DEVELOPMENTAL DYNAMICS 194231-238 (1992)
Development of Olfactory Nerve Glia Defined by a Monoclonal Antibody Specific for Schwann Cells ROBERT B. NORGREN, JR., NANCY RATNER, AND ROBERT BRACKENBURY Division of Neuroscience, Department of Psychiatry (R.B.N.),and Department of Anatomy and Cell Biology (N.R., R.B.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
ABSTRACT Although there is considerable eter (0.2 pm) olfactory nerve axons (Gasser, 1956; De interest in the possible role of olfactory glia in the Lorenzo, 1957; Cuschieri and Bannister, 1975; pathfinding abilities of olfactory nerve axons, the Kreutzberg and Gross, 1977; Farbman and Squinto, complete development of these glia in vivo has not 1985; Daston et al., 1990; Burd, 1991). The close conbeen described. Using a specific Schwann cell tact between axons resulting from this pattern of enmarker, the 1E8 antibody, we have found that ol- sheathment is not observed in mature peripheral factory nerve glia can be identified throughout de- nerves containing either myelinating or nonvelopment. These glia appear to originate in the myelinating Schwann cells (Webster et al., 1973). In olfactory placode and migrate initially into the pe- addition, electron microscopic studies (Doucette, 1989; riphery of the olfactory nerve, and later into the Marin-Padila and Amieva B, 1989) and tissue culture center of the nerve. Olfactory nerve glia enter the studies (Fields and Dammerman, 1985) suggest that presumptive olfactory bulb with the olfactory re- olfactory nerve glia are present both in the peripheral ceptor neuron axons and distribute themselves nervous system (the olfactory nerve) and within the along the edge of the olfactory nerve layer. The central nervous system (the olfactory nerve layer of the fact that olfactory nerve glia are specifically im- olfactory bulb). munostained by the lE8 monoclonal antibody, Several issues regarding olfactory nerve glia, which which recognizes the Schwann cell-specific pro- may relate to olfactory nerve development and regentein Po, suggests that these cells more closely re- eration, are currently the subject of much study and semble Schwann cells than astrocytes or enteric debate. First, although most Schwann cells are derived glia. These results support and extend previous from neural crest, it appears that olfactory nerve glia findings suggesting that olfactory nerve glia have originate in the olfactory placode (Couly and Le distinctive developmental and anatomical fea- Douarin, 1985; Marin-Padila and Amieva B, 1989; tures which may be important to the regenerative Chuah and Au, 1991). Second, the temporal and spatial capacity of the olfactory system. relationships between olfactory nerve glia and olfac0 1992 Wiley-Liss, Inc. tory nerve outgrowth have not been established. Third, the distribution within the olfactory bulb of glia that Key words: Olfactory, Glia, Development, are derived from the olfactory nerve is currently unreSchwann cell, Po, Ensheathment, Re- solved (Doucette, 1990). The studies reported here addressed these issues generation through the use of an antigenic marker that recognizes INTRODUCTION olfactory nerve glia throughout development. We have recently developed a monoclonal antibody (1E8) that The olfactory epithelium is one of the few areas of the recognizes the major myelin protein Po and labels both adult nervous system that continually produces new neurons (Graziadei and Monti-Graziadei, 1978; Barber, myelinating and non-myelinating Schwann cells in the 1982a; Brunjes and Frazier, 1986; Shepherd, 1988- chick (Bhattacharyya et al., 1991). 1E8 does not recog1989). As the axons of these newly generated neurons nize astrocytes, oliogodendrocytes, or neurons (Bhattamust continually re-establish specific connections with charyya et al., 1991). In the present study, we found the olfactory bulb, there is considerable interest in the that 1E8 marks olfactory nerve glia both in early chick role that glia may play in the pathfinding abilities of embryos and in mature chickens, supporting the hyolfactory nerve axons during development and regen- pothesis that olfactory nerve glia are more closely reeration (Barber, 1982b; Cancalon, 1986; Barber and lated to non-myelinating Schwann cells than to other Dahl, 1987; Doucette, 1990). Previous studies have established that glial cells of the olfactory nerve differ from glia that ensheath axons in peripheral nerves. Olfactory nerve glia do not myReceived August 14, 1992. elinate and rarely ensheath individual axons (see reprint requestsicorrespondence to Robert Norgren, Dept. Raisman, 1985, for exceptions). Instead, each olfactory of Address Cell Biology and Anatomy, University of Nebraska Medical Center, nerve glial cell ensheaths bundles of many small-diam- 600 S. 42nd Street, Omaha, NE 68198-6395. 0 1992 WILEY-LISS. INC
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glial cell types. The developmental distribution of olfactory glia as determined by 1E8 immunostaining also supports the view that these glia are derived from the olfactory placode rather than the neural crest. Further, we determined that 1E8-immunostained glia are found first around the perimeter of the olfactory nerve and are not observed in the center until later in development. Finally, we found that these 1E8-immunoreactive glia appear to enter the olfactory bulb via the olfactory nerve. In the adult olfactory bulb, these cells form a thin rim around the edge of the olfactory nerve layer.
