Expression of the barrier-associated proteins EAP-300 and claustrin in the developing central nervous system

Developmaztal Brain Research, 70 (1992) 9-24 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00

BRESD 51514

Expression of the barrier-associated proteins EAP-300 and claustrin in the developing central nervous system C r a i g F. M c C a b e a n d G r e g o r y J. Cole Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, SC 29425 (USA) (Accepted 2 June 1992)

Key words: EAP-300; Claustrin; Central nervous system barrier; Neural development; Radial glia; Keratan sulfate proteoglycan

Immunohistochemistry of embryonic chick central nervous system (CNS) and immunocytochemistry of retinal cells were performed to compare and map the expression of two barrier-associated molecules. EAP-300 (embryonic avian polypeptide of 300 kDa) and claustrin (a 320 kDa extracellular matrix keratan sulfate proteoglycan) were both transiently expressed in CNS regions that are considered non-permissive to either neuron migration or axon growth. In the developing spinal cord, EAP-300 and claustrin were both expressed by the marginal zone early in development and by the roof plate later in embryogenesis. In the developing rhombencephalon, immunoreactivity for both molecules was also observed first in the marginal zone, and later expression was restricted mostly to the midline. In the mesencephalon, EAP-300 and claustrin were also localized to the midline, and this expression represented a continuation of the expression observed in the spinal cord roof plate and hindbrain ventral midline. In the developing retina and cerebellum, EAP-300 and claustrin were differentially expressed. In retina, EAP-300 and claustrin were expressed by Miiller cells and the optic fiber layer, respectively. In cerebellum at embryonic day 12 (El2), EAP-300 was expressed by Bergman gila, but claustrin was not expressed until El5. Immunocytochemicai staining of retinal and cerebellar cultures indicated that EAP-300 was expressed by a subset of radial astrocytes, as confirmed by double labeling experiments with a specific marker for radial astrocytes. These data indicate that in the absence of claustrin expression, EAP-300 was expressed specifically by radial astrocytes during developmental periods of neuron migration. Also, the coexpression of EAP.300 and claustrin in CNS regions considered to be non-permissive to neurite extension suggests that these two devOopmentally regulated proteins may be associated with barrier function in the developing CNS.

INTRODUCTION During the development of the central nervous system (CNS), neurons migrate in a vectorial pattern, stop and extend their axons directionally to innervate specific target cells. The migration of embryonic neurons and axons follows a consistent developmental map, indicating their movements to be directed and non-random. Specialized, radially elongated glial cells have been reported to guide both the migration of neuronal cells to their terminal areas 2'26'35'47 and to direct the path taken by growing axons ~2'3LS°'Sm'59.In addition, the extracellular matrix (ECM) surrounding radial astrocytes contains sulfated proteoglycans 36'56 which have been shown to be inhibitory to axon growth7'9'39'54. The association of neurons with radial-like gila during CNS development was first observed by Ramon y CajaP 9, and the migration described in detail by

Rakic 45'4~. The cell to cell contacts made between migrating neurons and radial astrocytes involve specialized 'interstitial migration junctions '2°, specific cell adhesion molecules 11.15(CAMs) and ECM molecules 27''~7. However, neurons are not selective for the specific radial astrocyte upon which to migrate, because neurons from a specific brain locus will migrate equally well on radial glia from the same and different brain regions 24. Furthermore, radial astrocytes do not always pattern themselves after the spokes of a wheel as in the cerebellum, but may present curvilinear, palisade or funnel patterns as seen in the hippocampus 4s, brainstem 3s or optic fiber layer ~3 entering the optic stalk. The morphology of different rad',al glia, which in turn describes the neuroarchitecture of different brain areas, may also be determined by the region of neuroepithelium from which they were derived, instead of the type of neuron that migrates along them 25. During cell

Correspondence: G.J. Cole, Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA. Fax: (1) (803) 792-0664.

