Early embryonic quail dorsal root ganglia exhibit high affinity nerve growth factor binding and NGF responsiveness — absence of NGF receptors on migrating neural crest cells

DeL,elopmental Brain Research, 75 (1993) 55-64 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

55

BRESD 51679

Early embryonic quail dorsal root ganglia exhibit high affinity nerve growth factor binding and NGF responsiveness - absence of NGF receptors on migrating neural crest cells Joycelyn L. Speight, Lihua Yao, Irma Rozenberg and Paulette Bernd Department of Anatomy and Cell Biology, State Unicersity of New York, Health Science Center at Brooklyn, Brooklyn, NY 11203 (USA) (Accepted 2(1 April 1993)

Key words: Nerve growth factor (NGF); Neurotrophin; Nerve growth factor receptor; trk; Dorsal root ganglion: Neural crest; Quail: Avian

Dorsal root ganglia (DRG) midway through development require nerve growth factor (NGF) for survival and differentiation. These studies investigated when avian neural crest cells or D R G first exhibit high affinity NGF receptors in situ, and whether early embryonic cells expressing high affinity NGF receptors are responsive to NGF. Unfixed cryostat sections of quail embryos were exposed to varying concentrations of [125I]NGF to distinguish between high and low affinity binding. Radioautography revealed an absence of [125I]NGF binding on migrating neural crest cells in situ. Both high and low affinity NGF receptors were first detected in differentiating DRG at E3.5 (stage 23). The presence of high affinity receptors was additionally confirmed by identification of a high molecular weight complex on radioautographs of gels following cross-linking of [125I]NGF to dissociated DRG. The presence of high affinity NGF receptors in E3.5 DRG was unexpected since DRG have been reported to be unresponsive to NGF prior to the midpoint of development. Exposure of E3.5 DRG neuron-enriched cultures to exogenous NGF resulted in approximately 30% more neurons after 24 h in vitro. The effect of NGF was blocked by anti-NGF and was shown to be dose dependent. It remains to be determined whether the increase in cell number is due to a survival or mitogenic effect.

INTRODUCTION

proto-oncogene (also known as trk), a tyrosine kinase receptor ( p l 4 0 P r ° t ° t r k ) 28'37"75.

N G F is known to be required for the survival and differentiation of D R G neurons during development 45. This N G F dependency has been shown to vary depending upon embryonic age, with a maximal response in neurite outgrowth and survival occurring midway through development (i.e. avian embryonic days 7 to 13; E7 t o E 1 3 ) 2'3"12'15'24'77. Embryonic NGF-responsive neurons have been shown to possess specific NGF receptors on their cell surfaces with two apparent classes of receptors (site I, high affinity or p140 pr°t°trk, K d = 0.02 nM; site II, low affinity or p75 N°vR, K a = 1.2 n M ) 1'22'5157'5~s'65'72. The presence of high affinity NGF receptors is indicative of a potentially responsive cell, since most of NGF's biological effects appear to be mediated by the high affinity subtype 5'44. Recent studies have demonstrated that high affinity N G F binding requires the presence of the product of the t r k A

Relatively little is known about the role of NGF in the growth and differentiation of early D R G (i.e. avian E3 to E4), or its embryonic predecessor, the neural crest. Comparatively undifferentiated avian neural crest cells, either at the time of explantation or after 24 h in culture, do not exhibit NGF receptors v'25, suggesting that neural crest cells are unresponsive to NGF. However, N G F receptor m R N A has been detected by in situ hybridization in migrating neural crest cells 3°. D R G first exhibit binding of [12SI]NGF as early as E4 in the chick ~'°, but neither receptor subtype nor cell phenotype were distinguished. Although [1251]NGF binding was detected at this early stage of embryonic development, several studies have demonstrated that NGF does not promote neurite outgrowth in explants of these ganglia 13'15'4~'5°, nor does it appear to accelerate the maturation of sensory neuroblasts vs. It is less clear

Correspondence: P. Bernd, Department of Anatomy and Cell Biology, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, USA. Fax: (1)(718) 270-3732.

56 whether N G F plays a role in the survival of E3 to E4 D R G neurons. The results of Kalcheim et al. 36 would suggest that N G F does not influence survival, because N G F cannot rescue D R G neurons that have been physically separated from the neural tube by a silastic membrane in vivo. Furthermore, Ernsberger and Rohrer ~9 have shown that neuronal precursors from D R G do not initially require N G F for survival in culture. These studies were performed to determine when avian neural crest cells or D R G first exhibit high affinity N G F receptors in situ, and, furthermore, to determine whether early embryonic ceils expressing high affinity N G F receptors are responsive to NGF. High affinity N G F receptors were detected in early embryonic D R G by two independent methods; radioautography following exposure of unfixed cryostat sections to low concentrations of [t25I]NGF, and crosslinking to distinguish between the two N G F receptor subtypes by virtue of their molecular weights. These D R G were also shown to be responsive to NGF, as indicated by an increase in neuronal cell number in vitro. MATERIALS AND METHODS

Preparation of"quail embryo frozen sections Quail embryos were removed from their shells after varying lengths of incubation, dissected free of surrounding membranes, and staged according to morphological criteria used for chick embryos 27. Entire embryos were then coated in Tissue Tek (Miles Laboratories, Elkhart, IN), and quickly frozen in 2-methylbutane (Aldrich, Milwaukee, WI) that had been chilled in liquid nitrogen. Serial 10/~m tissue sections were cut on a Leitz cryostat and placed on slides coated with 3-aminopropyltriethoxysilane (2% in acetone; Sigma, St. Louis, MO). Sections to be processed for immunocytochemistry were fixed immediately after sectioning in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4). All slides were stored with dessicant at - 7 0 ° C until use (within 7 days).

