Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons

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1 Development 112, (1991) Printed in Great Britain The Company of Biologists Limited Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons ALFREDO RODRIGUEZ-TEBAR* and HERMANN ROHRERt Max-Planck-Institute for Psychiatry, Department of Neurochemistry, D-8033 Planegg-Martinsried, Federal Republic of Germany Author's address: Institute Cajal, CSIC, Doctor Arce, 37, Madrid, Spain t Author for correspondence: Max-Planck-Institute for Brain Research, Department of Neurochemistry, DeutschordenstraBe 46, D-6000 Frankfurt/M. 71, Federal Republic of Germany Summary An important step in the development of peripheral sensory and sympathetic neurons is the onset of the survival response and dependence on the presence of nerve growth factor (NGF) or other neurotrophic factors. We have recently observed that immature sympathetic neurons from 7-day-old chick embryos are unable to become NGF-responsive in vitro and we have now used these cells to identify molecules that induce NGF-dependent neuronal survival. We found that retinoic acid (RA) induces the ability of these cells to survive in the presence of NGF. At RA concentrations of 10~ 9-10~ 8 M virtually all neurons survived in the presence of NGF. RA was found to also induce the biologically active, high-affinity NGF receptor: highaffinity receptors were undetectable on dissociated E7 sympathetic neurons and were observed in vitro only in RA-treated neurons. These findings suggest that the induction of high-affinity NGF receptors may be sufficient to activate the survival response in sympathetic neurons and imply an important role for RA during neuron differentiation hi the peripheral nervous system. Key words: retinoic acid, nerve growth factor, NGF receptor, sympathetic neuron, neuron survival. Introduction At the time when sensory and sympathetic neurons are generated, neither survival nor neurite outgrowth is influenced by or dependent on nerve growth factor (NGF) (Davies and Lumsden, 1984; Coughlin and Collins, 1985; Davies et al. 1987; Ernsberger and Rohrer, 1988; Ernsberger et al. 1989). However, as the neurites reach their peripheral targets, the neurons require neurotrophic factors and die in their absence (Davies and Lumsden, 1984). The appearance of NGFresponsiveness and NGF-dependence of peripheral neurons is paralleled by the expression of NGF which is undetectable during initial neurite outgrowth and starts in peripheral targets at the time of innervation (Davies et al. 1987). We have previously demonstrated that neurons from the lumbosacral sympathetic chain ganglia of 7-day-old chick embryos represent the early, NGF-independent stage of neuron development, as they proliferate and survive in culture in the absence of added neurotrophic factor (Ernsberger et al. 1989). Interestingly, it was also observed that these neurons are unable to become NGF-responsive with time in culture, whereas in vivo the majority of lumbosacral sympathetic neurons has become NGF-responsive at embryonic day 11. Thus the acquisition of NGF-responsiveness, albeit crucial for neuronal survival, seems not to be predetermined but rather to depend on cellular interactions. Similar conclusions have also been obtained from the in vitro analysis of rat sympathoadrenal cells (Anderson and Axel, 1986; Stemple et al. 1988). We have used these immature E7 chick sympathetic neurons to identify the signals and cellular mechanisms leading to NGF-mediated neuronal survival. It was found that RA, which is known to affect the development of neuronal and non-neuronal tissues (Roberts and Sporn, 1984; Durston et al. 1989; Tickle et al. 1982; Maden, 1982; Shenefelt, 1972; Tamarin et al. 1984; Thorogood et al. 1982), induces the biologically active high-affinity NGF receptor and the ability of these cells to survive in the presence of NGF. Materials and methods Cell culture E7 sympathetic neuron cultures from lumbosacral sympathetic chain ganglia were prepared, plated on a laminin substratum and cultivated as previously described (Ernsberger et al. 1989). Medium was changed every 3-4 days. If not otherwise indicated, cells were cultivated in 35 mm dishes (Greiner) at a cell density of cells/dish. Clonal cell