10 migration, the neuronal soma is preceded by a leading process, which in some ways resembles a growth cone extending and contracting its filopodia along the radial astrocyte's rider, in an apparent effort to process a sum of positive and inhibitory cell adhesion molecules 2°. After the waves of neuronal migration along radial gila have subsided, radial astrocytes show both structural 38 and biochemical 3'4't° changes. It has also been demonstrated that the radial astrocytes lining the ependymal layer and external limiting membrane disappear before birth 34, although midline radial astrocytes survive into adulthood 3s. Radial astrocytes are also associated with CNS brain regions that are inhibitory to axon growth9'36'52'54'56. The factors responsible for the axon deflection characteristics of these midline barrier regions appear to be sulfated proteoglycans. Radial astrocytes can secrete proteoglycans into the ECM that will prevent the extension of growing neurites '~'54'56. CNS barrier regions containing both radial astrocytes and sulfated proteoglycans include the subplate of the telencephalon which delays the crossing of cortical, striatal and thalamic axons into the developing forebrain 16'36'4°, the glial knot which prevents the mixing of optic and olfactory axons "at''~2 and the dorsal and ventral midlines of the spinal cord and brainstem, respectively, which separate left- and right-side fiber tracts '~''~t'.Interestingly, at later developmental times, when the expression of these sulfated proteoglycans has been down-regulated, grow. ing axons will penetrate through these barriers. Thus, it appears that the ability of an axon to grow across a barrier structure depends on the Droportion of growth-promoting CAMs to inhibitory adhesion molecules, The growth of axons in vitro can be modulated by varying the ~tmounts and types of CAMs and sulfated proteoglycans provided as a substrate 9'54'55. Radial astrocytes located in barrier regions do possess adhesive molecules, such as SSEA-1, L1, HNK-1 and NCAM on their cell surface 5t', suggesting that the levels of positive adhesion molecules and sulfated proteoglycans may determine the permissiveness of a developing brain region to axon growth in vivo. The purpose of the present study was to map the distribution of EAP-300 and claustrin, two barrier-associated molecules, in the developing chick C N S 9'36. EAP-300 (embryonic avian polypeptide of 300 kDa) is a glycoprotein that is transiently expressed during chick embryogenesis. Claustrin is a 320 kDa keratan sulfate proteoglycan (KSPG) which is highly enriched in nervous tissue, copurifies with EAP-300 under non-dissociating conditions and inhibits axon extension in vitro. Since both molecules are present in the glial knot 3t', it was of interest to compare their expression during

development in other barrier regions. In addition~ we previously reported EAP-300 expression by Bergmann fibers in the cerebellum and on purified cerebellar astrocytes in vitro 36. To determine if EAP-300 and claustrin may be involved in neuronal migration because of their synthesis by radial gila, we analyzed their expression during periods of cell migration in the cerebellum and retina. To determine if EAP-300 expression was characteristic of radial astrocytes outside the cerebellum, mixed cultures from embryonic retina were double-labeled with monoclonal antibodies (MAbs) against EAP-300 and R5, a marker for radial astrocytes 59. Here, we report the expression of EAP-300 on radial astrocytes during periods of neuronal migration, and the coexpression of both EAP-300 and claustrin in CNS barriers during periods of axon pathway formation. These data suggest that the transient expression of EAP-300 and claustrin may play a role in the formation of the cytoarchitecture of the embryonic CNS. MATERIALS AND METHODS

Animals and antibodies Fertilized white Leghorn chicken eggs (Pee Dee Hatchery, Hartsville, SC) were incubated in a Humidaire rotary incubator at 38°C for the times indicated. The anti-EAP-300 rat monoclonal antibody, A 2B! I, was prepared by immunizing Sprague-Dawley rats with intact cells taken from embryonic day 7 (ET) chick retinas and screened with a fluorescence-activated cell sorter s. Hybridomas secreting A2BI~ MAb were cultured in serum-free DMEM-RPMi 1640 supplemented with Nutridoma-SP (Boehringer Mannheim, Indianapolis, IN), and MAb was purified by precipitation in 50% w/v ammonium sulfate -''~. The anti-claustrin mouse MAb, AHI0, was prepared by immunizing Balb/c mice with an immunopurified preparation of EAP-300 protein containing a KSPG °. Mice were injected with approximately 2/zg of protein, and boosted twice at 15 day intervals. Three to four days after the second boost, the spleens were removed and fused with Sp 2/0 myeloma cells. Hybridomas secreting antibody to the immunogen were identified using an ELISA. Positive hybridomas were subcloned twice and then expanded in serum-free medium supplemented with Nutridoma-SP to obtain purified MAb. Immunohistoehemical loca!ization of F.4F.300 and claustrin Whole embryos (Stage 28) staged according to Hamburger and Hamilton a~, or dissected tissues from timed ,,cubated eggs (Stage 29 or older), were fixed in 4% paraformaldehyde for 4 h and dehydrated through a series of alcohols to xylene as described previously ~. After embedding in paraffin, I0 /~m sections were mounted onto Vectabond-coated slides (Vector Laboratories, Burlingame, CA). Hydrated sections were incubated in 3% H20 2 for 5 min, and blocked for 20 rain with a 1:500 dilution of normal sheep serum (Biodesign International, Kennebunkport, ME). All antibody incubations included 2% BSA, and were for I h at room temperature, followed by a 5 rain wash in PBS. Incubation with A2B n (4 p.~,,'ml) or AHI0 (I /zg/ml) MAb was followed by an incubation Jvith biotinylated sheep anti-rat IgM or anti-mouse IgG antiserum, respectively (I :500, Biodesign). After washing in PBS, streptavidin-HRP (I:I000, Calbiochem, La Jolla, CA) was added for I h. Antibody binding to EAP-300 or claustrin was then visualized by adding 1.3 mM 3,Y-diaminobenzidine (DAB) containing 3.3 mM H 2 0 2 for 3 min. In experiments to control for non-specific staining, the primary antibody incubation was omitted on sister sections processed under otherwise identical conditions. The substitution of normal rat or

