. Moreover, veils extend significantly sooner than on filopodia contacting laminin, which often detach without extending veils. The overall behavioral responses of the growth cone, such as increased area and turning, result from integrating multiple discrete responses. Cycles of veil induction enlarge the growth cone and often lead it onto the cell. Invariant veil induction is abolished by blocking N-cadherin signaling. We propose an axonal guidance model in which different guidance cues act by inducing different but discrete and invariant responses.

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PubMed1999

Contact with isolated sclerotome cells steers sensory growth cones by altering distinct elements of extension.

During pathfinding, growth cones respond to guidance cues by altering their motility. This study shows that motile responses can be highly specific: filopodial contact with two different, physiologically relevant cells differentially alters discrete elements of motility. With each cell type, the responses to contact are invariant. Each cell induces a distinct response in sensory growth cones with every filopodial contact. Contact with an inhibitory cell, posterior sclerotome, alters a discrete motile characteristic; contact locally inhibits the ability of veils to extend down contacting filopodia. The inhibition is precise. Contact fails to alter other individual veil characteristics such as initiation frequency or extension rate. Moreover, despite local veil inhibition, the general level of extension across the growth cone is retained, as though protrusive activity is regulated to some set point. Contact with a stimulatory cell, anterior sclerotome, elicits a biphasic response. First, contact stimulates extension generally, altering the set point of protrusion. Contact increases veils and filopodia throughout the growth cone persistently. Then contacting processes consolidate, forming neurite. Filopodia contacting either cell type have similar lifetimes but different fates. Filopodia contacting posterior cells show morphological indications of structural instability, likely related to their inability to support veil extension. Filopodia contacting anterior cells branch, become morphologically complex, and ultimately consolidate into neurite. The invariance and precision of these responses suggests they are the steering components elicited by contact. These steering components, when integrated with other motile events, modulate growth cone trajectory. The discreteness of these responses suggests that guidance cues affect equally discrete elements in signaling cascades.

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PubMed1996

Cellular interactions that guide sensory and motor neurites identified in an embryo slice preparation.

We used cultured cross sections ("slices") of avian embryos to identify interactions that guide neurites during their encounters with seven tissues that impose a stereotyped gross anatomical nerve pattern. We show that cultured slices retain tissue morphology, molecular distribution patterns, and guidance cues. They also allow us to directly visualize responses of labeled sensory and motor neurons deposited on the slice's surface. This assay has high predictive power. Contact-mediated avoidance or stimulation and long-range attraction or repulsion are each distinguishable because each predicts different neurite lengths and trajectories. The analysis shows that all but one of these mechanisms contributes to guidance. Three tissues similarly stimulated neurite elongation, suggesting common responses to a contact-mediated stimulation. Four tissues similarly elicited avoidance on contact, suggesting a common contact-mediated inhibition. Neurite orientations implicate a previously unsuspected long-distance attraction to one tissue, dorsal anterior sclerotome. Long-range repulsion plays no detectable role. Each tissue elicits the same response in two different neural populations, sensory and motor neurons. These results suggest that a small set of repeated mechanisms mediates responses to tissues that axons contact serially during pathfinding.

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PubMed1996

Embryo slices.

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Animals↗
PubMed1995

Specifying the target identity of motoneurons.

In the vertebrate spinal cord, motoneurons are clustered into longitudinal columns in agreement with the targets they innervate. Motoneurons within each column acquire properties early in development that ensure their axons navigate to appropriate targets, but how this target identity is specified is unknown. Recently, Tsuchida et al. described the expression of putative regulatory genes within motor columns in the chicken spinal cord. Combinations of LIM-family homeobox genes differentially mark columns that project to distinct target groups. Expression precedes column formation and axon outgrowth, making these genes candidates for specifying the target identity of motoneuron groups.

Animals↗
PubMed1994

Neural crest cells prefer the myotome's basal lamina over the sclerotome as a substratum.