2 814 A. Rodriguez-Tibar and H. Rohrer densities were obtained by limiting dilution and cells were plated in 96-well microtiter plates under the same culture conditions. Cell counts in 35 mm dishes were obtained by examining phase-bright cells in randomly selected fields of the culture dishes. Approximately 5 % of the total surface of the dish was examined and duplicates were counted for each parameter in each experiment. The number of independent experiments analyzed is indicated by n. In clonal cultures, the wells were analysed after 3 h and 10 days for the presence of single cells. NGF (prepared according to Suda et al. 1978) was present at saturating concentrations (longml" 1 ) if not otherwise indicated. Brain-derived neurotrophic factor (BDNF; generously provided by Dr Y.-A. Barde, Max-Planck-Institut fur Psychiatrie, Martinsried, FRG) was added in some experiments. The concentrations used were saturating with respect to the survival of chick DRG neurons (Ernsberger and Rohrer, 1988). Basic fibroblast growth factor (hrbfgf, a generous gift from Dr W. Knoerzer, Progen Biotechnik, Heidelberg, FRG) was used at longml" 1. The same batch of hrbfgf was active as a survival factor for chick motoneurons (Arakawa et al. 1990). Ah-trans retinoic acid, retinol and retinal (Sigma, MUnchen, FRG) were dissolved in DMSO at concentrations of 10~ 2-10~ 3 M and then serially diluted in medium. Cell-culture-tested dexamethasone and ^3-estradiol were dissolved in ethanol, 3,3',5-triiodo-L-thyronine in 0.02M NaOH at a concentration of 10~ 3 M and diluted in medium (all from Sigma). NGF-receptor-binding studies NGF binding to freshly dissociated neurons from E7 chick embryos: sympathetic chains from 40 embryos were dissected into ice-cold Ca 2+ - Mg^-free Gey's buffer. Ganglia were washed by centrifugation, resuspended in 5 ml Gey's buffer and incubated at 37 C with trypsin (0.12mgml, Worthington) and DNAasel (0.012 mg ml, Sigma) for lomin. Trypsinization was terminated by adding 0.5 mg ml" 1 soybean trypsin inhibitor (Sigma) and horse serum to 10% final concentration. Cells were dissociated by passing the ganglia 6-8 times through the wide bore of a 5 ml glass pipette. Undissociated ganglia and large fragments were allowed to sediment and the supernatant removed. The dissociation procedure was repeated until full dissociation was achieved. Combined supernatants were filtered through a 50 /zm pore nylon net and the cell suspension was centrifuged in Krebs- Ringer-Hepes buffer (Greene and Rein, 1977) containing lmgml" 1 BSA (KRH/A) at a concentration of xlo 6 cells ml. Binding experiments were under steady-state conditions, i.e. cells were incubated with the ligand until equilibrium was reached. The rationale of the methods used has been discussed previously (Vale and Shooter, 1980). Experiments with cells in suspension were done at 4 C using quadruplicates of 100 ja aliquots of the cell suspensions to which 125 I-NGF (Rodriguez-T6bar et al. 1990) was added at the concentrations indicated. Cells were incubated with labelled NGF for 60min under gentle shaking. Blanks were done in quadruplicates by adding a 200-fold excess of native NGF to the cell suspension 30min before the addition of 125 I- NGF. Binding was terminated in two different ways. (1) For detection of high-affinity binding sites, binding mixtures were layered on top of a two-step sucrose gradient (300 mm and 150 mm sucrose in KRH buffer) in a Beckman centrifuge tube. Gradients were centrifuged in a Beckman microfuge for 60s. After freezing, the bottoms of the tubes were cut and counted (bound radioactivity) separately from the rest of the tube (free radioactivity). The centrifugation through a two-step sucrose gradient allows an efficient separation between the free and bound L25 I-NGF and reduces the non-specific binding. During the 60 s of centrifugation, a negligible amount of bound 125 I-NGF was lost, as the release of the ligand from its high-affinity receptors is very slow at this temperature (Sutter et al. 1979). (2) For detection of low-affinity binding sites, binding