Preparation of [ leSl]NGF 2.5S NGF was prepared from the salivary glands of adult male mice (Bioproducts for Science, Indianapolis, IN) 56, and iodinated as described previously using carrier-free NalZSI (15 Ci/mg, 100 mCi/ml; Amersham, Arlington Heights, IL) 8"7z. The reaction yielded [IzsI]NGF that was greater than 98% precipitable with trichloroacetic acid, had a specific activity of approximately 150 cpm/pg, and a concentration of 4 ng//zl.

time less than 1 min), and fixed as described above. Slides were thell processed for radioautography.

lmmunocytochemistry After thawing and fixation, sections were immunocytochemically stained using an avidin-biotin-peroxidase technique :~5. Migratory neural crest cells, as well as their derivatives undergoing gangliogenesis, were identified by Leu-7, a hybridoma ascites containing antiHNK-1 antibodies (Becton Dickinson, Mountain View, CA) f'4. Serial sections in which the primary antisera was omitted were used as controls (data not shown).

Light microscopic radioautography Following exposure to [12Sl]NGF, sections were dipped in nuclear emulsion (Kodak NTB-2, diluted 1 : 1 in water at 42°C) and exposed for 3 to 7 days at 4°C. Slides were then developed (D-19, 5 min), fixed, rinsed in water, dehydrated and coverslipped with Permount. Silver grains were visualized using darkfield microscopy, and phase optics were used to identify morphological features.

Preparation of dissociated dorsal root ganglia DRG were isolated from E3.5 (stage 23) quail embryos in cold Hanks' balanced salt solution (HBSS) containing calcium (Ca 2÷ ) and magnesium (Mg 2+ ). Dissociation was accomplished by exposure of DRG to HBSS (without Ca 2+ or Mg 2+) containing Dispase (0.5%, 0.5 U / m g ; Boehringer Mannheim, Indianapolis, IN) for 60 min in a 37°C water bath. Following dissociation, cells were spun (1000 rpm, 2 min; Centra-7R, International Equipment Company, Needham Heights, MA).

Cross-linking Following centrifugation, the supernatant was discarded, and the pellet resuspended in 1 ml of ice-cold binding buffer composed of KH2PO 4 1.2 mM (pH 7.4), NaCI 137 mM, KCI 4.7 raM, CaCI e 2.5 mM, MgSO4 1.2 mM, glucose 1 mg/ml, BSA 1 mg/ml, Hepes 10 mM (pH 7.0), and teupeptin 4 /,g/ml. All binding buffer reagents were obtained from Sigma. Dissociated DRG were counted with a hemacytometer with the total number of cells ranging from 2- to 4 x 106. [1251]NGF was cross-linked to its receptor as described by Escandon and Chao 2°. Control samples of dissociated DRG were pre-incubated with an excess of non-radioactive NGF (50/zg/ml) in ice-cold binding buffer (described above) for 30 min to 1 h, prior to incubation with [1251]NGF. Aliquots of dissociated cells were then added to an equal volume of binding buffer containing [1251]NGF (final concentration of 25 ng/ml; 2 h, on ice). At the end of incubation, the cross-linker was added at a final concentration of 50 /zM (2.25x 10 -3 M N-hydroxysuccinimidyl-4-azidobenzoate in dimethylsulfoxide, HSAB; Pierce, Rockford, 1L), and the mixture exposed to long-wave UV illumination (365 nm, 125 V, 60 Hz) for 7 rain on ice. Following incubation, the reaction was quenched with 50 mM lysine in PBS, and cells were washed twice in tris-saline (10 mM; pH 7.4). Dissociated DRG were resuspended in sample buffer, heated (5 min, 100°C), and frozen (-70°C) until electrophoresis. Sample buffer was composed of 50 mM Tris-HCl (pH 6.8), I00 nM dithiothreitol, 2% sodium dodecylsulfate (SDS), 10% glycerol, and 0.1% Bromophenol blue.

SDS-polyacrylamide gel electrophoresis Localization of receptors for NGF NGF receptors were localized as described by Richardson et al.63. Unfixed cryostat sections were briefly warmed on a hot plate at low heat to remove condensation and insure maximal adhesion of the tissue to the slide. The sections were then exposed to either a low (2 ng/ml; 80 pM) or high (20 ng/ml; 800 pM) concentration of [125I]NGF for 90 min at room temperature in phosphate-buffered saline (PBS) containing cytochrome C (1 mg/ml; Sigma), and leupeptin (4 p~g/ml; Sigma). Control slides with adjacent cryostat sections were incubated with an excess of non-radioactive NGF (l /*g/ml), in addition to [1251]NGF. Following exposure to [125I]NGF, slides were rapidly rinsed in 3 to 4 changes of ice-cold PBS (total

Gel electrophoresis was performed according to the procedure of Laemmli 39. The samples were thawed and equal amounts of cells (ranging from 2- to 4 x 105 cells depending on the experiment) were loaded on each lane of an SDS-polyacryiamide slab gel (4% stacking gel, 10% separating gel). Non-radioactive high molecular weight standards (Bio-Rad, Melville, NY) were run on an adjacent lane, including myosin (200 kDa), E. coli /3-galactosidase (116 kDa), phosphorylase B (97 kDa), BSA (66 kDa), and ovalbumin (45 kDa). Following etectrophoresis, gels were stained (025% Coomasie blue, 45.5% methanol, 9% acetic acid), destained (5% methanol, 5% acetic acid), dried under vacuum and heat, exposed to Kodak XOMAT AR film for 2 to 3 days, and developed in an Kodak X-OMAT processor.

57

Preparation of neuron-enriched dorsal root ganglia cultures E3.5 (stage 23) DRG were dissociated as described above. Neuron-enriched cultures were prepared by taking advantage of the differential attachment rate of neuronal and non-neuronal cells, according to the method of McCarthy and Partlow s4. Following centrifugation, the pellet was resuspended in complete medium (Eagle's minimal essential medium with Earle's salts and glutamine, 5% chicken embryo extract (CEE), 15% fetal bovine serum, 50 U / m l penicillin, 25 U / m l streptomycin, and 0.15% sodium bicarbonate; all reagents, except CEE, were obtained from GIBCO, Grand Island, NY; CEE was prepared from E9 whole chick embryos). Cells were counted with a hemacytometer and approximately 1.5 × 106 cells were added to uncoated 35 mm tissue culture dishes for 4 h at 37°C with intermittent agitation, after which cells remaining in suspension were transferred to 17 mm wells coated with 0.75 mg/ml polyornithine and 15 /xg/ml laminin (Sigma) 19 at a density of 250(I cells/well. Cultures were maintained at 37°C in a water-saturated atmosphere containing 7% CO 2 in the presence or absence of NGF (0.1 to 300 ng/ml) and/or a monoclonal anti-NGF antibody raised against mouse NGF (100 ng/ml; Boehringer Mannheim). In some studies, 250 ng of anti-NGF was pre-absorbed with 2.8 tzg of NGF (24 h, 4°C).