modified Eagle's media (DME) supolemented with 20% fetal calf serum on 30 mm Lissue culture dishes in a 37°C incubator. All antibody incubations included 2% BSA, were for 1 h at room temperature, and were followed by 3 rinses for 3 min in PBS. In the double label experiments with A2Bin and R5 MAbs, cells were briefly rinsed in PBS and then fixed in 4% paraformaldehyde for l0 min at room temperature. After rinsing in PBS, the cells were incubated with A2Bnn monoclonal antibody (4 p.g/ml) in PBS followed by an incubation with rhodamine isothiocyanate-conjugated goat anti-rat IgM antiserum (American Qualex, No A104 RN) diluted 1:500 in PBS. Next, the cells were permeabilized with PBS containing 0.2% Triton X-100 for 10 rain at room temperature

mouse serum for A2Bxx or AH10 MAb, respectively, also resulted in no non-specific staining. All experiments were repeated at least 3 times, and an effort was made to compare EAP-300 and claustrin expression patterns on sections that were _< 100 /zm apart in the embryo. Sections were viewed on a Zeiss Universal microscope equipped with differential interference contrast optics and photographed on Kodak Technical Pan 2415 film.

EAP-300 expression in retinal gila Retina cells from E6 chicken embryos were cultured according to published methods 9. Briefly, the cells were grown in Dulbecco's

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Fig. 1. EAP-300 and claustrin are both expressed in the funiculi or marginal layer of the early developing spinal cord. Cross sections of E5 (A,B)

and E8 (C-F) chick cervical spinal cord were stained with either AzBnt (A,C,E) or AHI0 (B,D,F) MAbs. A: EAP-300 was expressed in the funiculi (F) and skeletal muscl,. (M), but roof plate (R) expression was not consistently detected (DRG, dorsal root ganglia; N, notocord; V, vertebrae; *, floor plate; final magnification = 150x). B: claustrin was expressed by the funiculi, as well as spinal nerves (S) and roots (R) (150 x ). C: at E8, EAP-300 was expressed in a radial pattern in funiculi, but was not detected in roof (R) or floor (*) plates (240 x ). D: claustrin was expressed in a diffuse, punctate pattern in funiculi at the same stage of development (240×). E,F: at higher magnification (1,500×), EAP-300 was expressed along fibers (arrowheads) of apparent radial astrocytes (arrow), and claustrin appeared to be located in the ECM, displaying a diffuse expression pattern.

12 followed by 3 rinses in PBS. To identify the cu!tured radial astrocytes, R5 culture supernatant (1:10) was added, followed by an incubation with a fluorescein isothiocyanate-conjugated goat antimouse IgG antiserum (American Qualex, No AI02 FN) diluted 1:500 in PBS. After rinsing, the cells were coverslipped with 50% glycerol in PBS. As control experiments for antibody crossreactivity and spectral spillover between the rhodamine and fluorescein filters, primary antibodies (A-,BI~ and R5)were omitted in separate experiments using identical conditions. No crossreactivity or spectral spillover was observed. The coverslipped cells were photographed with Kodak Tmax 3200 ASA film at 40× magnification using a Nikon Diaphot microscope equipped for epifluorescence.