mixtures were put in a 0.5 ml Eppendorf tube and centrifuged as before. Supernatants were carefully removed and the bottoms of the tubes were cut off and counted (bound radioactivity). Free radioactivity was calculated by subtracting the bound radioactivity from the total input. This direct centrifugation method was chosen for the accurate determination of low-affinity receptors because the release of NGF bound to this receptor is very fast (Sutter et al. 1979). During the centrifugation the concentration of free ligand does not change and thus no bound 125 I-NGF is lost. NGF binding to cultured sympathetic neurons: cultured cells ( cells/35 mm dish) were washed with fresh medium lacking NGF for 6h. The washing procedure was repeated and the incubation extended for lh. Finally, the medium was removed and replaced by 2 ml/dish of KRH/A. This procedure ensured that all NGF, either free or bound to the neurons, was washed off. Binding experiments were done at room temperature. Cultured neurons were labelled with increasing concentrations of ^I-NGF, (10" u to 5xl0~ n M) in order to detect high-affinity receptors. All experimental points were made in triplicate. Blanks were made by prior addition of a 200-fold excess of native NGF to ^I-NGF. After the addition of 125 I-NGF, the cultures were incubated for 60min with gentle swirling. Then the binding buffer was removed and the cells washed four times with 3 ml ice-cold KRH/A. The washing step was completed within less than 2 min per dish. During this time period a negligible amount of bound 125 I-NGF was lost (see above). After binding, neurons appeared undamaged as judged by light microscopic observation. No cell loss was apparent. Cells were solubilized by extracting twice with 1 ml of 4 % SDS and the radioactivity was measured in a y-counter. Kinetic data were analysed by Scatchard plots (Scatchard, 1949) to determine both the dissociation constants (K d ) of the NGF receptors and the number of binding sites per neuron. Results Retinoic acid induces NGF-dependent neuronal survival Chick sympathetic neurons from 7-day-old chick embryos proliferate and survive in culture for a period of about 4 days in the absence of added neurotrophic factors. After this initial proliferation period, which leads to an increase in cell numbers (Ernsberger et al. 1989), the vast majority of the cells die and are not rescued by addition of nerve growth factor (NGF), the neurotrophic factor required for survival of older (E8-E12) sympathetic neurons (Figs 1 and 2). Retinoic acid (RA) induced the ability of E7 sympathetic neurons to survive in the presence of NGF (Figs 1 and 2). The addition of RA alone did not result in long-term neuronal survival, but the combination of RA and NGF together resulted in virtually complete survival for 10 days in culture. Neuronal cultures displayed an extensive network of fibres and could be maintained up to 4 weeks (latest time period analysed). The survival effect of RA might be either due to a

3 Effects of retinoic acid on sympathetic neurons 815 Fig. 1. RA induces NGF-dependent neuronal survival. E7 sympathetic neurons were grown for 10 days in the presence of NGF (longml"1) (A); RA ( 5 X 1 0 " 9 M ) (B); or both NGF and RA (C). Bar, 10/an. 140-, 160 E 2»120- r 140 t Sioo 1!~ i"2 903 "5? Q o o o 60 10" c o n c e n t r a t i o n of retinoids I M ] time in culture 7 (days ] 10 Fig. 2. RA induces NGF-dependent neuronal survival. Effect of NGF, RA and both NGF and RA on the number of E7 sympathetic neurons in vitro. Cells were kept in the presence of NGF ( - V - ), RA ( - X - ) and both NGF and RA ( O ) as in Fig. 1. The number of neurons determined after 3h, 3 days, 7 days, and 10 days are expressed as a percentage of 3 h values and represent the mean (±S.D.) of three independent determinations. direct action on sympathetic neurons or due to an indirect mechanism involving non-neuronal cells. E7 sympathetic ganglia contain only a low number of nonneuronal cells (Rohrer and Thoenen, 1987) and as the proportion of non-neuronal cells decreased continuously from 3.5±1% after 1 day in culture to 2±0%, 1.7±1% and0.9±0.5% (mean±s.d. of three independent experiments) after 3 