Assessment of neuronal cell number

f r o m t h e d o r s a l s u r f a c e o f t h e n e u r a l t u b e 42. A r e l a tively h i g h c o n c e n t r a t i o n ng/ml),

thereby

o f [125I]NGF was u s e d (20

labeling both

high and

low affinity

N G F r e c e p t o r s 72. E x a m i n a t i o n o f light m i c r o s c o p i c radioautographs

revealed a homogenous

distribution of

silver grains, e x c e p t f o r a slight i n c r e a s e in d e n s i t y o v e r t h e e c t o d e r m (Fig. 1A). T h e s a m e p a t t e r n o f b i n d i n g , i n c l u d i n g t h e i n c r e a s e d d e n s i t y o v e r t h e e c t o d e r m , was o b s e r v e d u n d e r c o n t r o l c o n d i t i o n s in w h i c h a n a d j a c e n t s e c t i o n w a s i n c u b a t e d w i t h an e x c e s s o f n o n - r a d i o a c t i v e NGF

(1 t x g / m l ) , in a d d i t i o n to [1251]NGF (Fig. 1B).

T h e s e r e s u l t s s u g g e s t t h a t m i g r a t i n g n e u r a l c r e s t cells in situ d o n o t e x h i b i t e i t h e r h i g h o r low affinity N G F r e c e p t o r s . T h e p r e s e n c e o f m i g r a t i n g n e u r a l c r e s t cells was

confirmed

in an

adjacent

section

stained

with

a n t i - H N K - 1 a n t i b o d i e s (Fig. 1C). H N K - I

is a cell sur-

f a c e d e t e r m i n a n t s h o w n to b e p r e s e n t a v i a n n e u r a l c r e s t cells 74.

on migrating

After 24 h in vitro, neuronal cell number was determined by counting the number of neurons along 4 different diameters, each pair separated by 45 °, and intersecting at the middle of the well. In this manner, approximately 11% of the well was randomly counted. Neurons were identified by virtue of phase bright cell bodies and possession of at least one neurite (see Fig. 4). For each set of experiments, neuronal cell number was expressed as a percentage of the maximal neuronal cell number obtained under the various culture conditions. Statistical significance (P < 0.001) was determined using an analysis of variance (Systat program for the IBM).

b i n d i n g at e i t h e r a h i g h c o n c e n t r a t i o n (20 n g / m l ) o r a

RESULTS

b o t h [~25I]NGF c o n c e n t r a t i o n s , a c c u m u l a t i o n s o f silver

Time course o f appearance o f N G F receptors on differentiating D R G M i g r a t i n g n e u r a l c r e s t cells a g g r e g a t e to f o r m D R G at a p p r o x i m a t e l y s t a g e s 20 to 214°. E3.5 ( s t a g e 23) was t h e e a r l i e s t s t a g e at w h i c h D R G

e x h i b i t e d [125I]NGF

r e l a t i v e l y low c o n c e n t r a t i o n (2 n g / m l ) o f [ 125 I ] N G F . A t g r a i n s w e r e s e e n o v e r D R G (Fig. 2A; h i g h e r c o n c e n t r a -

Absence o f N G F receptors on migrating neural crest cells"

tion not shown).

No such

accumulations

were

seen

r e g i o n o f s t a g e 19

u n d e r c o n t r o l c o n d i t i o n s in w h i c h an a d j a c e n t s e c t i o n

( e m b r y o n i c day 2; E 2 ) q u a i l e m b r y o s w e r e e x a m i n e d for t h e ability to b i n d [~2SI]NGF; a s t a g e at w h i c h

h a d b e e n e x p o s e d to an e x c e s s o f n o n - r a d i o a c t i v e N G F (1 / x g / m l ) , in a d d i t i o n to [~25I]NGF (Fig. 2B; h i g h e r

n e u r a l c r e s t cells a r e in t h e p r o c e s s o f m i g r a t i n g a w a y

c o n c e n t r a t i o n n o t shown). D R G w e r e also v i s u a l i z e d in

Cryostat seotions of the trunk

7

Fig. 1. Binding of [125I]NGF to stage 19 quail embryos. A,B: darkfield images of radioautographs (5 day exposure) prepared from adjacent unfixed cryostat sections following exposure to 20 ng/ml [I25I]NGF alone (A) or [t251]NGF plus an excess of non-radioactive NGF (1 #g/ml; B). Note the homogenous distribution of silver grains under both experimental (A) and control (B) conditions. There is a slight increase in grain density over the ectoderm (arrows; A,B) that is evident in both conditions, indicating that it is not due to specific binding of [1251]NGF. Marker = 100 tzm. C: brightfield image of HNK-1 immunoreactivity in a section adjacent to those in A and B. Note the presence of HNK-l-positive cells in areas through which neural crest cells would be expected to be migrating (arrows). The magnification is the same as in A and B. NT, neural tube: No, notochord.

