Ephrin-A3 is required for tonotopic map precision and auditory functions in the mouse auditory brainstem.

Tonotopy is a prominent feature of the vertebrate auditory system and forms the basis for sound discrimination, but the molecular mechanism that underlies its formation remains largely elusive. Ephrin/Eph signaling is known to play important roles in axon guidance during topographic mapping in other sensory systems, so we investigated its possible role in the establishment of tonotopy in the mouse cochlear nucleus. We found that ephrin-A3 molecules are differentially expressed along the tonotopic axis in the cochlear nucleus during innervation.

Ephrin-B/EphB Signaling Is Required for Normal Innervation of Lingual Gustatory Papillae.

The innervation of taste buds is an excellent model system for studying the guidance of axons during targeting because of their discrete nature and the high fidelity of innervation. The pregustatory epithelium of fungiform papillae is known to secrete diffusible axon guidance cues such as BDNF and Sema3A that attract and repel, respectively, geniculate ganglion axons during targeting, but diffusible factors alone are unlikely to explain how taste axon terminals are restricted to their territories within the taste bud. Nondiffusible cell surface proteins such as Ephs and ephrins can act as receptors and/or ligands for one another and are known to control axon terminal positioning in several parts of the nervous system, but they have not been studied in the gustatory system.

Neurotrophin-4 is more potent than brain-derived neurotrophic factor in promoting, attracting and suppressing geniculate ganglion neurite outgrowth.

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.

Brain-derived neurotrophic factor attracts geniculate ganglion neurites during embryonic targeting.

Geniculate axons are initially guided to discrete epithelial placodes in the lingual and palatal epithelium that subsequently differentiate into taste buds. In vivo approaches show that brain-derived neurotrophic factor (BDNF) mRNA is concentrated in these placodes, that BDNF is necessary for targeting taste afferents to these placodes, and that BDNF misexpression disrupts guidance. We used an in vitro approach to determine whether BDNF may act directly on geniculate axons as a trophic factor and as an attractant, and whether there is a critical period for responsiveness to BDNF.

Distinct roles for Sema3A, Sema3F, and an unidentified trophic factor in controlling the advance of geniculate axons to gustatory lingual epithelium.

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.

Neurotrophic factor receptor expression and in vitro nerve growth of geniculate ganglion neurons that supply divergent nerves.

We investigated which neurotrophic factors may contribute to the divergence of two peripheral nerves emanating from the geniculate ganglion. We compared receptor mRNA profiles of the neurons that supply the nerves, and also the growth of their neurites in response to neurotrophic factors in culture. Three mRNAs, Gfra2, TrkA, and TrkC, were differentially expressed.

Developmental expression of neurotrophin receptor genes in rat geniculate ganglion neurons.

Individual neurons dissected from immunohistochemically stained paraffin sections of the developing rat geniculate (VIIth cranial) ganglion were assayed for their content of mRNA of the neurotrophin receptor genes, p75 , trkA , trkB and trkC. Fetal and postnatal rats, from the 13th embryonic day (E13) until the 20th postnatal day (P20), were used. Single cells were subjected to RNA amplification, followed by treatment with reverse transcriptase and DNA amplification by the polymerase chain reaction (PCR).

Sema3A regulates the timing of target contact by cranial sensory axons.

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.

Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and 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.

Polymerizing microtubules activate site-directed F-actin assembly in nerve growth cones.

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.

Trigeminal ganglion axons are repelled by their presumptive targets.

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.

Microtubule stability decreases axon elongation but not axoplasm production.

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.

Localization of myosin II A and B isoforms in cultured neurons.

Tension generated by growth cones regulates both the rate and the direction of neurite growth. The most likely effectors of tension generation are actin and myosins. We are investigating the role of conventional myosin in growth cone advance.

The influence of AChR clustering stimuli on the formation and maintenance of AChR clusters induced by polycation-coated beads in Xenopus muscle cells.

Nerve, polycation-coated beads, and electric fields not only induce acetylcholine receptors (AChRs) to cluster, but they also reduce the number of spontaneous AChR patches (hotspots) away from the induced cluster sites on embryonic Xenopus myotomal muscle cells grown in tissue culture (the global effect). In vivo, the ability of an AChR clustering stimulus to depress cluster formation elsewhere on the muscle cell may influence both the site at which the neuromuscular junction develops as well as which axons survive during synapse elimination. Since the causes of hotspot formation may be variable and cannot be controlled, we have further characterized the global effect by using AChR-clustering stimuli that can be controlled by the experimenter.

Localization of intracellular proteins at acetylcholine receptor clusters induced by electric fields in Xenopus muscle cells.

Electric fields cause acetylcholine receptor (AChR) patches to form on the cathodal sides of cultured muscle cells. These patches are stable for several hours following cessation of an electric field treatment, indicating that the receptors are anchored to the cluster sites. Furthermore, at the ultrastructural level, AChR patches induced by electric fields are marked by an accumulation of extracellular matrix material and a sarcolemmal density.

The relationship between talin and acetylcholine receptor clusters in Xenopus muscle cells.

Talin is involved in mediating the cytoskeleton-extracellular matrix interaction at focal contacts in cultured fibroblasts. Recently this protein has been localized at both the myotendinous junction (MTJ) and the neuromuscular junction (NMJ) in skeletal muscle. At the MTJ, talin may mediate the insertion of myofibrils into the plasma membrane, thus serving a function similar to that seen at focal contacts.