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KIM HUNTER*t, MALCOLM MADEN*t, DENNIS SUMMERBELL§, ULF ERIKSSON¶, AND ..... Council project grant to N.H. and M.M.; K.H. was supported by the.
Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3666-3670, May 1991 Neurobiology

Retinoic acid stimulates neurite outgrowth in the amphibian spinal cord (retinol/cellular retinol- and retinoic acid-binding proteins/glia)

KIM HUNTER*t, MALCOLM MADEN*t, DENNIS SUMMERBELL§, ULF ERIKSSON¶, AND NIGEL HOLDER* *Anatomy & Human Biology Group, Division of Biomedical Science, King's College, Strand, London, WC2R 2LS, United Kingdom; §Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom; and ILudwig Institute for Cancer Research, Stockholm Branch, Box 60202, S-10401 Stockholm, Sweden

Communicated by Pierre Chambon, January 7, 1991

ABSTRACT There is increasing evidence that retinoic acid (RA), a vitamin A metabolite, plays a role in the development of the nervous system. Here we specifically test this notion by examining the effect of RA on neurite outgrowth from explanted segments of the axolotl spinal cord. We show that there is a threshold concentration in the region of 0.1-1 nM above which neurite outgrowth is stimulated 4-5 fold. Retinol, by contrast, only stimulated the migration of glial cells from the explants. Using HPLC we demonstrate that RA and retinol are present endogenously in the axolotl spinal cord. In addition, we have identified by immunocytochemistry with antipeptide antibodies the cells of the spinal cord that contain the binding proteins for RA (cellular RA-binding protein; CRABP) and retinol (cellular retinol-binding protein; CRBP). CRABP is found in the axons and CRBP is found in the ependyma and glial cells. These results provide strong evidence for a role for RA in the developing nervous system, and we propose a specific hypothesis involving CRBP, CRABP, retinol, and RA in the control of axon outgrowth in the spinal cord.

Endogenous neurotrophic factors have been found in CNS tissue extracts, and a role has been suggested for some of these in controlling directional guidance of axons. For example, the ventral floor plate of the rat embryo spinal cord produces a diffusable factor in vitro that attracts the commissural axons within a cord explant-behavior compatible with developmental patterns of growth in vivo (19). As described above, these neurons express high levels of CRABP, suggesting that RA may play a role in controlling axon outgrowth, a view that has recently been supported by data showing that the chicken ventral floor plate produces and releases RA (20). We have used the spinal cord of the axolotl, Ambystoma mexicanum, because this animal is a continuously growing larva that adds neurons at postembryonic stages (21) and that retains its embryonic character. This allows us to collect larger amounts of tissue and perform more precise dissections than is possible with embryos. As a result we also demonstrate by HPLC that RA and its metabolic precursor retinol are present endogenously in the axolotl spinal cord and by immunocytochemistry that CRABP and cellular retinol-binding protein (CRBP) are expressed in precise patterns within the CNS which may relate to their action in the spinal cord.

Exogenous administration of retinoic acid (RA), a biologically active metabolite of vitamin A, has profound effects on developing and regenerating limbs, causing the production of extra elements in one or several of the limb axes (1, 2). RA has also been detected endogenously in the chicken embryo limb bud at a concentration and with a distribution compatible with its being a morphogen that organizes pattern across the anteroposterior axis of the limb bud (3). Therefore, RA may be an important signaling molecule involved in the development of the embryo. The mechanism of action of RA involves two classes of proteins within the cell, one class in the cytoplasm and one in the nucleus. The cytoplasmic proteins are cellular RA-binding proteins I and II (CRABP I and CRABP II) (4-6), and the nuclear proteins are two families of RA receptors, the RARs (7-9) and the RXRs (10), which are members of the superfamily of steroid hormone receptors. RA also may be involved in the development of the nervous system because its exogenous application changes the anteroposterior pattern of the Xenopus central nervous system (CNS) and RA is present in the Xenopus embryo (11). RA is well known as a mammalian teratogen, the CNS being a major target (12, 13). Furthermore CRABP shows a precise localization to the neurons of the commissural system in the developing chicken (14) and mouse embryo (15, 16) spinal cord and shows temporally and spatially regulated expression in the rhombomeres of the hindbrain in these embryos (17). Here we provide further evidence for a role of RA in neural development by showing that it stimulates neurite outgrowth from explants of the spinal cord ofthe amphibian Ambystoma