58

Fig. 2. Binding of [125I]NGFto E3.5 (stage 23) quail embryos. A,B: darkfield images of radioautographs (6 day exposure) prepaled from adjacent unfixed cryostat sections following exposure to 2 ng/ml [t25I]NGF alone (A) or [1251]NGFplus an excess of non-radioactive N(;F ~I #g/ml; B). Note the accumulation of silver grains over DRG (large arrows) under experimental conditions (A) and the absence of silver grain accumulations under control conditions (B). The presence of specific [125I]NGFbinding is also seen over an area of the neural tube corresponding to the dorsal horn (small arrow). Marker=250 ~m. C: in an adjacent section, DRG (arrows) are demonstrated by ItNK-I immunoreactivity. The magnification is the same as in A and B. NT, neural tube; No. notochord. an adjacent stage 23 section by anti-HNK-1 antibodies (Fig. 2C). The presence of specific [125I]NGF binding at a relatively low concentration of [~25I]NGF (2 n g / m l ) suggests the presence of high affinity receptors, because high affinity N G F receptors are preferentially labeled at lower concentrations 72. It should also be noted that specific [125I]NGF labeling was seen in a portion of the neural tube corresponding to the dorsal horn (Fig. 2A). The absence of binding on the contralateral side is probably due to a combination of tissue loss and the section not being perfectly transverse. G o o d morphology is difficult to maintain in unfixed sections which are processed for [~25I]NGF binding. In contrast, sections processed for immunocytochemistry were fixed immediately after sectioning and exhibit fairly good morphology (i.e. Fig. 2C). Previous studies have demonstrated the presence of N G F receptors in the dorsal horn of embryonic chicken, fetal human tissue, as well as postnatal and adult rat, with respect to [~25I]NGF binding, 192-IgG immunoreactivity, and trkA m R N A expression 18,59,6(i,76,79 Raivich et al. 6° have indicated that [125I]NGF binding appears as early as E4 in the chick dorsal root entry zone, correlating with our findings. The above studies postulate that N G F receptors are primarily localized on the fibers and terminals of dorsal root ganglion neurons, which would suggest that these fibers and terminals should express the same subtypes of N G F receptor as the D R G itself (i.e. both high and low affinity). In support of this hypothesis, Riopelle et al. 66 have shown that the dorsal spinal cord of adult rats exhibits high affinity [~25I]NGF binding.

Determination of NGF receptor molecular weight An independent method was used to confirm the presence of high affinity N G F receptors in E3.5 (stage 23) D R G . N G F receptor subtypes can be differentiated from each other on the basis of molecular weight; the

l i g a n d - r e c e p t o r complex being 158 kDa for the high affinity site and 90-110 kDa for the low affinity N G F receptor ~°'34"5~. D R G isolated from E3.5 (stage 23) quail were dissociated and incubated with a relatively high concentration of [IzsI]NGF (25 ng/ml), followed by exposure to the lipid soluble cross-linker, HSAB, and SDS-polyacrylamide gel electrophoresis. Previous studies have determined that the enzyme used for dissociation, dispase, does not affect [I25I]NGF binding 7. Examination of a radioautograph of a gel (Fig. 3A) revealed the presence of a high molecular weight band between the 200 and 116 kDa markers, as well as a low molecular weight band located slightly below the 66 kDa marker. The presence of an [125I]NGF-receptor complex at approximately 158 kDa confirms the presence of high affinity N G F receptors in stage 23 D R G . Binding of [~25I]NGF to both high and low affinity receptors was shown to be specific; considerably less [125I]NGF-receptor complex was seen in lanes in which duplicate aliquots of dissociated E3.5 D R G

AB

Fig. 3. Cross-linking of [t2SI]NGF to its receptor. Dissociated DRG from E3.5 (stage 23) quail embryos were incubated with [IzSIINGF alone (A) or [lzSI]NGF plus an excess of non-radioactive NGF (50 /xg/ml; B), followed by cross-linking with HSAB and SDS-polyacrylamide gel electrophoresis. Radioautographs of gels revealed the presence of two bands (arrows), whose intensity was significantly reduced under control conditions (B). The position of molecular weight markers is indicated at the arrowheads.

59 were incubated with an excess of non-radioactive NGF (50 /xg/ml), in addition to [125I]NGF (Fig. 3B). It is unclear why the low affinity receptor (p75 NG~R) is detected at a lower molecular weight than previously reported 5.

Effect of NGF on neuronal cell number Neuron-enriched cultures prepared from E3.5 quail DRG were maintained for 24 h in complete medium in the presence or absence of NGF (100 ng/ml), and/or anti-NGF antibodies (100 ng/ml). Neuron-like cells

Fig. 4. Appearance of E3.5 D R G (stage 23) neuron-enriched cultures. Neuron-enriched cultures were prepared from dissociated E3.5 (stage 23) D R G and maintained for 24 h in vitro u n d e r the following conditions: (A) + N G F - A b , (B) - N G F + A b , (C) + N G F + A b , (D) + N G F + a A b (absorbed anti-NGF antibodies), (E) - N G F - A b . Neurons with phase bright cell bodies bearing neurites were present under all conditions (examples shown at arrows). Non-neuronal cells were also seen (arrowheads). The magnification is the same throughout this figure. Marker ~ 100/~m.

60 with phase-bright cell bodies bearing one or more neurites were present under all conditions (Fig. 4). Some non-neuronal cells were also present; these were identified as fiat phase-dark cells In the presence of N G F and in the absence of anti-NGF antibodies (Fig. 5; + N G F - A b ) , there were approximately 30% more neurons present after 24 h in vitro, as when compared to cultures maintained in the absence of N G F and in the presence of anti-NGF antibodies (Fig. 5; - N G F + A b ) . This difference in neuronal cell number was statistically significant ( P < 0.001). There was no statistically significant difference in the number of neurons observed, however, in cultures grown in either the presence or absence of exogenous NGF, if anti-NGF antibodies were not present (Fig. 5; + N G F - A b v s - N G F - A b ) . This suggests that complete medium, which contains both fetal bovine serum and chicken embryo extract (CEE), has levels of N G F or NGF-like substances sufficient to mask the effect of exogenous NGF. Another possible interpretation is that anti-NGF antibodies were toxic to cultures, resulting in a lower neuronal cell number. This seems unlikely for two reasons. First, as shown in Fig. 4, neurons grown in complete medium in the presence of anti-NGF antibodies ( - N G F + A b ) have a similar appearance to those grown in the absence of anti-NGF antibodies ( + N G F - A b or - N G F - A b ) . Second, cultures exposed to anti-NGF antibodies that were

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NGF Concentration (ng/ml) Fig. 6. Dose response of N G F on neuronal n u m b e r in E3.5 (stage 23) D R G neuron-enriched cultures. Neuron-enriched cultures were prepared from dissociated E3.5 (stage 23) D R G and maintained for 24 h in vitro in the presence of 0.l to 300 n g / m l NGF. Cultures were grown in the presence (solid line) and absence (dotted line) of chicken embryo extract (CEE). Note the dose-dependent effect of N G F on neuronal cell n u m b e r when cultures are grown without CEE. Neuronal cell n u m b e r was expressed as a percentage of the maximal neuronal cell n u m b e r obtained u n d e r the various culture conditions ( + S.E.M., n = 3; for those concentrations without error bars, n = 2).

preabsorbed with NGF (Fig. 5; + N G F +aAb), had neuronal numbers similar to those grown in the absence of anti-NGF antibodies (Fig. 5; + NGF - A b or - N G F - Ab). The specificity of N G F ' s effect was demonstrated by blockage with anti-NGF antibodies (Fig. 5; + N G F +Ab). Furthermore, NGF's effect was shown to be dose dependent (Fig, 6). As discussed above, NGF's effects are masked by complete medium, therefore, for this study cultures were maintained in complete medium with and without CEE. As shown in Fig. 6, NGF's dose dependency was only evident in the absence of CEE (dotted line).

