Members of the BMP, Shh, and FGF morphogen families promote chicken statoacoustic ganglion neurite outgrowth and neuron survival in vitro

Mechanosensory hair cells of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Members of several morphogen families are expressed within and surrounding the chick inner ear during stages of SAG axon outgrowth and pathfinding. On the basis of their localized expression patterns, we hypothesized that bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and sonic hedgehog (Shh) may function as guidance cues for growing axons and/or may function as trophic factors once axons have reached their targets. To test this hypothesis, three-dimensional collagen cultures were used to grow Embryonic Day 4 (E4) chick SAG explants for 24 h in the presence of purified proteins or beads soaked in proteins. The density of neurite outgrowth was quantified to determine effects on neurite outgrowth. Explants displayed enhanced neurite outgrowth when cultured in the presence of purified BMP4, BMP7, a low concentration of Shh, FGF8, FGF10, or FGF19. In contrast, SAG neurons appeared unresponsive to FGF2. Collagen gel cultures were labeled with terminal dUTP nick-end labeling and immunostained with anti-phosphohistone H3 to determine effects on neuron survival and proliferation, respectively. Treatments that increased neurite outgrowth also yielded significantly fewer apoptotic cells, with no effect on cell proliferation. When presented as focal sources, BMP4, Shh, and FGFs -8, -10, and -19 promoted asymmetric outgrowth from the ganglion in the direction of the beads. BMP7-soaked beads did not induce this response. These results suggest that a subset of morphogens enhance both survival and axon outgrowth of otic neurons.     

Analysis of a repulsive axon guidance molecule, draxin, on ventrally directed axon projection in chick early embryonic midbrain

The mesencephalic V neurons and tectobulbar axons in chick embryo project over long distances that appear during the early development of the chick optic tectum. The mesencephalic V neuron and tectobulbar axonal growth begin at Hamburger and Hamilton stage 14 and stage 18, respectively. Both fibers proceed downward from the dorsal to the ventral side of the lateral wall of the optic tectum and then turn caudally and join the medial longitudinal fasciculus. Their axons appear in the most superficial layer of the tectum at early stages and do not cross the dorsal midline of the tectum. Here, we report the role of draxin, a recently identified axon guidance protein, in the formation of the ventrally directed tectum axonal tracts in chicken embryo. draxin is expressed in a high dorsal to low ventral gradient in chick optic tectum. In vitro experiments show that draxin repels neurite outgrowth from dorsal tectum explants. In vivo overexpression resulted in inhibition or misrouting of axon growth in the tectum. Therefore, draxin may be an important member of the collection of repulsive guidance molecules that regulate the formation of the ventrally directed tectum axon tracts.     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.     

Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.     

Promotion of retinal neurite outgrowth by substratum-bound fibronectin

We have examined conditions under which aggregates of embryonic chick neural retina will extend neurities in vitro. Trypsin-dispersed cells from 7-day embryonic chick neural retina were aggregated in rotation culture for 8 hr and maintained in serum-free medium on a variety of standard culture substrate. Aggregates extend few neurites on untreated plastic, glass, or collagen substrata. However, pretreatment of these substrata with human plasma fibronectin enhances their capacity to support retinal neurite outgrowth. Aggregates cultured on fibronectin-treated substrata extend long, radially oriented neurites within 36 hr in vitro. The morphology of these neurites is distinct from that seen when aggregates are cultured on polylysine-treated substrata. In the latter case, neurites are highly branched and grow concentrically around the aggregate perimeter. Addition of fibronectin to polylysine-treated substrata stimulates radial neurite outgrowth. Promotion of neurite outgrowth is dependent on the amount of fibronectin bound to the culture substratum and on the pH at which binding occurs. The requirements for fibronectin-mediated neurite outgrowth are more stringent than those previously reported for fibroblast attachment and spreading.     

Sensory neurons influence the expression of cell adhesion factors by cutaneous cells in vitro and in vivo