Anterior sclerotome is presumed to be the only somitic tissue that guides neural crest cells as they migrate ventrally. In contrast, we report here that crest cells prefer the myotome's basal lamina over the sclerotome as a substratum. This conclusion stems from four observations. First, crest cells migrating between the neural tube and somite invade lumbar and thoracic somites only after the myotome has formed a basal lamina, as though they use this basal lamina to penetrate the somite. Second, crest cells alter their trajectories dramatically when they contact this basal lamina. They abruptly turn laterally and align closely with the myotome's basal surface. Third, crest cells invade sclerotome only when they fail to contact this basal lamina. For instance, the lateral half of each myotome is initially devoid of basal lamina. When the first crest cells reach the lateral myotome, they depart from the myotome's basal surface and penetrate lateral sclerotome. Only later, when a higher population density prevents some cells from contacting the basal lamina, do crest cells penetrate medial sclerotome. Conversely, crest cells that migrate between somites do not have access to myotome and fail to turn laterally. Fourth, when we prevent myotome development by surgically removing its precursor (the dermamyotome), crest cells fail to turn laterally within the somite. Instead, they move directly ventrally and colonize medial sclerotome. The preference for myotomal basal lamina implies that anterior sclerotome is a suboptimal environment for neural crest migration. The myotome's basal lamina may facilitate rapid migration through the somite before impediments to ventral migration develop.

Animals↗
PubMed1994

Glycoconjugates mark a transient barrier to neural crest migration in the chicken embryo.

We report that two molecular markers correlate with a transient inhibition of neural crest cell entry into the dorsolateral path between the ectoderm and the somite in the avian embryo. During the period when neural crest cells are excluded from the dorsolateral path, both peanut agglutinin lectin (PNA)-binding activity and chondroitin-6-sulfate (C6S) immunoreactivity are expressed within this path. Both markers decline as neural crest cells enter. Moreover, both markers are absent after an experimental manipulation that accelerates neural crest entry into this path. Specifically, dermamyotome deletions abolish expression of both markers and allow neural crest cells to enter the dorsolateral path precociously. After partial deletions, dermatome remnants remain. These remnants retain PNA and C6S labeling and impede migration locally. Local glycoconjugate expression thus correlates directly with avoidance responses. Since both PNA-binding activity and C6S expression also typify inhibitory somitic tissues, molecules indicated by these markers (or co-regulated molecules) are likely to inhibit both neural crest and axon advance.

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PubMed1993

Contact-mediated mechanisms of motor axon segmentation.

In the chick embryo, the segmental pattern of motor outgrowth depends on guidance cues provided by sclerotome cells. Motor axons preferentially invade the anterior sclerotome but avoid the posterior sclerotome. To determine how motor growth cone motility is influenced by these cells, we used videomicroscopy to analyze the behavior of motor growth cones as they confronted identified sclerotome cells in vitro. After contact, motor growth cones invariably avoided posterior sclerotome cells by either branching or turning. Both types of avoidance behavior were initiated by a local inhibition of veil protrusion: veils failed to progress along the contacting filopodia. This inhibition was specific to veils since contact failed to alter the number of filopodia protruded. Moreover, motor growth cones turned away from posterior cells despite more persistent filopodial contacts with these cells than with the laminin substratum. In no case did contact with posterior cells cause a complete loss of growth cone motility or a complete collapse of growth cone structure. In contrast, motor growth cones exhibited a selective affinity for anterior cells, preferring the surfaces of these cells to the laminin substratum. Contact with anterior cells stimulated a generalized increase in protrusive activity: contact caused a net increase in the extension of veils and filopodia both locally and at sites distant from the site of contact. Contact also elicited a localized thickening of contacting processes, suggesting that contact with anterior cells promotes neurite consolidation. This behavior of motor growth cones in vitro suggests that both an inhibition of veil formation by posterior cells and an enhancement of motility and axon consolidation by anterior cells contribute to the preferential advance of motor axons into anterior sclerotome in vivo. We suggest that patterned outgrowth results from the juxtaposition of two contrasting environments that differentially influence growth cone motility.

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PubMed1992

Descriptive and experimental analysis of the dispersion of neural crest cells along the dorsolateral path and their entry into ectoderm in the chick embryo.

We have characterized the dispersion of neural crest cells along the dorsolateral path in the trunk of the chicken embryo and experimentally investigated the control of neural crest cell entry into this path. The distribution of putative neural crest cells was analyzed in plastic sections of embryos that had been incubated for 24 hr in HNK-1 antibody, a procedure that we show successfully labels neural crest cells in the dorsolateral path and ectoderm. In accord with earlier observations, crest cells delay entering the dorsolateral path until a day or more after their counterparts have colonized the ventral path. However, once crest cells enter, they disperse rapidly through the path dorsal to the somite but still delay migrating dorsal to the intersegmental space. During dispersion, crest cells invade the ectoderm at sites associated with local disruptions in the basal lamina which may be caused by crest cells. Finally, deleting the dermamyotome releases an inhibition of neural crest cell migration: crest cells enter the dorsolateral path precociously. We speculate that the epithelial dermatome may transiently produce inhibitory substances and that emerging dermis may provide a long-distance, stimulatory cue.