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DISCUSSION

.....,.,.,.. ,.....,..... ,........... :..: ... •

'":'"'"" e,.,

I

I

÷ ÷

Culture Conditions Fig. 5. Effect of N G F and anti-NGF antibodies on neuronal number in E3.5 (stage 23) D R G neuron-enriched cultures. Neuronal cell n u m b e r was determined in E3.5 (stage 23) D R G neuron-enriched cultures after 24 h in vitro in the presence or absence of N G F and anti-NGF antibodies (n = 6 for each condition, except for + N G F + a A b in which n = 2). Neuronal cell n u m b e r was expressed as a percentage of the maximal neuronal cell n u m b e r obtained under the various culture conditions (+_S.E.M., + range for + N G F +aAb). The culture conditions which had significantly different numbers of neurons are indicated ( * : P < 0,00l )

This study has demonstrated the absence of [125I]NGF binding to migratory neural crest cells in situ. This confirms the results of previous in vitro studies 7'25, but is in contrast with those of Heuer et al. 3° which demonstrated NGF receptor m R N A on migrating neural crest cells. The apparent lack of correlation between the expression of NGF receptor m R N A with functional [t25I]NGF binding may be due to a greater sensitivity of in situ hybridization, as suggested by Heuer et al. 3°. An alternative possibility is that NGF receptor m R N A in neural crest cells is not translated. It should also be noted that the probe used

61 for in situ hybridization probably recognizes p75 N°FR, which is insufficient for high affinity binding. The trk A proto-oncogene product, p140 pr°t°trk, is required for high affinity N G F binding 2s'37'75, and does not appear to be present in migrating neural crest cells, at least in the mouse s3. Our results indicate that high affinity N G F receptors appear on early embryonic D R G by E3.5 (stage 23). This finding correlates with a recent report that t r k A m R N A appears on mouse DRG, during early development, just as they are condensing (equivalent to avian stage 20-21) 53. Verge et al. 75 have demonstrated in adult rat D R G that t r k A m R N A and high affinity N G F binding sites are closely co-localized. Although a previous study detected [125I]NGF binding in E4 chick D R G 6°, high and low affinity N G F receptors were not distinguished from one another, because a high concentration of [LzsI]NGF was used (20 ng/ml). Since high affinity N G F receptors are unique to neurons, it was impossible to ascertain, therefore, whether the [I25I]NGF binding detected by Raivich et al. 6° was on neurons or non-neuronal support cells. Both neurons and non-neuronal support cells exhibit the low affinity subtype ~1,67,68,72,73,80,81. The presence of high affinity N G F binding suggests that neurons from E3.5 (stage 23) D R G should be responsive to a n d / o r dependent upon N G F 44. Previous studies suggest that early embryonic D R G are unresponsive to N G F with respect to neurite outgrowth and survival 13"15"19'36"48"50. Although N G F causes an increase in neurite outgrowth and survival in D R G midway through development, there is no reason to assume that N G F would cause the same effects in younger ganglia, or necessarily exhibit its full range of effects. Our results have demonstrated that the presence of NGF increases the number of neurons in neuron-enriched cultures prepared from E3.5 DRG. This effect, although statistically significant, is relatively small (approximately 30%). This is not surprising since D R G are composed of subpopulations of neurons that have differing requirements for growth factors 3'16"19'7x. Whether this increase is due to an increase in cell survival or an increase in cell proliferation remains to be determined. Studies have shown that N G F can act as a mitogen for early embryonic cochleovestibular ganglia 6~, neonatal sympathetic ganglia 29'46, neuroblastoma cells 62, PC12 cells 9'm, and neonatal adrenal medullary cells 47. However, recent evidence suggests that NGF has no mitogenic effect upon sympathetic neurons from E15.5 rat ganglia ~7, indicating that NGF's mitogenic effect upon sympathetic ganglia must be purely due to effects on non-neuronal support cells.

Although the presence of high affinity NGF receptors and the identification of an NGF response in vitro suggests that NGF is important in the early development of DRG, it does not prove that NGF is playing a physiological role in vivo. NGF is typically described as a target-derived growth factor u; NGF responsive neurons, bearing high affinity N G F receptors, project a distance to an NGF-producing target (i.e. heart, skin). It is unclear, therefore, what purpose would be served by the presence of high affinity receptors in D R G at E3.5 (stage 23) since neurons have not yet contacted their targets and have no apparent source of NGF. However, recent evidence suggests that local sources of N G F do exist, at [east at later stages of development. N G F has been shown to be produced by support cells and developing neuronal cells in the peripheral and central nervous systems 2~3~4'). It has recently been demonstrated that NGF is actually a member of a 'family' of neurotrophins that are structurally similar to one another. Other members of the family include brain-derived neurotrophic factor (BDNF) 4'43, neurotrophin-3 (NT-3) 3~'52"~'~, and neurotrophin-4/5 (NT-4/5Y '2". It has been shown that early embryonic D R G appear to be somewhat responsive to BDNF and NT-3. Explants of E4 D R G exhibit a slight amount of neurite outgrowth in response to BDNF ~6, and at least some of the neurons, or their precursors, can be rescued by BDNF and appear to require the factor for survival v~~2"3~. Both BDNF and NT-3 accelerate maturation of developing sensory neurons 7s. It is not known whether NT-3 or N T - 4 / 5 effect neurite outgrowth from early embryonic DRG, however these factors are able to elicit neurite outgrowth a n d / o r support survival of avian D R G midway through