Dorsal root ganglion (DRG) neurons co-cultured with skin-derived fibroblast-like cells (FLCs) show a strong neurite outgrowth. However, when physical contact between FLCs and neurons is prevented with membrane inserts, the DRG neurons exhibit a low survival and a deficient neurite growth. This indicates that cell adhesion molecules influence neuronal survival and neurite growth in co-cultures. The aim of the present study is to find out if selected adhesion molecules are expressed by cultivated FLCs with and without nervous influences, and/or by normal and denervated whole skin. RT-PCR data show that cultured FLCs and denervated skin express L1, N-CAM, N-cadherin and ninjurin, but not neurofascin or TAG-1. However, cultured FLCs exposed to DRG homogenates and innervated skin express N-cadherin only. Following application of neutralizing L1-, N-cadherin- and ninjurin-antibodies (but not N-CAM-antibodies) in the culture medium the mean number of surviving neurons is decreased. Co-cultures incubated with L1-, N-cadherin- or ninjurin-antibodies all show significantly less neurite outgrowth compared to controls. In conclusion, the findings in this paper indicate (i) that FLCs cultured in vitro and denervated whole skin express the cell adhesion factors L1, N-CAM, N-cadherin and ninjurin, (ii) that FLCs treated with neural molecules and innervated whole skin express N-cadherin only, (iii) that L1, N-cadherin and ninjurin are important for DRG neurons co-cultured with FLCs in vitro in terms of survival and neurite extension and (iv) that there may exist subpopulations of DRG-neurons with different sensitivities for N-cadherin- and ninjurin-antibodies.     

N-cadherin, NCAM, and integrins promote retinal neurite outgrowth on astrocytes in vitro

Retinal ganglion neurons extend axons that grow along astroglial cell surfaces in the developing optic pathway. To identify the molecules that may mediate axon extension in vivo, antibodies to neuronal cell surface proteins were tested for their effects on neurite outgrowth by embryonic chick retinal neurons cultured on astrocyte monolayers. Neurite outgrowth by retinal neurons from embryonic day 7 (E7) and E11 chick embryos depended on the function of a calcium-dependent cell adhesion molecule (N-cadherin) and beta 1-class integrin extracellular matrix receptors. The inhibitory effects of either antibody on process extension could not be accounted for by a reduction in the attachment of neurons to astrocytes. The role of a third cell adhesion molecule, NCAM, changed during development. Anti-NCAM had no detectable inhibitory effects on neurite outgrowth by E7 retinal neurons. In contrast, E11 retinal neurite outgrowth was strongly dependent on NCAM function. Thus, N-cadherin, integrins, and NCAM are likely to regulate axon extension in the optic pathway, and their relative importance varies with developmental age.     

Retinoic acid as a survival factor in neuronal development of the grasshopper,Locusta migratoria

Based on experience with cell cultures of adult insect neurons, we develop a serum-free culture system for embryonic locust neurons. Influences of trophic substances on survival and neurite outgrowth of developing neurons are investigated. For the first time, a positive trophic effect of 9-cis retinoic acid (9-cis RA) was shown in vitro on embryonic neurons of an insect. We observed longer cell survival of 50 % developmental stage neurons in cultures supplemented with 0.3 nM 9-cis RA. Furthermore, an influence on neuron morphology was revealed, as the addition of 9-cis RA to cell culture medium led to an increase in the number of neurites per cell. Although an RA receptor gene, LmRXR (Locusta migratoria retinoid X receptor), was expressed in the central nervous system throughout development, the influence of 9-cis RA on neuronal survival and outgrowth was restricted to 50 % stage embryonic cells.     

S100 is present in developing chicken neurons and schwann cell and promotes motor neuron survival in vivo

We used polyclonal antisera recognizing S100, a small acidic protein highly enriched in nervous tissue, to stain sections of embryonic chicken lumbosacral spinal cord and hindlimb. S100 immunoreactivity was detected in developing sensory neurons of the dorsal root ganglia (DRG) and motor neurons of the ventral spinal cord as early as embryonic day (E) 5, and staining persisted through hatching. In contrast, expression of S100 first became apparent in Schwann cells at E13, just before myelination, and was not detected in developing skin or muscle. Since S100β was present in motor and sensory neurons and is known to promote neuronal survival and neurite extension in vitro (Winningham-Major, Staecker, Barger, Coats, and Van Eldik, 1989), we tested the ability of S100 to promote neuron survival in an in ovo survival assay. Addition of S100 to chick embryos in ovo during the period of naturally occurring motor neuron cell death resulted in a significant increase in motor neuron survival, but had no effect on the in vivo survival of sensory neurons in the DRG. The findings that S100 is present in spinal motor neurons and that the addition of S100 enhances the survival of these cells in vivo are consistent with the possibility that S100 may act as a naturally occurring neuron survival factor during development. © 1992 John Wiley & Sons, Inc.     

Primary Neuron Culture for Nerve Growth and Axon Guidance Studies in Zebrafish (Danio rerio)

Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr⁻¹ s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day⁻¹ growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca²⁺-imaging revealed local elevation of cytoplasmic Ca²⁺ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca²⁺ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.     

