Nerve Growth Factor Gradient Research Articles

Evidence for nerve growth factor-mediated paracrine effects in human epidermis.

Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.

Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.

Recent studies have shown that developing nerve fibres grow directly to their targets and are guided by specific cues, but the nature of these cues and the mechanism of guidance remain unknown. The growth of sympathetic axons towards an artificial source of nerve growth factor (NGF) in vivo and of sensory neurites up a concentration gradient of NGF in vitro has supported the hypothesis that NGF, produced by target tissues, acts as a chemotactic attractant for these nerve fibres during development. Both these studies and those of the influence of NGF or target tissues on neurite growth in vitro were conducted late in development when, following target encounter, the neurones had become dependent on NGF or target for survival. Here we have co-cultured embryonic mouse sensory neurones and their peripheral target tissue at a stage preceding their contact in vivo. Neurites grew directly and exclusively towards their own target but not to regionally inappropriate peripheral tissue. Antiserum to isogeneric NGF did not reduce this outgrowth but did reduce undirected neurite outgrowth which occurred in co-cultures of older neurones with denervated target tissue. These results demonstrate that agents other than NGF guide neurites of NGF-responsive neurones in development.

Characterization of the turning response of dorsal root neurites toward nerve growth factor.

This study reports that chick dorsal root ganglion neurites undergo a rapid (20 min) reorientation of their direction of growth in response to nerve growth factor (NGF) concentration gradients in vitro. Dorsal root ganglia from chick embryos were explanted onto a collagen-poly-L-lysine substrate. After 24-48 h in culture, NGF gradients were applied to individual growth cones via a micropipette containing 50 biological units NGF/ml. The growth cones turned and grew toward these NGF sources. This turning response was not caused by the trophic effects of NGF on neurite initiation, survival, or growth rate. Dorsal root neurites also grew toward sources of mono- and dibutyryl cyclic adenosine monophosphate (dB cAMP), cyclic guanosine monophosphate (cGMP), and elevated calcium in the presence of the calcium ionophore A23187. These results are consistent with the hypothesis that intracellular levels of cAMP and /or cGMP and calcium may play a role in the turning response of dorsal root neurites toward NGF, but do not establish a causal relationship between the mechanisms of action of NGF, cyclic nucleotides and calcium. Total growth cone adherence to the substrate was measured using a timed microjet of perfusion medium. NGF increased the adherence of growth cones to the substrate, but caffeine and dB cAMP which also elicit the positive turning response, decreased growth cone adherence. Calcium, which did not elicit the positive turning response, produced a greater growth cone adherence to the substrate than that observed with NGF. Although these results do not rule out a role of adhesion changes in axonal turning to NGF, they show that a general increase in adherence does not correlate well with the rapid turning response observed in this study.

Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture.

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.

Chemotactic response of nerve fiber elongation to nerve growth factor

These studies seek to determine whether the locomotory behavior of the extending tip of growing nerve fibers exhibits chemotaxis to nerve growth factor (NGF). Sensory neurons from dissociated dorsal root ganglia of chick embryos were cultured within semisolid agar matrices containing concentration gradients of NGF which diffused from an adjacent source. A preferential orientation of approximately 60% of the tips of nerve fibers and an enhanced extension of fibers up NGF gradients were observed. The initiation sites of nerve fibers on cell bodies were not consistently oriented by these NGF gradients. Orientation of nerve fiber extension toward the NGF source was observed in chambers with NGF sources ranging from 25 to 1000 ng/ml of β-NGF and from 400 to 5000 ng/ml of 7 S NGF, but no orientation was observed when the concentration of the NGF source was 15000 ng/ml of β-NGF. This oriented response apparently is not a concentration-dependent trophic response to NGF. The evidence supports the notion that chemotaxis is a regulatory factor in neuronal morphogenesis.