RESULTS Several studies indicate that olfactory nerve axons first exit the chick olfactory epithelium on E3 (Simonetta, 1933; Windle and Austin, 1935). Although 1E8 immunostaining was observed in cranial nerves (trigeminal and occulomotor) a t E3, no 1E8 immunostaining was visible in the olfactory nerve until E4. Early on E4,1E8 immunostaining within the olfactory nerve was very faint, while immunostaining within nearby nerve twigs (presumably of the ophthalmic portion of the trigeminal nerve) was very intense (Figs. lA, 2B). Later on E4, immunostaining was more distinct. At this time, immunostaining in the olfactory nerve near the lateral edge of the telencephalon was often in the shape of a ring surrounding a nonimmunostained center (Fig. 2A). Near the OE, immunostaining of the olfactory nerve remained very faint. In this region, 1E8 immunoreactivity was never seen in cells that were not associated with nerve fascicles. On E5, 1E8 immunostaining of cells within the olfactory nerve was more evident than a t E4, but the immunostaining in the olfactory nerve was still less prominent than in the trigeminal nerve. Immunostaining of cells in the olfactory nerve was observed both on fascicles of olfactory receptor neuron axons a s they left the olfactory epithelium and on more distal parts of the olfactory nerve in which many fascicles had become bundled together (Figs. lB, 2 0 . At this stage of development, the olfactory nerve is close to, but has not yet entered, the lateral edge of the telencephalon. The ring of immunostaining encircling the olfactory nerve, first observed on late E4, was still prominent, but some immunostaining was also apparent in the center of the nerve (Fig. 1B). Beginning at E6, 1E8 staining in the olfactory nerve was as intense a s in the nearby cranial nerves. At this age, the 1E8-immunostained olfactory nerve begins to surround both the lateral and medial edges of the rostral pole of the telencephalon (Figs. lC, 2D). In E7 animals, the spread of 1E8-immunostained cells both medially and laterally along the rostral telencephalon continued (Fig. 2E). 1E8 immunostaining was also observed in a fiber bundle in close proximity to the olfactory nerve in a n E7 animal (Fig. 1D). This fiber bundle is probably a branch of the ophthalmic trigeminal nerve.
On E8, a n evagination formed in the rostral telencephalon. 1E8 immunostaining was found along the edge of this immature olfactory bulb (Fig. 2F). 1E8 immunostaining in the olfactory bulb was more intense than that observed in the olfactory nerve. The spread of olfactory nerve glia around the enlarging olfactory bulb continued on E9 (Fig. 2G). On E l 0 (Figs. l E , 2H), the gross appearance of the olfactory bulb closely resembled that seen in hatchlings and in the adult chicken. In hatching chicks, the olfactory nerve layers and glomerular layers have developed sufficiently to be distinguished. As shown in Figure l F , 1E8 immunoreactive cells were clearly seen along the peripheral edge of the olfactory nerve layer, but not in the region of the olfactory nerve layer near the glomerular layer. No 1E8 immunostaining was ever observed in any brain region other than the olfactory nerve layer of the olfactory bulb. The fact that a Schwann cell-specific marker labelled cells in the olfactory nerve implied that they were glia. To corroborate this result, 1E8-immunostained cells were visualized a t high magnification in 1km cross-sections of hatchling olfactory nerve. Numerous immunostained processes surrounded unstained nerve bundles (Fig. 3). This pattern corresponds to the known distribution of olfactory glia in the olfactory nerve, confirming the specificity of the 1E8 antibody for olfactory nerve glia.
DISCUSSION The results reported here indicate that the 1E8 monoclonal antibody can be used as a specific marker for glial cells in the olfactory nerve a t all stages of development. The following discussion focuses on the use of this antibody to describe the development and distribution of olfactory nerve glia a s well as to examine their relationship to other glial types. Two lines of evidence indicated that the 1E8 monoclonal antibody specifically reacted with olfactory nerve glia. First, 1E8 has been previously shown to react with the major myelin protein Po and to specifically immunostain Schwann cells in peripheral nerves (Bhattacharyya et al., 1991). Second, the pattern of immunostaining in 1 km cross-sections of the olfactory nerve was congruent with the distribution of glial processes, which surround unstained axon bundles (Fig. 3). 1E8 appeared to immunostain the majority of olfactory nerve glia, but we cannot rule out the possibility that a small subpopulation of glia within the olfactory nerve is not immunostained by 1E8. In fact, electron microscopic and immunocytochemical experiments suggest that olfactory nerves may contain more than one glial cell type within the olfactory nerve (Barber and Lindsay, 1982; Pixley, 1992). Origin and Development of Olfactory Nerve Glia The available evidence suggests that, unlike enteric glia and Schwann cells in peripheral nerves, which are derived from the neural crest (Harrison, 1924;
DEVELOPMENTOFOLFACTORYNERVEGLIA
Fig. 1. Photomicrographs of 1E8 immunostaining in sections of embryonic and hatchling tissue. 1E8 immunostaining was visualized with DAB (brown reaction product). Some sections were counterstained with cresyl violet (blue staining). A: At E4, 1E8 immunoreactive cells are associated with nerve twigs, which presumably contribute to the ophthalmic branch of the trigeminal, in close proximity to the olfactory epithelium (oe). lmmunostaining of olfactory nerve glia is too faint to detect in this photomicrograph. Scale bar = 100 pm. B: By E5, 1E8 immunoreactive cells are associated with olfactory nerve (on) fascicles. Scale bar = 50 pm. C: On E6, the 1E8 immunoreactive cells in the olfactory nerve have begun to spread along the rostral telencephalon. Arrows indicate immunopositive cells along medial portion of the rostral telencephalon (tel). Magnification same as A. D: On E7, 1E8 immunolabeling in olfactory
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nerve (on) fascicles and another nerve, presumably the ophthalmic branch of the trigeminal (V) can be observed. Magnification same as B. E: By E l 0, 1E8 immunostaining is evident in the olfactory nerve (on) and the olfactory nerve layer of the presumptive olfactory bulb. The brown staining in the lower lefl of figure 1E represents endogenous peroxidase activity found in blood cells. Magnification same as A. F: In a hatchling, 1 E8 immunostaining is clearly visible along edge of the olfactory nerve layer of the olfactory bulb. Dashes indicate border between the olfactory nerve layer (onl) and the glomerular layer (gl). Scale bar = 25 pm. Abbreviations: gl = glomerular layer of the olfactory bulb, oe = olfactory epithelium; on = olfactory nerve; on1 = olfactory nerve layer of the 01factory bulb; tel = telencephalon; V = trigeminal nerve fascicles.
