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.

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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.

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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In this study, adhesions on individual filopodial shafts were shown to control veil (lamellar) advance and to be modulated by guidance cues. Adhesions were detected in individual filopodia of sensory growth cones using optical recordings, adhesion markers, and electron microscopy. Veils readily advanced along filopodia lacking shaft adhesions but rarely advanced along filopodia displaying shaft adhesions. Experiments altering adhesion showed that this relationship is not caused by veils removing adhesions as they advanced. Reducing adhesion with antibodies decreased the proportion of filopodia with shaft adhesions and coordinately increased veil advance. Moreover, the inhibitory relationship was maintained: veils still failed to advance on individual filopodia that retained shaft adhesions. These results support the idea that shaft adhesions inhibit veil advance. Of particular interest, guidance cues can act by altering shaft adhesions. When a cellular cue was contacted by a filopodial tip, veil extension and shaft adhesions altered in concert. Contact with a Schwann cell induced veil advance and inhibited shaft adhesions. In contrast, contact with a posterior sclerotome cell prohibited veil advance and promoted shaft adhesions. These results show that veil advance is controlled by shaft adhesions and that guidance signal cascades can alter veil advance by altering these adhesions. Shaft adhesions thus differ functionally from two other adhesions identified on individual filopodia. Tip adhesions suffice to signal. Basal adhesions do not influence veil advance but are critical to filopodial initiation and dynamics. Individual growth cone filopodia thus develop three functionally distinct adhesions that are vital for both motility and navigation.

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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