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PubMed1991

Spatial and temporal patterns of muscle cleavage in the chick thigh and their value as criteria for homology.

Regions of lower cell density, called cleavage zones, emerge within the dorsal and ventral muscle masses in the vertebrate limb to separate distinct muscles. In the chick thigh, the stereotyped patterns of separation have been broadly outlined, but differences in interpretation exist because no criteria for separation have been defined, and the tissues of the limb are indistinct early in development. We have examined the cleavage process using modern applications of light microscopy and immunocytochemistry to completely detail the spatial and temporal progression of cleavage in stage 27-32 embryos. We find that each muscle has a complex but characteristic pattern of separation along the proximodistal axis. The complex pattern of separation is not related to the positions of muscles within the thigh, locations of blood vessels, activity patterns of muscles, or innervation patterns. The initial separation patterns are more straightforward than later separations and may be of value in determining the phylogenetic history of limb muscles since the same patterns are common to many tetrapods. Our detailed documentation clarifies the ontogeny of the thigh musculature and reveals more complex separation patterns between muscles than previously described.

Animals↗
PubMed1991

Ultrastructural and morphometric analysis of the separation of two thigh muscles in the chick.

Limb muscles separate from one another in a complex but highly stereotyped sequence and spatial pattern. The process of separation is characterized by the progression of a region of increased extracellular space, the cleavage zone, along the proximodistal axis between the individual muscle anlagen. We analyzed ultrastructurally the muscles and cleavage zone during the separation of two representative muscles, the developing sartorius and iliotibialis in the chick thigh, to establish an accurate baseline for an analysis of the mechanisms of separation. Comparisons of the morphology and distribution of cells before and after separation show no evidence that muscles became separated by the massive influx of an exterior cell population; if populations invade the cleavage zone, they are small. We do find characteristic transitions within the cell population of the cleavage zone in situ that could accomplish cleavage without invoking massive cell movements. These progressive transitions within the cleavage zone include a loss of close cell-cell interactions, an increase in extracellular space, the assumption of a more stellate morphology by mesenchyme cells, and a gradual alteration in the composition of the extracellular matrix from one typical of early muscle to one typical of loose connective tissue. Myotubes do differentiate between the incipient muscles, ruling out the possibility that the location where muscles will separate is defined by sites where myotubes fail to differentiate. Instead, the myotubes in the cleavage zone gradually diminish in number and appear to be specifically recognized and removed from the cleavage zone by phagocytes. We suggest that the transitions within the cleavage zone, including the loss of muscle cells, are a result of the progressive differentiation of loose connective tissue. If so, then the spatial pattern and process of cleavage is a consequence of spatially programmed cell differentiation.

Animals↗
PubMed1991

Cells and cell-interactions that guide motor axons in the developing chick embryo.

A considerable challenge confronts any developing neuron. Before it can establish a functional and specific connection, it must extend an axon over tens and sometimes hundreds of microns through a complex and mutable environment to reach one out of many possible destinations. The field of axonal guidance concerns the control of this navigation process. To satisfactorily identify the cell interactions and molecular mechanisms that mediate axonal guidance, it is essential to first identify the pertinent cell populations. Embryonic surgeries have provided solid information on which tissues are critical and which are irrelevant to the navigation of motor axons within the chick embryo. The gross anatomical nerve pattern is established as axons respond to both positive (path) and negative (barrier) tissue environments. Analysis of the interactions of motoneurons with these tissues reveals that several cellular interactions--chemotaxis, substratum preference, and perhaps contact paralysis--are important to the common patterns of motor axon advance. Axons simultaneously interact with population-specific cues that have begun to be identified on the tissue level.

Animals↗
PubMed1991

Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo.