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Fig.2.
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Fig. 3. A 1 pm cross-section through the olfactory nerve of a hatchling demonstrates that 1E8 immunostaining is found in glia associated with the nerve, not axons. Scale bar = 10 p.m.
D’Amico-Martel and Noden, 1983; Le Douarin et al., 1991), olfactory nerve glia are derived from the olfactory placode. Investigators have noted the migration of two types of cells from the developing olfactory epithelium a t the electron microscopic level (Robecchi, 1972; Mendoza et al., 1982; Farbman and Squinto, 1985; Farbman and Menco, 1986; Doucette, 1989; MarinPadila and Amieva B, 1989). Cells of one type correspond to migrating LHRH neurons (Wray et al., 1989; Daikoku-Ishido et al., 1990; Ronnekleiv and Resko, 1990; Schwanzel-Fukuda and Pfaff, 1990; Norgren and Lehman, 1991). However, several experiments suggest that glial cells also arise in the olfactory epithelium and migrate into the olfactory nerve. First, when quail tissue from the olfactory placode region was transplanted into a chick which had its olfactory placode removed, the glial cells of the olfactory nerve were of quail origin, indicating a placodal source for these glia
Fig. 2. Drawings of horizontal sections of chick embryos illustrating 1E8 immunostaining. Rostra1 is down, caudal is up. Black labeling indicates 1E8 immunostaining. Stippling indicates neural tissue, either telencephalon or olfactory epithelium. In A-C, the gray border outlines the head. In D-H, only the rostral telencephalon and immature olfactory bulb are shown. Scale bar = 1 mm.A: Dorsal section of an E4 embryo head. A ring of immunostaining can observed in the olfactory nerve (on). tel, telencephalon. B: A more ventral section of the same chick head shown in A. At this level, the olfactory epithelium (oe) is present. Intense labeling in nerve twigs, presumably of the trigeminal nerve (V), can be observed. This drawing represents a section similar to the photomicrograph seen in A. C: Section through an E5 embryo head. 1 E8 immunopositive fascicles leaving the olfactory epithelium (oe) as well as staining in the olfactory nerve (on) in close proximity to the telencephalon (tel) can be observed. V, trigeminal nerve twigs. : Sections illustrating 1E8 immunostaining in the rostral telencephalon and/or immature olfactory bulb of E6 (D), E7 (E), E8 (F), E9 (G), E l 0 (H) chick embryos.
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(Couly and Le Douarin, 1985). Second, electron microscopic studies suggest that some cells migrating out of the olfactory epithelium ensheath bundles of axons in a manner similar to that observed in adult olfactory nerve glia (Farbman and Menco, 1986; Marin-Padila and Amieva B, 1989). Finally, cells expressing the glial marker GFAP migrate from E l 4 rat olfactory placode explants in tissue culture (Chuah and Au, 1991). Our results are inconsistent with the alternative view that olfactory nerve glia originate in the neural crest because 1E8-immunopositive cells were not observed migrating through extracellular space to the olfactory nerve. 1E8 does immunostain a subpopulation of migratory neural crest cells (presumptive Schwann cell precursors) (Bhattacharyya et al., 19911, suggesting that migrating crest cells would have been visible in our preparations if they had been present. Some cells close to the olfactory nerve were intensely immunostained by 1E8 (Figs. l A , C), but these cells appeared to be on the nasal portion of the ophthalmic branch of the trigeminal nerve which innervates the olfactory epithelium. Thus, although i t remains possible that there might be some mixing of Schwann cells in the trigeminal and olfactory nerves, the intense labeling of trigeminal glia compared to the weak immunostaining observed at early ages in the olfactory nerve supports the view that olfactory nerve glia arise in the olfactory placode. Although the olfactory nerve glia appear to arise in the olfactory placode, we never observed 1E8-immunopositive cells within the placode, indicating that the 1E8 antigen is not expressed until after the glia migrate into the nerve. This result is similar to the finding that the 1E8 antibody does not immunostain premigratory cells in neural crest but does immunostain cells shortly after they migrate away from the crest (Bhattacharyya et al., 1991). Although olfactory nerve axons begin extending from the olfactory placode to the telencephalon on E3, 1E8 immunostaining was not observed until E4. One interpretation of this finding is that olfactory nerve glia follow olfactory nerve axons out of the olfactory placode. This pattern of development, axon outgrowth followed by Schwann cell migration, is observed in other peripheral nerves (Speidel, 1964; Prestige and Wilson, 1980). However, i t is also possible that olfactory nerve glia migrate out before or at the same time as olfactory axons, but do not express the 1E8 antigen until later in development. The first cells immunostained for 1E8 were found around the perimeter of the olfactory nerve and only later were located throughout the nerve. There are two possible explanations for this pattern of immunostaining. It may be that the first glia to migrate into the olfactory nerve occupy the edge region, while later migrating glia from the olfactory epithelium occupy the center region of the nerve. A second possibility is that only the edge of the olfactory nerve receives migrating glia from the olfactory epithelium; the glia in the center of the nerve may derive from actively dividing pro-
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genitors in the edge region of the nerve. A similar pattern of glial development, i.e., glial cells first found along the edge and later in the center, has been observed in other peripheral nerves (Peters and Muir, 1959).