MATERIALS AND METHODS Cultures. Small pieces of spinal cord (-2 mm2) were dissected from 4- to 8-cm axolotls and placed on a glass coverslip adjacent to a piece of nitrocellulose paper previously soaked for 30 min in solutions (1 pg/ml to 10 mg/ml) of all-trans-RA (Sigma) dissolved in ethanol and washed in explant medium [a defined medium of Lebowitz L-15 medium/Ham's F-12 medium adapted from that of Olsen and Bunge (22)]. The explant and paper were then covered in a collagen solution, which gelled to provide a semisolid environment (adapted from ref. 23). Cultures were flooded with medium and maintained at 24°C in 2.5% C02/97.5% air for 18-24 hr. They were then examined with phase-contrast microscopy. For immunocytochemical examination of the cultures, they were dehydrated in an alcohol series, rehydrated in phosphate-buffered saline (PBS), and incubated with antibody for 2 hr. For the identification of neurites, the anti-neurofilament monoclonal antibody RT97 (a gift of Brian Anderton, Institute of Psychiatry, London) was used at a dilution of 1 in 15. For the identification of glial cells the monoclonal antibody RGE53 (a gift of Birgitte Lane, Uni-

mexicanum.

Abbreviations: RA, retinoic acid; CRABP, cellular RA-binding protein; CRBP, cellular retinol-binding protein; CNS, central nervous

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

tPresent address: Department of Anatomy, St. George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 ORE, United Kingdom. tTo whom reprint requests should be addressed.

system.

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Neurobiology: Hunter et al. versity of Dundee, Scotland) was used undiluted. RGE53 is known to stain glial cells in the axolotl spinal cord, recognizing a single 45-kDa band on immunoblots, the axolotl equivalent of human keratin 18 (24). After antibody incubation, cultures were rinsed in PBS, incubated in fluoresceinconjugated goat anti-mouse antibody (Sigma) for 2 hr, rinsed again in PBS, and mounted in XAM (B.D.H., Poole, U.K.). HPLC Analysis. Extractions and HPLC analysis were performed as established for the chicken embryo limb bud (3). Briefly, spinal cords from 15-20 12-cm axolotls were dissected, sonicated in stabilizing buffer, and extracted three times with 8:1 (vol/vol) ethyl acetate/methyl acetate containing butylated hydroxytoluene. The organic phases were pooled and dried over nitrogen, and 100 Al of methanol was added for HPLC analysis. As an internal radioactive standard, 250 pg of all-trans-[3H]RA was added to the sonicated tissues. A Beckman gold HPLC system was used. For reverse phase, an XL ODS C column was used with methanol/acetonitrile/water, 40:40:20 (vol/vol), at pH 6 as the solvent. For normal phase, a LicroSpher SI60 5-gm column was used with dichloromethane/acetonitrile, 95:5 (vol/vol), as a solvent. Immunoblotting and Immunocytochemistry with CRABP and CRBP. For Western blotting, tissue samples were homogenized in solubilization buffer for SDS/PAGE and centrifuged to remove debris. Aliquots (40 Al) of the extracts were electrophoresed on 12.5% gels and transferred to nitrocellulose paper. Immunoreactivity was detected by general blocking of protein binding sites with 5% milk powder/ 0.1% Tween in PBS, incubating with the affinity-purified antipeptide antibody and then with a biotinylated anti-rabbit antibody, and visualizing by the avidin-biotin technique with a kit from Vector Laboratories. For immunocytochemistry on sections of spinal cord, tissues were fixed in Perfix (Fisher Scientific) for 3 hr, dehydrated, cleared in xylene and embedded in wax. Sections (7 ,m) were cut and treated overnight at 4°C with the appropriate antibody at 5 ,ug/ml. The color was developed by using the avidin-biotinylated peroxidase complex with a kit from Vector Laboratories, and sections were counterstained with hematoxylin.

RESULTS Effect of RA on Spinal Cord Explants. The culture method involved placing explants in a semisolid collagen gel and making RA available to the explant on a carrier of nitrocellulose paper placed adjacent to the explant prior to covering with the collagen gel mixture. This system of delivery was used because (i) RA added to the medium at concentrations used in this study interfered with the setting of the collagen gel and (ii) we were attempting to duplicate in vivo conditions whereby RA may be provided by a local source. A total of 306 segments of spinal cord were cocultured adjacent to pieces of nitrocellulose paper that had been soaked in concentrations of RA varying from 1 pg/ml to 10 mg/ml. In such an environment, control explants showed no outgrowth of neurites or migration of cells. In the presence of RA, however, a substantial amount of neurite outgrowth was promoted (Figs. 1 and 2), with a pronounced threshold effect at a soaking concentration of 0.1 ,uM. Below this concentration there was a minimal response (neurites typically less than 0.4 mm long), but above it neurite outgrowth was stimulated 4- to 5-fold to reach a typical length of 1-2 mm (Fig. 1). RA soaking concentrations above 10 mg/ml were toxic, and no outgrowth was seen. Examples of neurite outgrowth in response to RA are shown in Fig. 2. Neurites were recognized by the anti-neurofilament antibody RT97 (Fig. 2 C and D). An estimate of the concentration of RA in the gel (rather than the soaking concentration into which the nitrocellulose