Axon outgrowth between the spinal cord and the hindlimb of the chick embryo is constrained by three tissues that border axon pathways. Growth cones turn to avoid the posterior sclerotome, perinotochordal mesenchyme, and pelvic girdle precursor during normal development and after experimental manipulation. We wanted to know if these functionally similar barriers to axon advance also share a common molecular composition. Since the posterior sclerotome differentially binds peanut agglutinin (PNA) and since PNA binding is also typical of prechondrogenic differentiation, we examined the pattern of expression of PNA binding sites and cartilage proteoglycan epitopes in relation to axon outgrowth. We found that all three barrier tissues preferentially express both PNA binding sites and chondroitin-6-sulfate (C-6-S) immunoreactivity at the time when growth cones avoid these tissues. Moreover, both epitopes are expressed in the roof plate of the spinal cord and in the early limb bud, two additional putative barriers to axon advance. In contrast, neither epitope is detected in peripheral axon pathways. In the somites, this dichotomous pattern of expression clearly preceded the invasion of the anterior sclerotome by either motor growth cones or neural crest cells. However, in the limb, barrier markers disappeared from presumptive axon pathways in concert with the invasion of axons. Since this coordinate pattern suggested that the absence of barrier markers in these axon pathways requires an interaction with growth cones, we analyzed the pattern of barrier marker expression following unilateral neural tube deletions. We found that PNA-negative axon pathways developed normally even in the virtual absence of axon outgrowth. We conclude that the absence of staining with carbohydrate-specific barrier markers is an independent characteristic of the cells that comprise axon pathways. These results identify two molecular markers that characterize known functional barriers to axon advance and suggest that barrier tissues may impose patterns on peripheral nerve outgrowth by virtue of their distinct molecular composition.

Animals↗
PubMed1990

Distribution and projection pattern of motoneurons that innervate hindlimb muscles in the quail.

We characterized the motoneuron pool positions and projection patterns in the embryonic quail hindlimb and compared them to those in the chick to determine the degree of similarity and to form a baseline for future chimeric experiments. We find that the most similar parameters of pool position correlate with the major axonal pathway choices. First, the medial-lateral pool position, which is highly conserved among birds and mammals, is identical in the quail and chick and correlates with the dorsal-ventral pathway choice, the first and least plastic of the choices within the limb. Second, although quail pools were known to be compressed into seven rather than eight segments, we show that the map of pools is compressed about a central point (segment three) that preserves the spatial relationships between anterior pools and the crural plexus, and between posterior pools and the sciatic plexus. Access to guidance cues that are restricted to each plexus region is thus maintained between species. Third, pool position along the anterior-posterior axis is the least similar parameter between species. In fact, the entire lumbosacral motor complex may shift by +/- half a segment in individual quail. Despite the consequent differences in segmental projections, the specific projection pattern within each quail hindlimb is identical to that in the chick. There is no need to preserve the exact segmental pattern either phylogenetically or during development, because motoneurons accommodate to modest variations in their position along the anterior-posterior axis by sorting out at the limb base. The contrast between variable segmental and constant limb projections also demonstrates that neither the specification nor the precise projection of motoneurons is dependent upon the specification or development of somites.

Animals↗
PubMed1990

The perinotochordal mesenchyme acts as a barrier to axon advance in the chick embryo: implications for a general mechanism of axonal guidance.

To test the hypothesis that the perinotochordal mesenchyme (the sclerotome ventral to the spinal nerve pathway) is a barrier to axonal advance in the chick embryo, we determined whether axons directly confronted with perinotochordal mesenchyme would turn to avoid it. The initial direction of motor axon outgrowth was altered by rotating the right half of the neural tube after deleting the left half. Perinotochordal mesenchyme was identified histologically or by peanut agglutinin (PNA) binding. We found that axons turned to avoid the perinotochordal mesenchyme and traversed only the dorsal-anterior sclerotome at all stages of outgrowth. When the ventral root was positioned at the midline, axons projected around the perinotochordal mesenchyme and formed spinal nerves on both sides of the embryo. Furthermore, neural crest cells and sensory axons did not penetrate perinotochordal mesenchyme, even in the absence of motor axons. In contrast, perinotochordal mesenchyme did not exhibit inhibitory function and did not differentially bind PNA when the notochord was deleted; axons ramified widely within it. We conclude that the dorsal-anterior sclerotome is permissive and that the perinotochordal mesenchyme is relatively inhibitory for the advance of axons and neural crest cells. Two additional pairs of tissues provide similar permissive/inhibitory contrasts in the embryo, the anterior/posterior sclerotome and the plexus/pelvic girdle mesenchyme. We hypothesize that guidance by all three pairs is mediated by the same set of cellular interactions and has a common molecular basis. We further propose that the transient expression of substances characteristic of these contrasting tissue pairs could serve to guide axons elsewhere, in both the peripheral and the central nervous systems.

Animals↗
PubMed1989

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.

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