Relationship of Olfactory Nerve Glia to Other Glial Types The findings reported here may help resolve the ongoing controversy over the relationship between olfactory nerve glia and other glial cell types. Olfactory nerve glia have been identified by some as Schwann cells (Gasser, 1956; De Lorenzo, 1957; Cuschieri and Bannister, 1975; Mendoza et al., 1982; Couly and Le Douarin, 1985) and by others a s astrocytes (Kreutzberg and Gross, 1977; Barber and Lindsay, 1982; Doucette, 1984). In addition, enteric glia and olfactory nerve glia share several common attributes; both ensheath bundles of tightly packed axons, fail to form a basal lamina, and express GFAP (Jessen and Mirsky, 1980; Barber and Lindsay, 1982; Doucette, 1990; Gershon and Rothman, 1991).The fact that olfactory glia are labeled by 1E8, which is specific for myelinating and non-myelinating Schwann cells but does not label astrocytes or enteric glia (Bhattacharyya et al., 1991), suggests that olfactory nerve glia more closely resemble non-myelinating Schwann cells than other glial cell types. Glia Within the Olfactory Nerve Layer of the Olfactory Bulb The origin and identity of glial cells within the 01factory nerve layer of the olfactory bulb is of considerable interest, as these cells may facilitate axonal growth. In most peripheral nerves, astrocytes on the central nervous system (CNS) side of the CNSiperiphera1 nervous system (PNS) border zone are thought to prevent regenerating axons from crossing into the CNS. In contrast, regenerating olfactory nerve axons are able to re-innervate neurons in the CNS (Oley et al., 1975; Graziadei and Okano, 1979; Doucette et al., 1983), suggesting that glia in the olfactory bulb may have novel properties. In fact, olfactory bulb glia in culture have been shown to be more effective in stimulating outgrowth of substantia nigra neuron processes than either striatal or mesencephalic glia (Denis-Donini and Estenoz, 1988). Previous ultrastructural studies suggest that some of the glia in the olfactory nerve layer of the olfactory bulb do not originate within the brain, but are instead derived from the periphery. These studies indicate that during development, olfactory nerve glia enter the presumptive olfactory bulb with olfactory axons (Doucette, 1989; Marin-Padila and Amieva B, 1989). Our observation that 1E8-immunopositive cells were seen in the telencephalon only after the olfactory nerve reached the presumptive olfactory bulb provides additional support for this view. In addition, the only cells in the CNS that were labeled with the 1E8 antibody were found in the olfactory nerve layer of the olfactory bulb; a s was
previously mentioned, 1E8 is specific for Schwann cells and does not immunostain astrocytes (Bhattacharyya et al., 1991). Thus, 1E8-immunopositive cells of the olfactory nerve layer of the olfactory bulb exhibit ontogenetic and phenotypic characteristics of peripheral rather than central glia. As Schwann cells have been demonstrated to facilitate axonal regeneration while adult astrocytes retard regeneration (Perkins et al., 1980; Reier et al., 1983; Liuzzi and Lasek, 19871, similarities between Schwann cells and 1E8-immunostained cells of the olfactory nerve layer may be important in the unique ability of regenerating olfactory nerve axons to grow into the olfactory bulb. It is important to note that 1E8-immunostained cells were found only along the edge of the olfactory nerve layer and that other glia, deeper in the olfactory nerve layer, were not immunostained. Glia of the olfactory nerve layer of the olfactory bulb (and the nerve itself) exhibit characteristics of both astrocytes and Schwann cells (see Doucette, 1990, for review). It will be interesting to determine if the 1E8-immunopositive glia of the olfactory nerve layer exhibit any of the characteristics of astrocytes or whether astrocytic and Schwann cell characteristics are found in separate populations of cells of the olfactory nerve layer. In conclusion, the observations reported here strengthen the view that olfactory nerve glia arise in the olfactory epithelium, migrate within the olfactory nerve, and enter the olfactory bulb. The fact that these cells react with the 1E8 monoclonal, which recognizes Po, suggests that they are similar to peripheral glia, which may contribute to their ability to support the regeneration of olfactory axons within the CNS. Further studies, in which the 1E8 monoclonal can be used to identify, purify, or perturb olfactory nerve glia, may help to define the role of these cells in regeneration.
EXPERIMENTAL PROCEDURES Freshly fertilized chick eggs (N = 56) obtained from a commercial source (SPAFAS) were incubated a t 39°C for varying amounts of time. Embryos were removed from eggs, staged, and then either immersion-fixed overnight or perfused. Hatchlings (N = 3) and 3 year old hens (N = 3) were also perfused. Cardial perfusion with a 0.75% saline rinse was followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Brains with attached olfactory nerves were removed. After fixation, early embryo heads and brains from older animals were cryoprotected by infiltration with 20% sucrose in phosphate buffer. Cryostat sections (20 p.m) were incubated in primary antibody for 24 h r at 4°C. Purified 1E8 antibody was used a t a final concentration of 3 pg/ml in phosphate buffer with 0.2% Triton X-100. The avidin-biotin-HRP technique (Vectastain) and diaminobenzidine were used to visualize 1E8-immunoreactive cells. Some of these sections were counterstained with the Nissl stain cresyl violet. In addition, the olfactory nerves of hatchlings were embedded in gelatin, cross-sections were cut on a freezing
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microtome, and free-floating sections were immunostained a s above. These sections were subsequently embedded in EM bed-812 and 1 pm sections cut on a n ultramicrotome. Some sections were counter-stained with toludine blue.
ACKNOWLEDGMENTS We are grateful to Jan e Withrow and Xiao Gu for excellent technical assistance. This work was supported by NIH grant NS 30047 (RBN) and NS 27227 (RB) and (NR).
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Windle, W.F. and Austin, M.F. (1935) Neurofibrillar development in the central nervous system of chick embryos up to 5 days’ incubation. J. Comp. Neurol. 63:431-463. Wray, S., Grant, P., and Gainer, H. (1989) Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc. Natl Acad. Sci. USA 8623132-8136.