Enhancement of Neurite Outgrowth and Aspartate-Glutamate Uptake Systems in Retinal Explants Cultured with Chick Gizzard Extract

Chicken gizzard extract promoted a long and radially directed neurite outgrowth from retinal explants of 8-day-old chick embryo in cultures of 2–3 days. The neurite outgrowth from retinal explants cultured in the absence of gizzard extract was short and restricted to the explant perimeter. The neurite outgrowth promoted by gizzard extract depended strictly on several factors. (a) Fetal calf serum and polycationic substratum were required in this culture system, (b) Pretreatment of the polyornithine-coated substratum with gizzard extract allowed the retinal explants to extend neurites even in the absence of gizzard extract in the medium. (c) Maximal neurite outgrowth was observed in retinal explants dissected from 8-day embryos, but thereafter the explants’response to gizzard extract rapidly declined and was almost lost at the 12th day. As a biochemical parameter of differentiation of cultured neuroretina, uptake systems for neurotransmitter candidates were examined in homogenates of retinal explants cultured in the absence or presence of gizzard extract. After 3 days in culture with gizzard extract, the uptake increased for aspartate and glutamate 1.6- to 1.8-fold and for γ-aminobutyric acid to a lesser degree when examined at a concentration for high-affinity uptake (10^-6M). In contrast, the uptake capacity for glycine, choline, and dopamine was not altered in explants cultured with or without gizzard extract. Kinetic analysis showed that the enhanced capacity to accumulate aspartate was not due to an alteration of K^m, but to an increase of V^max. The results suggest that one or several factors in chick gizzard muscle promote not only neurite outgrowth but also the aspartate-glutamate uptake systems in the developing neuroretina, probably related to ganglion cells.     

Inhibition of branching and spine maturation by repulsive guidance molecule in cultured cortical neurons

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies in Xenopus and chick embryos revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos demonstrated its function in regulating cephalic neural tube closure. Moreover, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we demonstrate in vitro that RGMa, an RGM homolog, inhibits neurite growth and cortical neuron branching on mouse embryonic day 16. Further, exposure of cultured neurons to RGMa significantly reduced the number of colocalized immunoreactive clusters of synapsin 1 and PSD-95 in the spines. This RGMa-mediated inhibition of the assembly of presynaptic and postsynaptic components suggests a role of RGMa in inhibiting mature synapse formation. Thus, RGMa may negatively regulate neuronal network formation in cortical neurons.     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia

The ability of embryonic chick heart to elicit neuritic outgrowth in different ganglia was tested to examine (1) whether stimulative activity is possessed by the heart only at specific stages and (2) whether the ability of the ganglionic neurons to respond is limited to certain periods of development. As an assay, ganglia were explanted into thin collagen gels with ventricular tissue placed at a distance of about 1 mm. Neuritic outgrowth was measured after 2 days. Control ganglia and ganglia cultured with added nerve growth factor (NGF) were also scored. Four types of tested ganglia, including the ciliary ganglion, showed a peak in neuritic outgrowth when cultured with heart of embryonic Day 18, at about which age the heart becomes sympathetically innervated in ovo. No age-related size differences that could account for this temporal pattern were found among the heart explants when measuring their protein content. A peak in neuronal susceptibility to heart tissue was evident in the 6-day ciliary ganglion and in the 8-day paravertebral, Remak, and spinal ganglia, roughly coinciding with the onset of fibre outgrowth in ovo. Neurite extension is concluded to have been triggered by a factor spread from the heart explants and being distinct from the mouse type of NGF since anti-NGF did not at any stage block the events and since added NGF at all stages failed to evoke neurite formation in the ciliary ganglia. A testable hypothesis is that this factor regulates the growth of sympathetic and possibly parasympathetic and sensory fibres in the developing chick heart.     

The effect of cyclic AMP on neuritic outgrowth in explant cultures of developing chick olfactory epithelium

The effect of cyclic AMP (cAMP) analogs and phosphodiesterase (PDE) inhibitors on neurite outgrowth was studied in explant cultures of olfactory neurons. Nasal pits from 5- or 6-day-old chick embryos were minced, explanted into culture dishes, and grown in a serum-free medium. One of the cyclic AMP analogs, dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8-Br-cAMP), or one of the PDE inhibitors, theophylline or isobutylmethylxanthine (IBMX), was added to the culture medium. The explants were examined for neurite outgrowth after 2 days in vitro. Db-cAMP increased the number of explants expressing neurites by 25-35% over control cultures, whereas 8-Br-cAMP had essentially no effect at the same concentrations. Addition of dibutyryl cyclic GMP (dbcGMP) gave no increase in neurite outgrowth, thus indicating that the effect of enhancing neuritic growth is specific to cAMP and not cyclic nucleotides in general. The resulting increase in neurite outgrowth is due to the cyclic nucleotide component of dbcAMP, since both IBMX and theophylline, which elevate intracellular cAMP, also increased neurite outgrowth significantly. When forskolin was added to the culture medium, there was a trend to increased neurite outgrowth; this was significantly enhanced when a subthreshold concentration of theophylline was added in addition to the forskolin.     