Proc. Natl. Acad. Sci. USA 88 (1991)

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RA, log(ng/ml) FIG. 1. The response of larval axolotl spinal cord explants in collagen gels to nitrocellulose paper soaked in increasing concentrations of RA. The response measured was the maximum length of neurites in each culture. Each point represents data from 7-27 cultures, making a total of 306 cultures in all. A threshold soaking concentration at 0.1 /.M is apparent above which a 4- to 5-fold stimulation of neurite length occurs. Standard errors are marked.

was put), and thus a more realistic value of the maximum concentration of RA to which the explant would be exposed, can be obtained by calculating the volume of the nitrocellulose paper (assuming it is fully saturated with RA) and taking the volume of the collagen gel as 1 ml. This gives a value of 20 pg on the nitrocellulose paper at the threshold and a maximum concentration of RA to which the explants would be exposed of 0.4 nM. Of course this estimate is rough: it assumes that all the RA will diffuse out of the nitrocellulose paper, and it ignores any diffusion gradients that would surely be established within the gel (see ref. 25), which would only serve to further reduce the effective concentration of RA received by the explant. A more accurate estimate would involve determining the diffusion characteristics of RA in collagen, whether RA breaks down in collagen, and the rate of RA metabolism within the explant. As well as neurite outgrowth, cells were seen to migrate out from the explants in response to RA. These were seen only in explants that displayed neurite outgrowth, but migrating cells did not seem to be specifically associated with bundles of neurites (Fig. 2A). Migrating cells were RT97-, and therefore nonneuronal, but reacted with the anti-keratin 18 antibody RGE53 (Fig. 2 E and F) previously found to recognize intermediate filaments in radial and other glia in the axolotl spinal cord (24). For comparison we examined the effect of retinol, the metabolic precursor of RA. Even over a 7-fold range of soaking dilutions (10 ng/ml to 100 mg/ml), neurite outgrowth was stimulated in only a minority of cultures (in total, 25 of 110 = 22%), and there was no correlation with increasing dose. Those few neurites that did grow were short (less than 0.4 mm). Thus, in contrast to RA, retinol was not particularly effective at promoting neurite outgrowth. However, cell migration was seen in a high proportion of cases at each dose (minimum, 56%; maximum, 93%) and with no obvious dose

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c-f) and retinol (b). (A) Camera lucida drawing of an RA-treated

culture after 18 hr. Neurite bundles (arrows) are seen emanating from the explant (ex) and growing into the gel. Many individual cells are also seen (dots) leaving the explant. The concentration of RA on the nitrocellulose paper in the culture from which this drawing was taken was 3 ug/ml. (Bar = 0.5 mm.) (B) Camera lucida drawing of a retinol-treated culture after 18 hr. In contrast to A, no neurites have been extended from the explant (ex), but cell migration was stimulated (dots represent cells). (Bar = 0.5 mm.) (C) Immunocytochemical demonstration of neurites in a RA-treated (3 ,ug/ml soaking solution) explant culture. A small neurite bundle is stained with the anti-neurofilament monoclonal antibody RT97. (Bar = 100 ,um.) (D) Accompanying phase-contrast micrograph. (Bar = 100 Aum.) (E) Immunocytochemical demonstration of glial cells in a RA-treated culture (3 5Lg/ml soaking solution) stained with the monoclonal antibody RGE53, which is known to stain glial cells in the axolotl spinal cord (24). (Bar = 50 ,um.) (F) Accompanying phase-contrast micrograph. (Bar = 25 ,um.)

dependency. Because so few cultures showed neurite outgrowth, cell migration took place in the majority of cases in the absence of neurite outgrowth, in contrast to the situation with RA. HPLC Analysis. To examine whether these responses might be a normal part of the developmental repertoire of the spinal cord or simply a nonspecific response to a chemical, we looked for the presence of endogenous retinoids. Extraction, detection, and quantitation of retinoids by reversephase and normal-phase HPLC were performed by techniques used previously in the chicken embryo limb bud (3). On reverse-phase HPLC, the array of absorbance peaks detected at 354 nm from axolotl spinal cords was similar to that seen in the chicken limb bud. Each of the retinoid peaks was identified by coelution with both external standards and internal tritiated standards. By these criteria peak 1 in Fig. 3A was identified as all-trans-RA, peak 2 as all-trans-retinol, and peak 3 as 13-cis-RA. To further establish the identity of peaks 1, 2, and 3, extracts were chromatographed by normal-phase HPLC with a different buffer system. Again the same peaks, identified by coelution with tritiated and nonradioactive