Development of Olfactory Nerve Glia Defined by a Monoclonal Antibody Specific for Schwann Cells ROBERT B. NORGREN, JR., NANCY RATNER, AND ROBERT BRACKENBURY Division of Neuroscience, Department of Psychiatry (R.B.N.),and Department of Anatomy and Cell Biology (N.R., R.B.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
ABSTRACT Although there is considerable eter (0.2 pm) olfactory nerve axons (Gasser, 1956; De interest in the possible role of olfactory glia in the Lorenzo, 1957; Cuschieri and Bannister, 1975; pathfinding abilities of olfactory nerve axons, the Kreutzberg and Gross, 1977; Farbman and Squinto, complete development of these glia in vivo has not 1985; Daston et al., 1990; Burd, 1991). The close conbeen described. Using a specific Schwann cell tact between axons resulting from this pattern of enmarker, the 1E8 antibody, we have found that ol- sheathment is not observed in mature peripheral factory nerve glia can be identified throughout de- nerves containing either myelinating or nonvelopment. These glia appear to originate in the myelinating Schwann cells (Webster et al., 1973). In olfactory placode and migrate initially into the pe- addition, electron microscopic studies (Doucette, 1989; riphery of the olfactory nerve, and later into the Marin-Padila and Amieva B, 1989) and tissue culture center of the nerve. Olfactory nerve glia enter the studies (Fields and Dammerman, 1985) suggest that presumptive olfactory bulb with the olfactory re- olfactory nerve glia are present both in the peripheral ceptor neuron axons and distribute themselves nervous system (the olfactory nerve) and within the along the edge of the olfactory nerve layer. The central nervous system (the olfactory nerve layer of the fact that olfactory nerve glia are specifically im- olfactory bulb). munostained by the lE8 monoclonal antibody, Several issues regarding olfactory nerve glia, which which recognizes the Schwann cell-specific pro- may relate to olfactory nerve development and regentein Po, suggests that these cells more closely re- eration, are currently the subject of much study and semble Schwann cells than astrocytes or enteric debate. First, although most Schwann cells are derived glia. These results support and extend previous from neural crest, it appears that olfactory nerve glia findings suggesting that olfactory nerve glia have originate in the olfactory placode (Couly and Le distinctive developmental and anatomical fea- Douarin, 1985; Marin-Padila and Amieva B, 1989; tures which may be important to the regenerative Chuah and Au, 1991). Second, the temporal and spatial capacity of the olfactory system. relationships between olfactory nerve glia and olfac0 1992 Wiley-Liss, Inc. tory nerve outgrowth have not been established. Third, the distribution within the olfactory bulb of glia that Key words: Olfactory, Glia, Development, are derived from the olfactory nerve is currently unreSchwann cell, Po, Ensheathment, Re- solved (Doucette, 1990). The studies reported here addressed these issues generation through the use of an antigenic marker that recognizes INTRODUCTION olfactory nerve glia throughout development. We have recently developed a monoclonal antibody (1E8) that The olfactory epithelium is one of the few areas of the recognizes the major myelin protein Po and labels both adult nervous system that continually produces new neurons (Graziadei and Monti-Graziadei, 1978; Barber, myelinating and non-myelinating Schwann cells in the 1982a; Brunjes and Frazier, 1986; Shepherd, 1988- chick (Bhattacharyya et al., 1991). 1E8 does not recog1989). As the axons of these newly generated neurons nize astrocytes, oliogodendrocytes, or neurons (Bhattamust continually re-establish specific connections with charyya et al., 1991). In the present study, we found the olfactory bulb, there is considerable interest in the that 1E8 marks olfactory nerve glia both in early chick role that glia may play in the pathfinding abilities of embryos and in mature chickens, supporting the hyolfactory nerve axons during development and regen- pothesis that olfactory nerve glia are more closely reeration (Barber, 1982b; Cancalon, 1986; Barber and lated to non-myelinating Schwann cells than to other Dahl, 1987; Doucette, 1990). Previous studies have established that glial cells of the olfactory nerve differ from glia that ensheath axons in peripheral nerves. Olfactory nerve glia do not myReceived August 14, 1992. elinate and rarely ensheath individual axons (see reprint requestsicorrespondence to Robert Norgren, Dept. Raisman, 1985, for exceptions). Instead, each olfactory of Address Cell Biology and Anatomy, University of Nebraska Medical Center, nerve glial cell ensheaths bundles of many small-diam- 600 S. 42nd Street, Omaha, NE 68198-6395. 0 1992 WILEY-LISS. INC
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glial cell types. The developmental distribution of olfactory glia as determined by 1E8 immunostaining also supports the view that these glia are derived from the olfactory placode rather than the neural crest. Further, we determined that 1E8-immunostained glia are found first around the perimeter of the olfactory nerve and are not observed in the center until later in development. Finally, we found that these 1E8-immunoreactive glia appear to enter the olfactory bulb via the olfactory nerve. In the adult olfactory bulb, these cells form a thin rim around the edge of the olfactory nerve layer.