Influence of Olfactory Epithelium on Mitral/Tufted Cell Dendritic Outgrowth

Stereotypical connections between olfactory sensory neuron axons and mitral cell dendrites in the olfactory bulb establish the first synaptic relay for olfactory perception. While mechanisms of olfactory sensory axon targeting are reported, molecular regulation of mitral cell dendritic growth and refinement are unclear. During embryonic development, mitral cell dendritic distribution overlaps with olfactory sensory axon terminals in the olfactory bulb. In this study, we investigate whether olfactory sensory neurons in the olfactory epithelium influence mitral cell dendritic outgrowth in vitro. We report a soluble trophic activity in the olfactory epithelium conditioned medium which promotes mitral/tufted cell neurite outgrowth. While the trophic activity is present in both embryonic and postnatal olfactory epithelia, only embryonic but not postnatal mitral/tufted cells respond to this activity. We show that BMP2, 5 and 7 promote mitral/tufted cells neurite outgrowth. However, the BMP antagonist, Noggin, fails to neutralize the olfactory epithelium derived neurite growth promoting activity. We provide evidence that olfactory epithelium derived activity is a protein factor with molecular weight between 50–100 kD. We also observed that Follistatin can effectively neutralize the olfactory epithelium derived activity, suggesting that TGF-beta family proteins are involved to promote mitral/tufted dendritic elaboration.     

β-Site Amyloid Precursor Protein (APP)-cleaving Enzyme 1 (BACE1)-deficient Mice Exhibit a Close Homolog of L1 (CHL1) Loss-of-function Phenotype Involving Axon Guidance Defects

BACE1 is the β-secretase enzyme that initiates production of the β-amyloid peptide involved in Alzheimer disease. However, little is known about the functions of BACE1. BACE1-deficient mice exhibit mild but complex neurological phenotypes suggesting therapeutic BACE1 inhibition may not be completely free of mechanism-based side effects. Recently, we have reported that BACE1 null mice have axon guidance defects in olfactory sensory neuron projections to glomeruli in the olfactory bulb. Here, we show that BACE1 deficiency also causes an axon guidance defect in the hippocampus, a shortened and disorganized infrapyramidal bundle of the mossy fiber projection from the dentate gyrus to CA3. Although we observed that a classical axon guidance molecule, EphA4, was cleaved by BACE1 when co-expressed with BACE1 in HEK293 cells, we could find no evidence of BACE1 processing of EphA4 in the brain. Remarkably, we discovered that the axon guidance defects of BACE1^−/− mice were strikingly similar to those of mice deficient in a recently identified BACE1 substrate, the neural cell adhesion molecule close homolog of L1 (CHL1) that is involved in neurite outgrowth. CHL1 undergoes BACE1-dependent processing in BACE1^+/+, but not BACE1^−/−, hippocampus, and olfactory bulb, indicating that CHL1 is a BACE1 substrate in vivo. Finally, BACE1 and CHL1 co-localize in the terminals of hippocampal mossy fibers, olfactory sensory neuron axons, and growth cones of primary hippocampal neurons. We conclude that BACE1^−/− axon guidance defects are likely the result of abrogated BACE1 processing of CHL1 and that BACE1 deficiency produces a CHL1 loss-of-function phenotype. Our results imply the possibility that axon mis-targeting may occur in adult neurogenic and/or regenerating neurons as a result of chronic BACE1 inhibition and add a note of caution to BACE1 inhibitor development.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

L1-mediated axon outgrowth occurs via a homophilic binding mechanism

The molecular mechanism by which the L1 cell adhesion molecule mediates neurite outgrowth has been examined. Purified L1 from mouse and L1 from chick brain were attached to nitrocellulose dishes. Both chick and mouse neurons were able to adhere to purified mouse L1 and chick L1. Both molecules promoted neurite extension from chick and mouse neurons. Addition of Fabs specific for chick L1 to the cultures inhibited chick neurite outgrowth on both mouse L1 and chick L1. These findings suggest that L1-like molecules support neurite outgrowth via a "homophilic" binding mechanism.