development 6.2~,52,69. It has been shown that the members of the NGF 'family' of neurotrophins appear to exert their biological effects by binding to tyrosine kinase receptors which are products of the trk 'family' of proto-oncogenes. As mentioned in the Introduction, high affinity NGF binding requires the presence of the product of the t r k A proto-oncogene 2~3775. The trk B proto-oncogene product is a functional receptor for B D N F 2~'~'7°'71. while the trkC proto-oncogene product is a functional receptor for NT-3 41. It should be noted, however, that most neurotrophins bind to two or more of the trk tyrosine kinase receptors, although usually with lower affinity and lesser biological potency. For example, NT-3 also binds to t r k A and trkB 2~~'41"7°'71, and N T - 4 / 5 activates both trk A and trk B ~. The presence of high affinity NGF binding and N G F responsiveness in E3.5 DRG, as described in this study, suggests the presence of the t r k A proto-onco-

gene product. In view of the fact that both NT-3 and N T - 4 / 5 also bind to the trk A proto-oncogene product, it is possible that all, some or none of these three neurotrophins (NGF, NT-3, N T - 4 / 5 ) might exert physiological effects in vivo. Although the presence of receptor is a prerequisite for ganglionic responsiveness or dependence, it is not sufficient. It remains to be determined whether these neurotrophins are present during early development of the D R G and what specific roles they play in vivo.

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16 Acknowledgements. The authors are indebted to Dr. Valerie Verge and Dr. Peter Richardson for sharing their expertise in receptor radioautography. We would also like to thank Dr. Susan Meakin, Dr. Moses Chao, and Dr. Enrique Escandon for advice on cross-linking, Dr. Cheryl Dreyfus, Dr. Keiko Fukada, Dr. Robert Garofalo, Dr. Kiyomi Koizumi, and Dr. Richard Janeczko for helpful discussions, and Mr. Vincent Garofalo and Mr. Lou Dienes for excellent photographic work. This study was supported by a grant from the National Science Foundation (BNS-8896101) and the Dysautonomia Foundation.

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20 REFERENCES 2t 1 Andres, R.Y., Jeng, I. and Bradshaw, R.A., Nerve growth factor receptors: identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons, Proc. Natl. Acad. Sei. USA, 74 (1977) 2785-2789. 2 Banks, B.E.C., Banthorpe, D,V., Berry, A.R., Davies, H.F.F.S., Doonan, S., Lamont, D.M., Shipolini, R. and Vernon, C.A., The preparation of nerve growth factors from snake venoms, Biochem. J., 108 (1968) 157-158. 3 Barde, Y.A., Edgar, D. and Thoenen, H., Sensory neurons in culture: changing requirements for survival factors during embryonic development, Proc. Natl. Acad. Sci. USA, 77 (1980) 119912(~3. 4 Barde, Y.A., Edgar, D. and Thoenen, H., Purification of a new neurotrophic factor from mammalian brain, EMBO J., 1 (1982) 549-553. 5 Barker, P.A. and Murphy, R.A., The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins, Mol. Cell. Biochem., I l0 (1992) 1-15. 6 Berkemeier, L.R., Winslow, J.W., Kaplan, D.R., Nikolics, K., Goeddel, D.V. and Rosenthal, A., Neurotrophin-5: a novel neurolrophic factor that activates trk and trkB, Neuron, 7 (1991) 857-866. 7 Bernd, P., Appearance of nerve growth factor receptors on cultured neural crest cells, Dec. Biol., 112 (1985) 145-156. 8 Bern& P.. Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor, Dec. Blot,. 115 (1986) 415-424. 9 Boonstra, J., Moolenaar, W.H., Harrison, P.H., Moed, P., Van der Saag, P.T. and De Laat, S.W., Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells, J. Cell Biol., 97 (1983) 92 98. 10 Burstein, D.E. and Greene, L.A., Nerve growth factor has both mitogenic and antimitogenic activity, Dec. Biol., 94 (1982) 477482. 11 Carbonetto, S. and Stach, R.W., Localization of nerve growth factor bound to neurons growing nerve fibers in culture, Dec. Brain Res., 3 (1982) 463-473. 12 Charlwood, K.A., Griffith, M.J., Lamont, M.D., Vernon, C.A.

22

23

24

25

26

27

28

29 30

31

32 33

and Wilcock, J.C., Effects o! ne~,'e growth factor trom the venom of Vipera russelli on sensory and sympathetic ganglia from the embryonic chick in culture, J. Emffo,ol. l;,;rp. Morphol., 32 (1974) 239-252. Collins, F., Developmental time course of the eflect ot' nerve growth factor on the parasympathetic ciliary gangJion, Det. Brain Res., 39 (1988) 111-116. Davies, A.M., Bandtlow, C., Heumann, R., Korsching, S., Rohrer, tt. and Thoenen, H., Timing and site of nerve growth factor synthesis in developing skin in relation to innervafion and expres~ sion of the receptor, Nature, 326 (1987) 353-358. Davies, A.M. and Lindsay, R.M., The cranial sensory ganglia in culture: differences in the response of placode- derived and neural crest-derived neurons to nerve growth factor, Dec. Blot,, III (1985) 62-72. Davies, A.M., Thoenen, H. and Barde, Y.A., The response of chick sensory neurons to brain-derived neurotrophic factor, J. Neurosci., 6 (1986) 1897-1904. DiCicco-Bloom, E. and Black, I.B., Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts, Proc. Natl. Acad. Sci. USA, 85 (1988) 4066-4070. Eckenstein, F., Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord, Brain Res., 446 (1988) 149-154. Ernsberger, U. and Rohrer, H., Neuronal precursor cells m chick dorsal root ganglia: differentiation and survival in vitro, Dec. Biol., 126 (1988) 420-432. Escandon, E. and Chao, M.V., Identification of high- and low-affinity NGF receptors during development of the chicken central nervous system, Det. Biol., 142 (1990) 293-300. Finn, P.J., Ferguson, I.A., Wilson, P.A., Vahaviolos, J. and Rush, R.A., lmmunohistochemical evidence for the distribution of nerve growth factor in the embryonic mouse, J. Neuroeytol., 16 (1987) 639-647. Frazier, W.A., Boyd, L.F. and Bradshaw, R.A., Properties of the specnflc binding of [1,~ I]nerve growth factor to responsive peripheral neurons, J. Biol. Chem., 249 (1974) 5513-5519. Glass, D.J., Nye, S.H., Hantzopoulos, P., Macchi, M.L, Squinto, S.P., Goldfarb, M. and Yancopoulos, G.D., TrkB n~diates BDNF/NT-3-dependent survival and proliferation in fibroblasts lacking the low affinity NGF receptor, Cell, 66 (1991) 405-413. Greene, L.A., Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons, DeL. Biol., 58 (1977) 106-113. Greiner, C.A.M., Lloyd, A.T. and Guroff, G., Ontogeny of the nerve growth factor receptor in primary cultures of neural crest cells, Det,. Brain Res., 26 (1986) 145-150. ttallbook, F., lbanez, C.F. and Persson, H., Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary, Neuron, 6 (1991) 845-858. tlamburger, V. and Hamilton, H.L., A series of normal stages in the development of the chick embryo, J. Morphol., 88 (1951) 49-92. ttempstead, B.L., Martin-Zanca, D., Kaplan, D.R., Parada, L.F. and Chao, M.V., High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor, Nature. 350 (1991) 678-683. tlendry, I.A., Cell division in the developing sympathetic nervous system, Ji Neurocytol., 6 (1977) 299-309. Heuer, J.G., Fatemie-Nainie, S., Wheeler, E.F. and Bothwell, M., Structure and developmental expression of the chicken NGF receptor, Dec. Blot,, 137 (1990)287-304. Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H.. Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection, J. Cell Biol., 1(14 (1987) 1623--1631. ttofer, M.M. and Barde, Y.A., Brain-derived neurotrophic factor prevents neuronal death in viw), Nature, 331 (1988) 261-262, Hohn, A., Leibrock, J., Bailey, K. and Barde, Y.A., Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family, Nature, 344 (1990) 339-341.