RESULTS Several studies indicate that olfactory nerve axons first exit the chick olfactory epithelium on E3 (Simonetta, 1933; Windle and Austin, 1935). Although 1E8 immunostaining was observed in cranial nerves (trigeminal and occulomotor) a t E3, no 1E8 immunostaining was visible in the olfactory nerve until E4. Early on E4,1E8 immunostaining within the olfactory nerve was very faint, while immunostaining within nearby nerve twigs (presumably of the ophthalmic portion of the trigeminal nerve) was very intense (Figs. lA, 2B). Later on E4, immunostaining was more distinct. At this time, immunostaining in the olfactory nerve near the lateral edge of the telencephalon was often in the shape of a ring surrounding a nonimmunostained center (Fig. 2A). Near the OE, immunostaining of the olfactory nerve remained very faint. In this region, 1E8 immunoreactivity was never seen in cells that were not associated with nerve fascicles. On E5, 1E8 immunostaining of cells within the olfactory nerve was more evident than a t E4, but the immunostaining in the olfactory nerve was still less prominent than in the trigeminal nerve. Immunostaining of cells in the olfactory nerve was observed both on fascicles of olfactory receptor neuron axons a s they left the olfactory epithelium and on more distal parts of the olfactory nerve in which many fascicles had become bundled together (Figs. lB, 2 0 . At this stage of development, the olfactory nerve is close to, but has not yet entered, the lateral edge of the telencephalon. The ring of immunostaining encircling the olfactory nerve, first observed on late E4, was still prominent, but some immunostaining was also apparent in the center of the nerve (Fig. 1B). Beginning at E6, 1E8 staining in the olfactory nerve was as intense a s in the nearby cranial nerves. At this age, the 1E8-immunostained olfactory nerve begins to surround both the lateral and medial edges of the rostral pole of the telencephalon (Figs. lC, 2D). In E7 animals, the spread of 1E8-immunostained cells both medially and laterally along the rostral telencephalon continued (Fig. 2E). 1E8 immunostaining was also observed in a fiber bundle in close proximity to the olfactory nerve in a n E7 animal (Fig. 1D). This fiber bundle is probably a branch of the ophthalmic trigeminal nerve.
On E8, a n evagination formed in the rostral telencephalon. 1E8 immunostaining was found along the edge of this immature olfactory bulb (Fig. 2F). 1E8 immunostaining in the olfactory bulb was more intense than that observed in the olfactory nerve. The spread of olfactory nerve glia around the enlarging olfactory bulb continued on E9 (Fig. 2G). On E l 0 (Figs. l E , 2H), the gross appearance of the olfactory bulb closely resembled that seen in hatchlings and in the adult chicken. In hatching chicks, the olfactory nerve layers and glomerular layers have developed sufficiently to be distinguished. As shown in Figure l F , 1E8 immunoreactive cells were clearly seen along the peripheral edge of the olfactory nerve layer, but not in the region of the olfactory nerve layer near the glomerular layer. No 1E8 immunostaining was ever observed in any brain region other than the olfactory nerve layer of the olfactory bulb. The fact that a Schwann cell-specific marker labelled cells in the olfactory nerve implied that they were glia. To corroborate this result, 1E8-immunostained cells were visualized a t high magnification in 1km cross-sections of hatchling olfactory nerve. Numerous immunostained processes surrounded unstained nerve bundles (Fig. 3). This pattern corresponds to the known distribution of olfactory glia in the olfactory nerve, confirming the specificity of the 1E8 antibody for olfactory nerve glia.
DISCUSSION The results reported here indicate that the 1E8 monoclonal antibody can be used as a specific marker for glial cells in the olfactory nerve a t all stages of development. The following discussion focuses on the use of this antibody to describe the development and distribution of olfactory nerve glia a s well as to examine their relationship to other glial types. Two lines of evidence indicated that the 1E8 monoclonal antibody specifically reacted with olfactory nerve glia. First, 1E8 has been previously shown to react with the major myelin protein Po and to specifically immunostain Schwann cells in peripheral nerves (Bhattacharyya et al., 1991). Second, the pattern of immunostaining in 1 km cross-sections of the olfactory nerve was congruent with the distribution of glial processes, which surround unstained axon bundles (Fig. 3). 1E8 appeared to immunostain the majority of olfactory nerve glia, but we cannot rule out the possibility that a small subpopulation of glia within the olfactory nerve is not immunostained by 1E8. In fact, electron microscopic and immunocytochemical experiments suggest that olfactory nerves may contain more than one glial cell type within the olfactory nerve (Barber and Lindsay, 1982; Pixley, 1992). Origin and Development of Olfactory Nerve Glia The available evidence suggests that, unlike enteric glia and Schwann cells in peripheral nerves, which are derived from the neural crest (Harrison, 1924;
DEVELOPMENTOFOLFACTORYNERVEGLIA
Fig. 1. Photomicrographs of 1E8 immunostaining in sections of embryonic and hatchling tissue. 1E8 immunostaining was visualized with DAB (brown reaction product). Some sections were counterstained with cresyl violet (blue staining). A: At E4, 1E8 immunoreactive cells are associated with nerve twigs, which presumably contribute to the ophthalmic branch of the trigeminal, in close proximity to the olfactory epithelium (oe). lmmunostaining of olfactory nerve glia is too faint to detect in this photomicrograph. Scale bar = 100 pm. B: By E5, 1E8 immunoreactive cells are associated with olfactory nerve (on) fascicles. Scale bar = 50 pm. C: On E6, the 1E8 immunoreactive cells in the olfactory nerve have begun to spread along the rostral telencephalon. Arrows indicate immunopositive cells along medial portion of the rostral telencephalon (tel). Magnification same as A. D: On E7, 1E8 immunolabeling in olfactory
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nerve (on) fascicles and another nerve, presumably the ophthalmic branch of the trigeminal (V) can be observed. Magnification same as B. E: By E l 0, 1E8 immunostaining is evident in the olfactory nerve (on) and the olfactory nerve layer of the presumptive olfactory bulb. The brown staining in the lower lefl of figure 1E represents endogenous peroxidase activity found in blood cells. Magnification same as A. F: In a hatchling, 1 E8 immunostaining is clearly visible along edge of the olfactory nerve layer of the olfactory bulb. Dashes indicate border between the olfactory nerve layer (onl) and the glomerular layer (gl). Scale bar = 25 pm. Abbreviations: gl = glomerular layer of the olfactory bulb, oe = olfactory epithelium; on = olfactory nerve; on1 = olfactory nerve layer of the 01factory bulb; tel = telencephalon; V = trigeminal nerve fascicles.
