Papers by William Rochlin
Journal of Comparative Neurology
Journal of Cell Science
Tension generated by growth cones regulates both the rate and the direction of neurite growth. Th... more 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. In this paper we report the localization of the two most prominent isoforms of brain myosin II in growth cones, neurites and cell bodies of rat superior cervical ganglion neurons. Affinity purified polyclonal antibodies were prepared against unique peptide sequences from human and rat A and B isoforms of myosin heavy chain. Although each of these antibodies brightly stained nonneuronal cells, antibodies to myosin heavy chain B stained neurons with greater intensity than antibodies to myosin heavy chain A. In growth cones, myosin heavy chain B was most concentrated in the margin bordering the thickened, organelle-rich central region and the thin, actin-rich peripheral region. The staining colocalized with actin bundles proximal and distal to the...
Developmental neuroscience, Jan 2, 2016
The innervation of taste buds is an excellent model system for studying the guidance of axons dur... more 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. We report that ephrin-B2 linked β-galactosidase staining and immunostaining was present along the dorsal epithelium of the mouse tongue as early as embryonic day 15.5 (E15.5), but was not detected at E14.5, when a...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1998
Previous work suggested that in mouse, presumptive targets of the trigeminal ganglion, rather tha... more 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 arr...
Molecular Biology of the Cell, 1999
We identify an actin-based protrusive structure in growth cones termed “intrapodium.” Unlike filo... more 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 bur...
The Journal of Comparative Neurology, 2004
The trigeminal ganglion provides the somatosensory innervation for the anterior rat tongue. At ea... more 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]
The Journal of Comparative Neurology, 2000
Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tong... more 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 targetderived 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.
Developmental Neuroscience, 2010
BDNF beads did not advance beyond them. At E18, when axons would be penetrating pregustatory epit... more BDNF beads did not advance beyond them. At E18, when axons would be penetrating pregustatory epithelium in vivo, BDNF continued to exert a tropic effect on geniculate neurites. However, at postnatal and adult stages, the influence of BDNF was predominantly trophic. Our data support a role for BDNF acting as an attractant for geniculate axons during a critical period that encompasses initial targeting but not at later stages.
Developmental Neuroscience, 2005
We investigated which neurotrophic factors may contribute to the divergence of two peripheral ner... more 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. Only one ligand, Neurturin, promoted substantially different nerve regrowth from the nerves, and therefore may contribute to nerve divergence. Three receptor mRNAs were expressed in 100% of the neurons: TrkB, TrkB.T2 (kinase-lacking isoform), and NCAM-140. Ligands for these Trks and FRalpha-1 promoted more outgrowth than ligands for the other receptors. NT-3 and BDNF synergistically promoted outgrowth. Finally, receptors are coexpressed at random rates, arguing against the existence of neuronal subtypes defined by a combinatorial code of these receptors.
Developmental Neuroscience, 2012
ml NT4 or 200 ng/ml BDNF) suppress it. Only partial suppression was seen at E12 (when axons first... more 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.
Journal of Neurocytology, 2000
Individual neurons dissected from immunohistochemically stained paraffin sections of the developi... more 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). The identity of the PCR products was verified by subcloning and sequencing. A total of 227 neurons were examined, of which 212 (93%) gave a PCR signal for at least one neurotrophin receptor. We found: (1) Approximately half of the neurons expressed more than one receptor. (2) A truncated version of trkB, possessing the ligand-binding region but lacking the tyrosine kinase domain, occurred quite frequently, often in combination with the full-length trkB, with trkA or both. (3) The pattern of staining for trkB-like immunoreactivity was usually predictive that either its full length or truncated mRNA would be present. This was not the case for trkC-like immunoreactivity. Western blots on E15 brain tissue showed no band for full-length trkC (∼ 150 kDa), suggesting the antibody may have been immunoreactive with a truncated (∼ 120 kDa) but not a full-length version of the trkC receptor. (4) The pattern of neurotrophin receptor gene expression changed during development. (5) p75 expression occurred infrequently-in only 7 of the 212 neurons that gave a signal for any receptor.
Journal of Neurocytology, 2004
Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3-4 days be... more 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.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1996
Microtubules are a primary cytoskeletal constituent of axons and growth cones. In addition to ser... more 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 nocoda...
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Papers by William Rochlin