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FIG. 3. Chromatographs of reverse-phase (A) and normal-phase (B) HPLC analysis of extracts of spinal cord. -, Absorbance at 354 nm; ---, elution profile of the [ H]RA standard determined by scintillation counting. Arrows mark the elution positions of external standards: 1, all-trans-RA; 2, all-trans-retinol; and 3, 13-cis-RA. The coincidence of the internal standard radioactivity peak (---) and the position of authentic all-trans-RA deduced from external standards (peak 1) on both reverse-phase and normal-phase columns confirms the presence of this retinoid in the axolotl spinal cord.

standards were present (Fig. 3B). As an additional criterion of identification, the fractions including peak 1 from a normal-phase chromatogram (Fig. 3B) were pooled, dried, and rerun on the reverse-phase system. This resulted in coelution of a single peak with authentic all-trans-RA, thus confirming its presence in the axolotl spinal cord. DNA estimations were performed on these homogenates of spinal cords before extraction so that amounts of RA per unit of DNA could be calculated. The amount of RA on the chromatograms was estimated from peak heights by using previously established standard curves, and these calculations gave a value in the cord of 1-2 pg/,ug of DNA, a similar figure to that of the chicken embryo limb bud (3). In contrast to the limb bud, however, a similar amount of retinol was detected (compare peaks 1 and 2 in Fig. 3 A and B) rather than a 25-fold excess.

Immunocytocbemlstry of CRABP and CRBP in Set

of

Spinal Cord. We then considered the question of whether the specific binding proteins for retinol (CRBP) and RA (CRABP) might be associated with any particular cell types within the spinal cord. To do so we used affinity-purified antibodies on tissue sections of 4- to 8-cm axolotls. These antibodies were prepared from antisera raised against synthetic peptides corresponding to sequence stretches unique to CRBP and CRABP (26). To confirm that the antibodies each recognized a protein of the appropriate molecular weight in axolotl tissue

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Proc. Natl. Acad. Sci. USA 88 (1991)

and that they do not cross-react with each other, they were used on immunoblots (Western blots) prepared from axolotl CNS. The CRBP antibody detected a single band of -15 kDa (Fig. 4, lane 2) that comigrated with pure rat CRBP (lane 1). The CRABP antibody detected a single band (lane 5) that migrated at a position corresponding to a slightly higher molecular mass than CRBP and did not cross-react with pure CRBP protein (lane 4). We have found that the CRABP antibody does not blot as well as the CRBP antibody both in these experiments with the axolotl and in experiments with other species such as mouse. Nevertheless, the antibody gives excellent immunocytochemical results. Use of these antibodies on sections of axolotl spinal cord revealed a precise and complementary localization of CRBP and CRABP. CRBP immunoreactivity was detected in a subpopulation of cells in contact with the neural canal and in the dorsal and ventral midline (Fig. 5A). In the axolotl these cells were predominantly radial glia, which have processes running through the white matter to the periphery of the cord (24). Immunoreactivity was particularly clear in the processes of the ventral floor plate glia (Fig. 5A, thin arrow). No immunoreactivity was detected in the white matter of the spinal cord or in the peripheral nervous system. On the other hand, CRABP showed a complementary distribution, being present in axons in the white matter of the cord rather than in glial cells in the grey matter (Fig. 5B) and was evident in commissural neurons running through the ventral floor plate. Examining serial sections through the spinal cord and brain revealed that not all axons were immunoreactive to CRABP. In the hindbrain, for example, axons in the dorsal tracts were unlabeled (Fig. 5C), and also striking was the almost complete absence of immunoreactivity in the ventral midlinethe location of the ventral floor plate glia that were positive for CRBP (Fig. 5A). As in the spinal cord, commissural axons were evident here, passing through the ventral floor plate. Finally, since CRABP was present in the axons of the cord, we have examined sections of RA-treated explants, and it was clear that most axons present within the collagen gel were CRABP-positive.