days, 7 days and 10 days, respectively, an indirect effect of RA seemed to be rather unlikely. However, to prove this point, single cells were cultured in 96-well microtitre plates for 10 days and, as in the mixed cultures, RA induced NGFmediated survival: 90 % of the neurons plated survived Fig. 3. Concentration dependence of the effects of RA, retinol and retinal, on NGF-dependent neuronal survival. E7 sympathetic neurons were grown in the presence of NGF (longml"1) and additional concentrations of RA ( O ), retinol ( ) and retinal ( T ) as indicated. Cell numbers were determined after 3 h and 10 days-. Cell numbers are expressed as percentage of 3 h values and represent the mean (±S.E.M.) of three to four independent experiments. as compared to 4 % in the presence of NGF alone (96 and 72 cells analysed, respectively). Treatment of E7 sympathetic neurons with RA induced NGF-dependent survival, but the cells were unable to respond to the structurally related neurotrophic factor BDNF (Leibrock et al. 1989) (data not shown). Since E10 chick sympathetic neurons respond to NGF but not to BDNF (Barde et al. 1982), it seems that RA treatment specifically induces the neurotrophic response characteristic for sympathetic neurons. Basic fibroblast growth factor (bfgf) had no effect on the NGF-mediated survival of E7 sympathetic neurons (data not shown). The dose-response curve for the RA-induced survival response to NGF (Fig. 3) shows half-maximal effects at 10~ 9 M and maximal effects of RA at a concentration of 5xlO~9M. RA at high concentrations (10~ 7 M) caused neuronal death. Other retinoids tested, retinol (Vitamin A) and retinal had no effect up to

4 816 A. Rodriguez-Tibar and H. Rohrer Table 1. Effects of thyroid hormone (T3), estrogen (estradiol) and glucocorticoids (dexamethasone) on NGF-dependent survival Hormone concentration (M) 10"* lo" 7 10" 8 io- 9 Sympathetic (% of T ± ± ±2.5 neuron neurons Dex 3.6± ± ± ±1.2 survival (lod) plated) Estradiol 4.9± ± ± ±2,3 E7 chick sympathetic, neurons were cultivated in the presence of NGF (longml ) and hormones at the concentrations indicated. In control experiments, RA (5X10~ 9 M) and NGF maintained 119±25 % of the cells plated (data are expressed as the mean±s.d. of three independent experiments). concentrations of 10 M, however, in the presence of NGF and 10~ 6 M retinol or retinal, 42 % and 62 % of the cells were maintained, respectively. Since activated RA receptors have been shown to bind to thyroid hormone responsive elements (Graupner et al. 1989), we analysed the effects of other ligands for receptors of the steroid/thyroid hormone family. Neither thyroid hormone, estradiol nor glucocorticoid (dexamethasone) had any effect on the ability of the cells to survive in the presence of NGF. Neuronal survival never exceeded 5 % of the cells plated after culturing for 10 days in the presence of 10-10~ 9 M hormone (Table 1). Thus the response to NGF is due to RA and the low concentrations required for the effect indicate a specific effect via cellular RA receptors acting on their specific responsive elements. Expression of NGF receptors and effects of RA Neuronal survival and differentiation effects of NGF are initiated by binding of NGF to its specific receptor. Two types of NGF receptors have been described, lowaffinity (K d 2X10~ 9 M) and high-affinity receptors (K d 2X10~"M) (Sutter et al. 1979). Neuronal survival and fibre outgrowth correlate with the presence of highaffinity receptors (Green et al. 1986), which is therefore considered to be the biologically active receptor. Thus, the lack of NGF response during early PNS neuron development may be solely due to a lack of high-affinity receptors. To address this question, steady-state binding studies using suspensions of freshly dissociated E7 sympathetic neurons were carried out. They demonstrated the presence of low-affinity NGF receptors (K d =2.1 (±1.1) X10~ 9 M), but no high-affinity receptors were detectable (Fig. 4). As the presence of the biologically active NGF receptor is a prerequisite for the NGF response, we investigated whether E7 sympathetic neurons in culture are able to acquire high-affinity NGF receptors. The slow off-rate of high-affinity receptors made it possible to carry out binding studies using cells attached to the culture dish. It was found that after several days in culture only cells kept in the presence of RA expressed detectable levels of high-affinity NGF receptors (Fig. 5). Whereas low-affinity, fast-dissociating recep- 0) 0) 7.5? 