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Fig.2.
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Fig. 3. A 1 pm cross-section through the olfactory nerve of a hatchling demonstrates that 1E8 immunostaining is found in glia associated with the nerve, not axons. Scale bar = 10 p.m.
D’Amico-Martel and Noden, 1983; Le Douarin et al., 1991), olfactory nerve glia are derived from the olfactory placode. Investigators have noted the migration of two types of cells from the developing olfactory epithelium a t the electron microscopic level (Robecchi, 1972; Mendoza et al., 1982; Farbman and Squinto, 1985; Farbman and Menco, 1986; Doucette, 1989; MarinPadila and Amieva B, 1989). Cells of one type correspond to migrating LHRH neurons (Wray et al., 1989; Daikoku-Ishido et al., 1990; Ronnekleiv and Resko, 1990; Schwanzel-Fukuda and Pfaff, 1990; Norgren and Lehman, 1991). However, several experiments suggest that glial cells also arise in the olfactory epithelium and migrate into the olfactory nerve. First, when quail tissue from the olfactory placode region was transplanted into a chick which had its olfactory placode removed, the glial cells of the olfactory nerve were of quail origin, indicating a placodal source for these glia
Fig. 2. Drawings of horizontal sections of chick embryos illustrating 1E8 immunostaining. Rostra1 is down, caudal is up. Black labeling indicates 1E8 immunostaining. Stippling indicates neural tissue, either telencephalon or olfactory epithelium. In A-C, the gray border outlines the head. In D-H, only the rostral telencephalon and immature olfactory bulb are shown. Scale bar = 1 mm.A: Dorsal section of an E4 embryo head. A ring of immunostaining can observed in the olfactory nerve (on). tel, telencephalon. B: A more ventral section of the same chick head shown in A. At this level, the olfactory epithelium (oe) is present. Intense labeling in nerve twigs, presumably of the trigeminal nerve (V), can be observed. This drawing represents a section similar to the photomicrograph seen in A. C: Section through an E5 embryo head. 1 E8 immunopositive fascicles leaving the olfactory epithelium (oe) as well as staining in the olfactory nerve (on) in close proximity to the telencephalon (tel) can be observed. V, trigeminal nerve twigs. : Sections illustrating 1E8 immunostaining in the rostral telencephalon and/or immature olfactory bulb of E6 (D), E7 (E), E8 (F), E9 (G), E l 0 (H) chick embryos.
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(Couly and Le Douarin, 1985). Second, electron microscopic studies suggest that some cells migrating out of the olfactory epithelium ensheath bundles of axons in a manner similar to that observed in adult olfactory nerve glia (Farbman and Menco, 1986; Marin-Padila and Amieva B, 1989). Finally, cells expressing the glial marker GFAP migrate from E l 4 rat olfactory placode explants in tissue culture (Chuah and Au, 1991). Our results are inconsistent with the alternative view that olfactory nerve glia originate in the neural crest because 1E8-immunopositive cells were not observed migrating through extracellular space to the olfactory nerve. 1E8 does immunostain a subpopulation of migratory neural crest cells (presumptive Schwann cell precursors) (Bhattacharyya et al., 19911, suggesting that migrating crest cells would have been visible in our preparations if they had been present. Some cells close to the olfactory nerve were intensely immunostained by 1E8 (Figs. l A , C), but these cells appeared to be on the nasal portion of the ophthalmic branch of the trigeminal nerve which innervates the olfactory epithelium. Thus, although i t remains possible that there might be some mixing of Schwann cells in the trigeminal and olfactory nerves, the intense labeling of trigeminal glia compared to the weak immunostaining observed at early ages in the olfactory nerve supports the view that olfactory nerve glia arise in the olfactory placode. Although the olfactory nerve glia appear to arise in the olfactory placode, we never observed 1E8-immunopositive cells within the placode, indicating that the 1E8 antigen is not expressed until after the glia migrate into the nerve. This result is similar to the finding that the 1E8 antibody does not immunostain premigratory cells in neural crest but does immunostain cells shortly after they migrate away from the crest (Bhattacharyya et al., 1991). Although olfactory nerve axons begin extending from the olfactory placode to the telencephalon on E3, 1E8 immunostaining was not observed until E4. One interpretation of this finding is that olfactory nerve glia follow olfactory nerve axons out of the olfactory placode. This pattern of development, axon outgrowth followed by Schwann cell migration, is observed in other peripheral nerves (Speidel, 1964; Prestige and Wilson, 1980). However, i t is also possible that olfactory nerve glia migrate out before or at the same time as olfactory axons, but do not express the 1E8 antigen until later in development. The first cells immunostained for 1E8 were found around the perimeter of the olfactory nerve and only later were located throughout the nerve. There are two possible explanations for this pattern of immunostaining. It may be that the first glia to migrate into the olfactory nerve occupy the edge region, while later migrating glia from the olfactory epithelium occupy the center region of the nerve. A second possibility is that only the edge of the olfactory nerve receives migrating glia from the olfactory epithelium; the glia in the center of the nerve may derive from actively dividing pro-
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genitors in the edge region of the nerve. A similar pattern of glial development, i.e., glial cells first found along the edge and later in the center, has been observed in other peripheral nerves (Peters and Muir, 1959).