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DISCUSSION We describe here three experiments that together provide substantial evidence to suggest that retinoids are involved in 1

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FIG. 5. Immunocytochemical localization of CRBP (A) and CRABP (B and C) on sections of 4- to 8-cm axolotl spinal cord and hindbrain. (A) CRBP is seen in the cells surrounding the neural canal of the spinal cord, the location of radial glial cell bodies (thick arrow) and in the ventral floor plate glial processes (thin arrows). (Bar = 25 ,um.) (B) CRABP has a reciprocal distribution, being localized to axons in the white matter of the spinal cord (arrow). (Bar = 50 ,um.) (C) In the hindbrain there are dorsal axon tracts that are not immunoreactive to CRABP (thick arrow). The processes of the ventral floor plate glia also fail to label (thin arrow). (Bar = 100 tim.)

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FIG. 4. Detection of immunoreactive proteins in extracts of axolotl tissues. Extracts in lanes 1 and 2 were treated with CRBP antibody; those in lanes 4 and 5 were treated with CRABP antibody. Lanes: 1, pure rat CRBP; 2, axolotl CNS; 3, axolotl brain incubated with normal rabbit serum as a control for lane 2; 4, pure rat CRBP to show that the two antibodies do not cross-react; 5, axolotl CNS; 6, control for lane 5 incubated with normal rabbit serum. Positions of molecular

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the development of the nervous system. First, RA stimulates outgrowth of neurites from axolotl spinal cord explants with a threshold effect in the region of 0.1-1 nM, whereas retinol primarily stimulates outgrowth of glial cells with no apparent dose response. Second, RA and retinol can be detected endogenously in the spinal cord. Third, CRABP is present in vivo in most but not all axons in the white matter of the spinal cord, whereas CRBP is present in glial cells located in the grey matter and in their processes located in the ventral floor plate. As a result of these experiments our hypothesis for the function of RA in neurite outgrowth is that the CRBP+ glial and ependymal cells of the axolotl spinal cord sequester

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retinol, metabolize it to RA, and then release RA, which is required for promoting neurite outgrowth from neurons in the immediate environment. This hypothesis is also supported by data from the developing nervous system of the mouse and chicken embryo. In the mouse, in situ hybridization analysis ofCRBP and CRABP mRNA has shown that the corresponding genes are expressed in nonoverlapping regions (27). In the chicken embryo, the ventral floor plate produces RA from retinol at a higher rate than the rest of the neural tube, and most significantly, when the floor plate is grafted to the anterior margin ofthe chicken embryo limb bud, it mimics the patterning effects of exogenous RA by causing duplications (20). Only the floor plate cells of the chicken cord express CRBP (28), and CRABP is expressed by the commissural neurons at the time they are extending neurites toward the floor plate (14). Thus, here too RA may play a role in controlling the growth of axons, particularly commissural axons, that normally grow towards and cross the ventral floor plate. These suggested in vivo roles can be envisaged as extrapolations ofthe well-established demonstrations in vitro that RA induces mouse (29, 30) and human (31) embryonal carcinoma cells to differentiate into neurons. However, the physiological role of CRABP and CRBP remains to be established despite these correlations. One recent experiment of relevance concerning CRABP involved the administration of a radiolabeled derivative of RA to pregnant mice with the result that the radioactivity only localized to those regions of the embryo expressing CRABP (32), reinforcing the suggested role for this protein in the normal functioning of RA in the embryo. Of particular importance with regard to the action of RA in neurite outgrowth will be to distinguish the effects of RA on glial and neuronal populations and to establish whether RA is taken up by growing neurites and transported to the cell body to interact with the RA receptors located in the nucleus. Because of the highly extended nature of its cytoplasm, the neurite may be the ideal cell type with which to establish the role of CRABP. In conclusion, the development ofthe nervous system may be seen in terms of three phases: (i) neuronal specification, (ii) neurite outgrowth, and (iii) maintenance. There is now considerable evidence to suggest that RA is involved in each of these phases. Concerning neuronal specification, when administered at gastrula stages, RA causes an anteroposterior transformation of the Xenopus CNS whereby the forebrain and midbrain are reduced or absent and the hindbrain and spinal cord are exaggerated (11). Also, CRABP is expressed in specific rhombomeres early in the developing hindbrain of the chicken (17) and mouse embryos and at the same time as the expression of homeobox genes in these structures. It has already been established that homeobox gene expression can be regulated in a precise way by RA (18). Concerning neurite outgrowth, the results described here and those discussed above suggest a role for RA in this aspect. Concerning axonal maintenance, CRABP continues to be expressed in the white matter of the chicken embryo and axolotl spinal cord (Fig. 5B), suggesting a continuing role in the maintenance of axon tracts in the cord. Thus, from its initial formation, the nervous system may need to strictly regulate the levels of retinoids to maintain correct functioning. This work was supported by a Science and Engineering Research

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