5.0 o o x> NGF f / NGF bound. 10" 12 M Fig. 4. Scatchard plot of the binding of ^I-NGF to freshly dissociated neurons from paravertebral lumbar sympathetic ganglia of E7 chick embryos. Assays to detect high-affinity binding sites ( x ) and low-affinity binding sites ( ) were made as described in Materials and methods. The radioactivity measured in the assay for high-affinity binding sites most likely corresponds to residual I-NGF bound to the low-affinity receptors incompletely dissociated during the two-step gradient centrifugation procedure. The determination of low-affinity sites reveal 125 I-NGF bound to sites per cell with an affinity of 2.9X10~ 9 M, i.e. low-affinity NGF receptors. tors cannot be characterized using cells attached to culture dishes, the K d of the high-affinity receptor determined in the presence of RA (5.4 (±2.5) X10~ U M), is similar to previous determinations using cell suspensions (Sutter et al. 1979; Godfrey and Shooter, 1986). The most straightforward interpretation of our results is that the induction of high-affinity receptors by RA (Fig. 5) induces the survival response to NGF in E7 sympathetic neurons. Discussion The present results demonstrate that NGF-dependent neuronal survival is induced by RA in immature chick sympathetic neurons. RA was also found to induce the expression of the biologically active high-affinity NGFreceptor. These findings suggest that the induction of significant numbers of high-affinity receptors may be sufficient to induce the survival response to NGF and in addition imply an important role for RA during neuron differentiation in the peripheral nervous system. To investigate early stages in neuronal development, sympathetic neurons have considerable advantages, since these cells can be unambiguously identified as neurons from early stages onwards by the presence of several neuron-specific properties (Rothman etal. 1978; Rohrer and Thoenen, 1987) and since immature phenotypes can be maintained in culture (Rohrer and Thoenen, 1987). We have previously shown that the survival and proliferation of sympathetic neurons from E7 chick embryos depends on an appropriate substratum, but is not influenced by the presence of NGF (Ernsberger et al. 1989). Whereas during normal development NGF-dependence and NGF-responsiveness is detectable at E8-11, E7 sympathetic neurons did

5 Effects of retinoic acid on sympathetic neurons 817 o 2 c A 13 g B I-NGF bound, 10" 12 M Fig. 5. Binding of 125 I-NGF to cultured sympathetic neurons from E7 chick embryos. Neurons were dissociated and cultured in Falcon 35 mm dishes at densities of neurons/dish. After 4 days in culture, binding of labelled NGF was assayed as described in Materials and methods. A and B show two binding experiments each, which have been carried out with cells grown in parallel with identical cell densities. (A) Neurons were grown for 4 days in the absence ( ) or in the presence (A) of NGF (longmt 1 ). (B) 4-day culture in the absence ( ) or presence (D) of 5X10~ 9 M RA. The K d and number of receptors determined in B are: 3.7X10~ U M and 5020 sites. not acquire NGF-responsiveness with time in culture. This finding indicated that the appearance of NGFresponse is not genetically programmed, but can be influenced in vitro either by inhibitory or stimulatory signals. We now describe that RA is able to induce NGFresponsive neuronal survival. The effect is due to a direct effect of RA on the neuronal cell population, since we observed NGF-dependent survival also when single, isolated neurons were cultivated in clonal cell cultures. The RA concentration necessary to produce half-maximal effects (10~ 9 M) is in the range of the dissociation constant of at least one cellular retinoic acid receptor (Brand et al. 1988; Cavey et al. 1990) and the RA concentrations determined in RA-responsive tissues (Thaller and Eichele, 1987; Durston et al. 1989) are higher than those required to elicit an NGFresponse. Several distinct retinoic acid receptors are known at present, RARo- (Petkovich et al. 1987; Giguere et al. 1987), RAR/3 (Brand et al. 1988; Benbrook et al. 1988), RARy(Zelent et al. 1989; Krust et al. 1989) and RXRo- (Mangelsdorf et al. 1990; Rowe et al. 1991), which are differentially expressed during development (Zelent et al. 1989; Dolld et al. 1989; Ruberte et al. 1990; Rowe et a\. 