63 34 Hosang, M. and Shooter, E.M., Molecular characteristics of nerve growth factor receptors on PC12 cells, J. Biol. Chem., 260 (1985) 655-662. 35 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between A B C and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 36 Kalcheim, C., Barde, Y.A., Thoenen, H. and Le Douarin, N.M., In vivo effect of brain-derived neurotrophic factor on the survival of developing dorsal root ganglion cells, EMBO J., 6 (1987) 2871-2873. 37 Klein, R., Jing, S., Nanduri, V., O'Rourke, E. and Barbacid, M., The trk proto-oncogene encodes a receptor for nerve growth factor, Cell, 65 (1991) 189-197. 38 Klein, R., Nanduri, V., Jing, S., Lamballe, F., Tapley, P., Bryant, S., Cordon-Cardo, C., Jones, K.R., Reichardt, L.F. and Barbacid, M., The trk B tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3, Cell, 66 (1991) 395-403. 39 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (197(I) 680-685. 40 Lallier, T.E. and Bronner-Fraser, M., A spatial and temporal analysis of dorsal root and sympathetic ganglion formation in the avian embryo, Det,. Biol., 127 (1988) 99-112. 41 Lamballe, F., Klein, R. and Barbacid, M., trkC, a new m e m b e r of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3, Cell, 66 (1991) 967-979. 42 Le Douarin, N., The Neural Crest, Cambridge University Press, Cambridge, England, 1982. 43 Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B., Masiakowski, P., Thoenen, H. and Barde, Y.A., Molecular cloning and expression of brain-derived neurotrophic factor, Nature, 341 (1989) 149-152. 44 Levi, A. and Alema, S., The mechanism of action of nerve growth factor, Anne. Rec. Pharmacol. Toxicol., 31 (1991) 205-228. 45 Levi-Montalcini, R., The nerve growth factor: its mode of action on sensory and sympathetic nerve cells, Harl,ey Lect., 60 (1966) 217 259. 46 Levi-Montalcini, R. and Booker, B., Excessive growth of the sympathetic ganglia evoked by a protein isolated from mouse salivary glands, Proc. Natl. Acad. Sci. USA, 46 (1960) 373 384. 47 Lillien, L.E. and Claude, P., Nerve growth factor is a mitogen fl)r cultured chromaffin cells, Nature, 317 (1985) 632-634. 48 Lindsay, R.M. and Rohrer, H., Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF. lack N G F receptors but are supported by a liver-derived neurotrophic factor, Det,. Biol., 112 (19851 30-48. 49 Lu, B., Yokoyama, M., Dreyfus, C.F. and Black, 1.B., N G F gene expression in actively growing brain glia, J. Neurosci., 11 (1991) 318-326. 50 Luduena, M.A., Nerve cell differentiation in vitro, Det'. Biol., 33 (1973) 268-284. 51 Lyons, C.R., Stach, R.W. and Perez-Polo, J.R., Binding constants of isolated NGF-receptors from different species, Biochem. Biophys. Res. Commun., 115 (1983) 368 374. 52 Maisonpierre, P.C., Belluscio, L , Squinto, S., Ip, N.Y., Furth, M.E., Lindsay, R.M. and Yancopoulos, G.D., Neurotrophin-3: a neurotrophic factor related to N G F and BDNF, Science, 247 (199(I) 1446-1451. 53 Martin-Zanca, D., Barbacid, M. and Parada, L F . , Expression of the trk proto-oncogene is restricted to the sensory cranial and spinal ganglia of neural crest origin in mouse development, Genes Del'., 4 (1990) 683-694. 54 McCarthy, K.D. and Partlow, L.M., Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: a new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates, Brain Res., 114 (1976) 391 414. 55 Meakin, S.O. and Shooter, E.M., Molecular investigations on the high-affinity nerve growth factor receptor, Neuron, 6 (1991) 153163. 56 Mobley, W.C., Schenker, A. and Shooter, E.M., Characterization

