Microtubules are a primary cytoskeletal constituent of axons and growth cones. In addition to serving as a scaffolding for axon assembly, they also provide a means of transport of organelles that are essential for outgrowth and maintenance of synaptic function. Pharmacological manipulations that disrupt net assembly of microtubules also interfere with growth cone advance and axon extension. Less is known after the effects of disrupting microtubule dynamics without affecting net assembly. To investigate this, we studied the effects of low doses of nocodazole on axon extension and microtubule organization in rat superior cervical ganglion neurons. We report that 165-330 nM nocodazole significantly reduces axon extension rate and increases axon diameter without affecting the rate of production of axoplasm or microtubule polymer, and without decreasing the average length or number of microtubules. Two observations suggested that microtubule dynamics were depressed by this dose of nocodazole. First, this treatment eliminated the highly divergent lengths and positions of microtubules characteristic of normal growth cones, inducing an array in which each microtubule terminated at roughly the same position in the proximal regions of the growth cone. Second, there was a decrease in the proportion of microtubule length containing mostly tyrosinated (newly assembled) alpha-tubulin and an increase in the proportion of microtubule length containing mostly acetylated (older, more stable) alpha-tubulin. Together, these data suggest that a decrease in dynamic instability of microtubules is sufficient to disrupt axon extension but does not interfere with axoplasm production.

We identify an actin-based protrusive structure in growth cones termed "intrapodium." Unlike filopodia, intrapodia are initiated exclusively within lamellipodia and elongate in a continuous (nonsaltatory) manner parallel to the plane of the dorsal plasma membrane causing a ridge-like protrusion. Intrapodia resemble the actin-rich structures induced by intracellular pathogens (e.g., Listeria) or by extracellular beads. Cytochalasin B inhibits intrapodial elongation and removal of cytochalasin B produced a burst of intrapodial activity. Electron microscopic studies revealed that lamellipodial intrapodia contain both short and long actin filaments oriented with their barbed ends toward the membrane surface or advancing end. Our data suggest an interaction between microtubule endings and intrapodia formation. Disruption of microtubules by acute nocodazole treatment decreased intrapodia frequency, and washout of nocodazole or addition of the microtubule-stabilizing drug Taxol caused a burst of intrapodia formation. Furthermore, individual microtubule ends were found near intrapodia initiation sites. Thus, microtubule ends or associated structures may regulate these actin-dependent structures. We propose that intrapodia are the consequence of an early step in a cascade of events that leads to the development of F-actin-associated plasma membrane specializations.

The trigeminal ganglion provides the somatosensory innervation for the anterior rat tongue. At early embryonic stages (embryonic day [E] 12-13) pre-tongue explants repel trigeminal axon outgrowth, and this is mediated by Sema3A (Rochlin and Farbman [1998] J. Neurosci. 18:6840-6852; Rochlin et al. [2000] J. Comp. Neurol. 422:579-593). Despite a decrease in repulsion by E14 and older tongue explants, Sema3A mRNA persists throughout the dorsal epithelium through E18, after axons have begun to penetrate papilla epithelium. We investigated the hypothesis that Sema3A continues to act as a repellent and that subpopulations of trigeminal axons that penetrate the epithelium become unresponsive to Sema3A. Sema3A repelled trigeminal axons in vitro regardless of the neurotrophic factor used to stimulate axon outgrowth, but the minimum level of Sema3A required to repel depended on the neurotrophic factor. Thus, in vitro, trigeminal axons are repelled by Sema3A when they would be penetrating the Sema3A-mRNA rich epithelium in vivo. Whereas dorsal epithelium on tongue explants dissected at stages preceding target contact (E15) repelled trigeminal axons in vitro, explants dissected at later stages (E18), after axons would have penetrated the epithelium in vivo, were not repellent. To determine whether Sema3A prevents premature target penetration in vivo, we assessed the timing of target contact by sensory axons in Sema3A-/minus; and +/+ mice. Contact of the epithelium occurs prematurely in Sema3A-/minus; mice, but not penetration. Taken together, our data imply that Sema3A acts as a short-range repellent that regulates the timing of target contact by trigeminal axons.

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures.

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures.

Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3-4 days before penetrating fungiform papilla epithelium (E17). This latency may result from chemorepulsion by epithelial Sema3A (Dillon et al. (2004) Journal of Comparative Neurology 470, 13-24), or Sema3F, which we report is also expressed in this epithelium. Sema3A and Sema3F repelled or suppressed geniculate neurite outgrowth, respectively, and these effects were stage and neurotrophic factor dependent. BDNF-stimulated outgrowth is repelled by Sema3A until E17, but insensitive to Sema3F from E16. NT-4-stimulated neurite outgrowth is sensitive to Sema3A and Sema3F through E18, but NT-4 has not been detected in E15-18 tongue. E15-18 tongue explants did not exhibit net chemorepulsion of geniculate neurites, but the ability of tongue explants to support geniculate neurite outgrowth fluctuates: E12-13 (Rochlin et al. (2000), Journal of Comparative Neurology, 422, 579-593) and E17-18 explants promote and may attract geniculate neurites, but stages corresponding to intralingual arborization do not. The E18 trophic and tropic effects were evident even in the presence of BDNF or NT-4, suggesting that some other factor is responsible. Intrinsic neurite outgrowth capability (without exogenous neurotrophic factors) fluctuated similarly: ganglia deteriorated at E15, but exhibited moderate outgrowth at E18. The chemorepulsion studies are consistent with a role for Sema3A, not Sema3F, in restricting geniculate axons from the epithelium until E17, when axons penetrate the epithelium. The transient inability of tongue explants to promote geniculate neurite outgrowth may signify an alternative mechanism for restricting geniculate axons from the epithelium: limiting trophic factor access.

Previous work suggested that in mouse, presumptive targets of the trigeminal ganglion, rather than intermediate structures, attract pioneer axons from the time their growth cones exit the ganglion (Lumsden and Davies, 1986). In rat we find that some presumptive targets repel trigeminal axons. The repellant activity is concentrated in the anterior and ventral epithelium of the mandibular arch at embryonic day 12 (E12) and was also present in the maxillary arch. The activity is blocked by anti-neuropilin-1. E13 mandible explants repel trigeminal axons during the first day of outgrowth in vitro, but thereafter permit or attract trigeminal ganglion axon outgrowth. By E14, lingual nerve afferents first enter the tongue in vivo, and the repellant influence becomes restricted to the midline. The progressive restriction of the repellant influence may contribute to the in vivo progression of nerve development: the earliest afferents turn anteriorly lateral to the tongue, but subsequently arriving afferents advance into the tongue and then turn away from the midline. Thus, the repellant may influence the order of nerve branch development and the timing of innervation of epithelial and subepithelial targets. Heterochronic studies revealed that the loss of repellant influence from presumptive lateral tongue surface results from downregulation of the repellant activity, not of responsiveness to the repellant. Because presumptive targets repel trigeminal axons during the initial stages of advance from the trigeminal ganglion and do not have a net attractive influence until after afferents have arrived near the target, intermediate structures must guide these axons initially.

The geniculate ganglion, which provides innervation to taste buds in the anterior tongue and palate, is unique among sensory ganglia in that its neurons depend on both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF) for survival. Whereas BDNF is additionally implicated in taste axon guidance at targeting stages, much less is known about the guidance role of NT4 during targeting, or about either neurotrophin during initial pathfinding. NT4 and BDNF have distinct expression patterns in vivo, raising the possibility of distinct roles. We characterized the influence of NT4 and BDNF on geniculate neurites in collagen I gels at early embryonic through postnatal stages. During early pathfinding to the tongue (embryonic days 12–13; E12–13), NT4 and BDNF promote significantly longer outgrowth than during intralingual targeting (E15–18). NT4 is more potent than BDNF at stimulating neurite outgrowth and both factors exhibit concentration optima, i.e. intermediate concentrations (0.25 ng/ml NT4 or 25 ng/ml BDNF) promote maximal neurite extension and high concentrations (10 ng/ml NT4 or 200 ng/ml BDNF) suppress it. Only partial suppression was seen at E12 (when axons first emerge from the ganglion in vivo) and postnatally, but nearly complete suppression occurred from E13 to E18. We show that cell death is not responsible for suppression. Although blocking the p75 receptor reduces outgrowth at the optimum concentrations of NT4 and BDNF, it did not reduce suppression of outgrowth. We also report that NT4, like BDNF, can act as a chemoattractant for geniculate neurites, and that the tropic influence is strongest during intralingual targeting (E15–18). NT4 does not appear to act as an attractant in vivo, but it may prevent premature invasion of the epithelium by suppressing axon growth.