1991). It is unclear which of those is involved in the action of RA on chick sympathetic neurons. However, the recently described strong expression of chick RXRi* in the developing chick peripheral nervous system (Rowe et al. 1991), suggests the involvement of RXRaror a member of this receptor family in RA actions in the chick PNS. The lack of NGF response in E7 sympathetic neurons can be due to the absence of NGF receptors, or missing steps further downstream in the signal transduction pathway, or both. In an attempt to decide between these alternatives, the binding of NGF to specific receptors on E7 sympathetic neurons was investigated. Two types of NGF receptors have been described, lowaffinity (K d 2X10~ 9 M) and high-affinity receptors (K d 2X10~ U M) (Suiter etal. 1979). NGF dissociates rapidly from the low-affinity, but only very slowly from the high-affinity receptor. Since neuronal survival and fibre outgrowth elicited by NGF are mediated by highaffinity receptors (Green et al. 1986), the absence of high-affinity receptors during early stages of neuron differentiation would explain the lack of an NGFresponse. Steady-state binding studies demonstrated that this is indeed the case, i.e. freshly isolated E7 sympathetic neurons expressed low-affinity receptors but no high-affinity receptors were detected. The NGFresponse and expression of high-affinity NGF receptors correlated also in cultured neurons: whereas RAtreated sympathetic neurons acquired high-affinity receptors and survived with NGF, these receptors were never observed in cultures maintained without RA. The most parsimonious interpretation of our results is that E7 sympathetic neurons do not express significant numbers of high-affinity receptors and that the induction of these receptors by RA is sufficient to induce the survival response to NGF. More complicated schemes involving the induction of other members of the signal transduction pathway of NGF in addition to the highaffinity receptor are, however, also compatible with our data. Since the response of chick lumbosacral sympathetic neurons to NGF starts between E7 and E8 (Ernsberger et al. 1989) the ability to detect high-affinity receptors depends on the exact developmental stage of the ganglia. Thus the previous demonstration of a small number of high-affinity NGF receptors (Godfrey and Shooter, 1986) may be due to slight differences in the ages of the ganglia used. NGF receptors were detected previously by autoradiograpy in cultures of E7 sympathetic neurons (Rohrer et al. 1983), but it is not possible to distinguish high-affinity from low-affinity receptors under the conditions used (for discussion see Lindsay and Rohrer, 1985). The structure of the high-affinity NGF receptor is not known, but there is evidence that low-affinity receptors are a part of the high-affinity receptor complex (Hempstead et al. 1989) and are modified by the association of an additional protein (Hosang and Shooter, 1985) resulting in the increased affinity. Furthermore, it has been shown recently that the lowaffinity NGF receptor binds the structurally related neurotrophic factor BDNF with the same affinity (Rodriguez-Tdbar et al. 1990), suggesting that highaffinity receptors for different neurotrophic factors result from the association of different accessory proteins to a single low-affinity receptor. The present