and isolation of proteolytically modified heine growlh factor, Biochemisto', 15 (1976) 5543-5552. 57 Olender, E.J. and Stach, R.W., Sequestration of [l=51]labelled fi nerve growth factor by sympathetic neurons, ,I. Biol. ('henl., 255 (198(I) 9338 9343. 58 Olender, E.J.. Wagner, B J . and Stach, R.W.. Sequestration of [I2~l]labeled fl nerve growth faclor by embryonic sensory neurons, J. Neurochem., 37 ( 1981 ) 436-442. 59 Pioro, E.P. and Cuello, A.C., Dislribution of nerve growth faclor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergie system. It. Brainstem, cerebellum and spinal cord, Neuroscicncc, 34(199t))89 11(I. 6/) Raivich, G., Z i m m e r m a n n , A. and Sulter, A.. The spatial and temporal pattern of /3-NGF receptor expression in lhe developing chick embryo. EMBO J., 4 (19851 637-1~44. 61 Represa, J. and Bernd, P., Nci~.c grov¢lh faclor and scrum differentially reguhite development of lhc enlbryonic olic vesicle and cochleovestihular ganglion in vilro, /)el. Biol., 134 (198t~) 21 29. 62 Rcwlhclla, R.P. and Butler', R.tI., Nerve growth hlctor may stimuhite either division or diftk-~rcntiation of chined ('1300 neurobhistoma cells in serum-free cultures..I. ('ell. Phwiol., Ill4 (198/)) 27-33. 63 Richardson, P.M., Verge, V.M.K. and Riopcllc, R.,I., Ouantilative radioautography for N G F receptors. In R.A. Rush (Ed.). Nerce Growth Fuctors, Wiley, New York, NY. 1989, pp. 315 326. 64 Rickmann, M., Fuwcett. J.W. and Kcynes, R.,I., The migralion of neural crest cells and the growth of motor axons through the roslral half of the chick sornite, ,I. l-mhrvol, l:'tp. Motphol., t~(I (19851 437-455. 65 Riopelle, R.J., Klearman, M. and Stiller. A.. Nerve growth factor receptors: analysis of the interaction of fl-NGF with menlbrancs of chick embyro dorsal root ganglia, Brain Rcs., 199 (19~0) 63 77. 66 Riopelle, R.J., Verge, V.M.K. and Richardson, P.M., Propcrlies of receptors tk~r nerve growth factor in the malure rat ne~'ous system, Mol, Brain Res., 3 (1987) 45-53. 67 Rohrer, H., Nonneuronal cells from chick sympalhelic and dorsal root s e n s o ~ ganglia express calecholamint_' uptake and receptors for nerve growth factor during development, l)er. Biol., I11 (19851 95 107. 68 Rohrer, H. and Sommer, 1., Simullaneous expression of neuronal and glial properties by chick ciliary ganglion cells during development, J. Neurosci., 3 (19831 1683 1693. 69 Rosenthal, A., Goeddel, D.V., Nguyen, T., Lewis, M., Shih, A., l,aramee, G.R., Nikolics, K. and Winslow, J.W., Primary structure and biological activily of a novel human neurotrophic factor, Neuron, 4 (1990) 767 773. 7(1 Soppet, D., Escandon, E., Maragos, J., Middlemas, D.S., Reid. S.W., Blair, J., Burton, L.E., Stanton, B.R., Kaplan, D.R., 1hinter, T., Nikolics, K. and Parada, LF., The neurotrophic factors brain-derived neurotrophic factor and neurolrophin-3 are ligands for the trkB tyrosine kinase receptor, Ce//, 65 (1991) 895 903. 71 Squinto, S.P., Stilt, T.N., Aldrich. I".tt., Davis, S., Bianco, S.M.. Radziejewski, C., Glass, D.J.. Masiakowski, P., Furth. M.E., Valenzuela, D.M., DiStefano, P.S. and Yancopoulos, (LD., trk B encodes a functional receplor for brain-derived ncurotrophie factor and neurotrophin-3 but not nerve growlh factor, (k'll, 65 (1991)885 893. 72 Sutter, A., Riopelle, R.J., Harris-Warrick, R.M. and Shooter, E.M., Nerve growth factor receptors. ('haraeterization of lwo distinct classes of binding sites on chick cmbyro sensory ganglia cells, ,L Biol. Chem., 254 (1979) 5972-5982. 73 Sutter. A., Riopelle, R.J., FIarris-Warrick, R.M. and Shooter, E.M., The heterogeneity of nerve growlh faclor receplors. In M. Bitensky (Ed.), Transmembrane Si&vuUlin~. Liss, New York, NY, 1979, pp. 659-667. 74 Tucker, G.C., Aoyama, 11., Lipinski, M., Tursz, T. and Thiery, J.P., Identical reactivity of monochmal antibodies HNK-I and NC-I: conservation in vertebrates on cells derived from the neural primordium and on some lenkocylcs, ('ell Di['/:, 14 (19H41 223 23O.

64 75 Verge, V.M.K., Merlio, J.P., Grondin, J., Ernfors, P., Persson, H., Riopelle, R.J., Hokfelt, T. and Richardson, P.M., Colocalization of NGF binding sites, trk mRNA and low-affinity NGF receptor mRNA in primary sensory neurons: responses to injury and infusion of NGF, Z Neurosci., 12 (1992) 4011-4022. 76 Wetmore, C., Bygdeman, M. and Olson, L., High and low affinity NGF receptor mRNAs in human foetal spinal cord and ganglia, NeuroReport, 3 (1992) 689-692. 77 Winick, M. and Greenberg, R.E., Chemical control of sensory ganglia during a critical period of development, Nature, 205 (t965) 180-181. 78 Wright, E.M., Vogel, K.S. and Davies, A.M., Neurotrophic factors promote the maturation of developing sensory neurons be-

fore they become dependent on these factors lot survival, Neuron, 9 (1992) 139-150. 79 Yip, H.K. and Johnson, E.M., Nerve growth factor rcceptors in rat spinal cord: an autoradiographic and immunohistochemica[ study, Neuroscience, 22 (1987) 267--279. 80 Zimmermann, A. and Sutter, A., /3-Nerve growth factor (flNGF) receptors on glial cells, Cell-cell interaction between neurones and Schwann cells in cultures of chick sensory ganglia, EMBO J.~ 2 (1983) 879-885. 81 Zimmermann, A., Sutter, A., Samuelson, J. and Shooter, E.M., A serological assay for the detection of cell surface receptors of nerve growth factor, J. Supramol. Struct., 9 (1978) 351-361.