Relationship of Olfactory Nerve Glia to Other Glial Types The findings reported here may help resolve the ongoing controversy over the relationship between olfactory nerve glia and other glial cell types. Olfactory nerve glia have been identified by some as Schwann cells (Gasser, 1956; De Lorenzo, 1957; Cuschieri and Bannister, 1975; Mendoza et al., 1982; Couly and Le Douarin, 1985) and by others a s astrocytes (Kreutzberg and Gross, 1977; Barber and Lindsay, 1982; Doucette, 1984). In addition, enteric glia and olfactory nerve glia share several common attributes; both ensheath bundles of tightly packed axons, fail to form a basal lamina, and express GFAP (Jessen and Mirsky, 1980; Barber and Lindsay, 1982; Doucette, 1990; Gershon and Rothman, 1991).The fact that olfactory glia are labeled by 1E8, which is specific for myelinating and non-myelinating Schwann cells but does not label astrocytes or enteric glia (Bhattacharyya et al., 1991), suggests that olfactory nerve glia more closely resemble non-myelinating Schwann cells than other glial cell types. Glia Within the Olfactory Nerve Layer of the Olfactory Bulb The origin and identity of glial cells within the 01factory nerve layer of the olfactory bulb is of considerable interest, as these cells may facilitate axonal growth. In most peripheral nerves, astrocytes on the central nervous system (CNS) side of the CNSiperiphera1 nervous system (PNS) border zone are thought to prevent regenerating axons from crossing into the CNS. In contrast, regenerating olfactory nerve axons are able to re-innervate neurons in the CNS (Oley et al., 1975; Graziadei and Okano, 1979; Doucette et al., 1983), suggesting that glia in the olfactory bulb may have novel properties. In fact, olfactory bulb glia in culture have been shown to be more effective in stimulating outgrowth of substantia nigra neuron processes than either striatal or mesencephalic glia (Denis-Donini and Estenoz, 1988). Previous ultrastructural studies suggest that some of the glia in the olfactory nerve layer of the olfactory bulb do not originate within the brain, but are instead derived from the periphery. These studies indicate that during development, olfactory nerve glia enter the presumptive olfactory bulb with olfactory axons (Doucette, 1989; Marin-Padila and Amieva B, 1989). Our observation that 1E8-immunopositive cells were seen in the telencephalon only after the olfactory nerve reached the presumptive olfactory bulb provides additional support for this view. In addition, the only cells in the CNS that were labeled with the 1E8 antibody were found in the olfactory nerve layer of the olfactory bulb; a s was
previously mentioned, 1E8 is specific for Schwann cells and does not immunostain astrocytes (Bhattacharyya et al., 1991). Thus, 1E8-immunopositive cells of the olfactory nerve layer of the olfactory bulb exhibit ontogenetic and phenotypic characteristics of peripheral rather than central glia. As Schwann cells have been demonstrated to facilitate axonal regeneration while adult astrocytes retard regeneration (Perkins et al., 1980; Reier et al., 1983; Liuzzi and Lasek, 19871, similarities between Schwann cells and 1E8-immunostained cells of the olfactory nerve layer may be important in the unique ability of regenerating olfactory nerve axons to grow into the olfactory bulb. It is important to note that 1E8-immunostained cells were found only along the edge of the olfactory nerve layer and that other glia, deeper in the olfactory nerve layer, were not immunostained. Glia of the olfactory nerve layer of the olfactory bulb (and the nerve itself) exhibit characteristics of both astrocytes and Schwann cells (see Doucette, 1990, for review). It will be interesting to determine if the 1E8-immunopositive glia of the olfactory nerve layer exhibit any of the characteristics of astrocytes or whether astrocytic and Schwann cell characteristics are found in separate populations of cells of the olfactory nerve layer. In conclusion, the observations reported here strengthen the view that olfactory nerve glia arise in the olfactory epithelium, migrate within the olfactory nerve, and enter the olfactory bulb. The fact that these cells react with the 1E8 monoclonal, which recognizes Po, suggests that they are similar to peripheral glia, which may contribute to their ability to support the regeneration of olfactory axons within the CNS. Further studies, in which the 1E8 monoclonal can be used to identify, purify, or perturb olfactory nerve glia, may help to define the role of these cells in regeneration.
EXPERIMENTAL PROCEDURES Freshly fertilized chick eggs (N = 56) obtained from a commercial source (SPAFAS) were incubated a t 39°C for varying amounts of time. Embryos were removed from eggs, staged, and then either immersion-fixed overnight or perfused. Hatchlings (N = 3) and 3 year old hens (N = 3) were also perfused. Cardial perfusion with a 0.75% saline rinse was followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Brains with attached olfactory nerves were removed. After fixation, early embryo heads and brains from older animals were cryoprotected by infiltration with 20% sucrose in phosphate buffer. Cryostat sections (20 p.m) were incubated in primary antibody for 24 h r at 4°C. Purified 1E8 antibody was used a t a final concentration of 3 pg/ml in phosphate buffer with 0.2% Triton X-100. The avidin-biotin-HRP technique (Vectastain) and diaminobenzidine were used to visualize 1E8-immunoreactive cells. Some of these sections were counterstained with the Nissl stain cresyl violet. In addition, the olfactory nerves of hatchlings were embedded in gelatin, cross-sections were cut on a freezing
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microtome, and free-floating sections were immunostained a s above. These sections were subsequently embedded in EM bed-812 and 1 pm sections cut on a n ultramicrotome. Some sections were counter-stained with toludine blue.
ACKNOWLEDGMENTS We are grateful to Jan e Withrow and Xiao Gu for excellent technical assistance. This work was supported by NIH grant NS 30047 (RBN) and NS 27227 (RB) and (NR).
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Windle, W.F. and Austin, M.F. (1935) Neurofibrillar development in the central nervous system of chick embryos up to 5 days’ incubation. J. Comp. Neurol. 63:431-463. Wray, S., Grant, P., and Gainer, H. (1989) Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc. Natl Acad. Sci. USA 8623132-8136.