6 818 A. Rodriguez-Tibar and H. Rohrer observation that RA specifically induces the expression of high-affinity NGF receptors suggests that RA either regulates the expression of the accessory protein responsible for high-affinity NGF binding or stimulates its interaction with the low-affinity receptor. However, in a neuroblastoma cell line, RA treatment (10~ 5 M) has been previously observed to increase the number of both high- and low-affinity NGF receptors (Haskell et al. 1987). The acquisition of NGF-responsiveness and NGFdependence has also been analysed using cultures of sympathoadrenal precursor cells from embryonic and neonatal rat adrenal medulla (Doupe et al. 1985a,b\ Anderson and Axel, 1986; Stemple et al. 1988; Birren and Anderson, 1990) and has led to conclusions similar to those from studies in chick embryos. Precursor cells from E14.5 animals were found to survive and proliferate independently of the presence of NGF (Anderson and Axel, 1986). In cultures of such cells, as well as in a cell line (MAH) derived from these cells (Birren and Anderson, 1990), bfgf induced neurite outgrowth. Furthermore, induction of NGF receptor mrna in response to FGF was described in this precursor cell line. Neurons of a more advanced stage of differentiation, which rapidly extend processes in the absence of bfgf, were defined and isolated using a monoclonal antibody directed against the cell surface antigen B2. NGF-dependent survival was not observed in B2 + cells. B2 + cells display very similar properties to E7 chick sympathetic neurons, not only with respect to proliferation, neurite production, expression of tyrosine hydroxylase, but also by the lack of FGF-effects (D. Anderson, personal communication) and inability to survive with NGF. It is tempting to speculate that the lack of NGF-response in B2 + cells may also be due to the absence of high-affinity receptors. Taken together, these findings suggest a simple model for the onset of NGF-dependent survival during the early stages of sympathetic neuron development: proliferation, survival and initial neurite outgrowth of immature peripheral neurons are independent of NGF. FGF, or a related, secretable factor, stimulates initial neurite outgrowth and induces the expression of lowaffinity NGF-receptors. Low concentrations of RA, produced either in the environment or within peripheral ganglia, then induce the expression of high-affinity NGF receptors, allowing the cells to-respond to NGF. Thus, at the time when the neurites reach their targets, the neurons respond to peripheral, target cell-derived NGF and the number of neurons can be adjusted to the size and innervation density of the target. It is unclear if this hypothetical scheme also applies to other types of neurons. In contrast to sympathetic neurons, chick sensory neurons acquire survival response to NGF and BDNF in culture (Ernsberger and Rohrer, 1988). This might, however, be explained by exposure of sensory ganglia to RA in vivo before the onset of culture or by local supply of RA by non-neuronal cells present in larger numbers in cultures of sensory ganglia (Rohrer and Thoenen, 1987). It should also be pointed out that whereas the appearance of NGF-response may be explained by the acquisition of a functional signal transduction pathway, the mechanisms leading to dependence on neurotrophic factors are still unclear. At present, there is no direct evidence for an in vivo role of RA in trunk PNS development, as retinoidinduced teratogenic effects have not been described for trunk neural crest-derived ganglia (Shenefelt, 1972; Kochar, 1967). However, such effects may be less obvious as compared with gross craniofacial malformations, or remained undetected since the critical period for production of malformations may be later than for cranial crest. The model proposed for the role of RA in sympathetic neuron development, although being speculative, makes a number of predictions which can be tested; for example, that RA should (1) be present in the limbs, not only during early development (Thaller and Eichele, 1987), but also up to E10; (2) act retrogradely; and (3) induce the accessory protein responsible for the formation of the biologically active, high-affinity NGF receptor. Our data imply a role for retinoic acid in nervous system development not only in pattern formation during early developmental stages (Durston et al. 1989), but also later, during the stage of target field innervation. 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