Westent analysis of EAP-300 and clausobz proteins E9 chick brain and retinal tissues, and El5 cerebella were homogenized in PBS containing 0.5% NP-40, 1 mM EDTA, and a protease inhibitor cocktail containing aprotinin (1,000 Kallekrien inhibitor units/ml), PMSF (l raM), leupeptin (0.5/xg/ml) and pepstatin (I tzg/ml). Protein concentration was measured by the Coomassie Vrilliant blue "~ protein micro-assay (Bio-Rad, Richmond, CA) using bovine lgG as a standard. Total protein (40/zg/lane) was separated through 5% polyacrylamide Gels and electroblotted onto nitrocellulose filters ~'5~. To reduce background staining, the filter was incubated with 5% non-fat dry milk in Tris-buffered saline for 45 rain, followed by incubation first with either A2BIt MAb (4 ~ g / n d ) or AHI0 MAb (I ~ g / m l ) for 2 h, and second with biotinylated goat anti-rat or -mouse lgG Vecatastain ABC reagents (avidin and biotinylated horseradish peroxidase, Vector Laboratories), respectively, and DAB.

RESULTS In previous studies we have demonstrated that EAP-300 has a restricted distribution in embryonic chick. Tissues that have been shown to express EAP-300 include brain, spinal cord, spinal nerve and somite at'. The expression of EAP-300 was also shown to be down-regulated during embryogenesis. When EAP-300 was immunopurified from embryonic chick brain under non-dissociating conditions, a KSPG reproducibly eoprecipitated "at', suggesting that this KSPG and EAP300 may be functionally related. Although this KSPG, named claustrin, has been purified and shown to be inhibitory to growing axons ~, the function of EAP-300 remains to be determined. Monoclonal antibodies were generated against this KSPG and used for immunohistochemistry to compare the expression patterns of claustria and EAP-300 in the developing chick CNS. We have reported EAP-300 expression in putative barriers to neurite growth, such as the glial knot of the diencephalon and the midline regioI~s of the midbrain and spinal cord 36. Since KSPG expression has also been reported in midline regions of the midbrain and spinal cord -s~',we analyzed the cellular distributions of EAP-300 and claustrin in these barriers during embryogenesis.

EAP-300 and claustrin expression in the deceloping spinal cord EAP-300 and claustrin were both expressed in the developing chick spinal cord between the ages of E5

and El5, during the period of axon growth into and out of the c o r d 29 (Fig. 1). At E5, both EAP-300 and claustrin were expressed in the funiculi, or marginal layer, of the cord, as well as the spinal roots and nerves 36. Claustrin expression was restricted to the developing nervous system, while EAP-300 was also detected in primitive skeletal muscle (Fig. 1A, B). The restricted distribution of claustrin was not due to the use of AH10 MAb, since a polyclonal antiserum to a claustrin core protein gave identical staining patterns (data not shown). Detectable levels of invariable staining for either protein were not observed in dorsal root ganglion cells, the dorsal roof and ventral floor plates of the cord, and developing bone. At E8, the marginal layer of the cord continued to stain with both MAbs (Fig. 1C,D), but a decrease in AH10 signal could be seen between E5 and E8 (Fig. 1B,D). Interestingly at this age, neither the roof nor floor plates expressed EAP-300 or claustrin. Although the marginal layer expressed both molecules, their cellular patterns of expression were quite different (Fig. 1C-F). The radial-like pattern of EAP-300 expression contrasts with the diffuse pattern of claustrin expression. EAP-300 was present on radial fibers apparently anchored to the piai membrane of the cord, suggesting expression by radial astrocytes (Fig. 1E). In contrast, claustrin's diffuse expression pattern resembles that seen with many ECM molecules 14'27, including other KSPGs 41'56 (Fig. 1F). Thus, in the early developing spinal cord, EAP-300 and claustrin were both expressed in the marginal layer of the cord, but were absent in the glial roof and floor plates. In the late developing spinal cord, both EAP-300 and claustrin were expressed by the roof plate, again with distinct expression patterns (Fig. 2). Roof plate expression of these proteins has been observed by E9 of cervical spinal cord development, when the roof plate exhibits a wedge-shaped morphology (data not shown). By El3, EAP-300 expression was restricted to the septum-like roof plate, with a down-regulation of expression to undetectable levels in other spinal cord regions (Fig. 2A,B). In contrast, the funiculi retained their expression of claustrin, although claustrin was also expressed in the roof plate. Neither the ventral floor plate nor the surrounding glial limiting membrane of the spinal cord expressed EAP-300 or claustrin. At El5, there was no change in the expression patterns of either molecule (Fig. 2C,D), but at E20, claustrin expression was strongly down-regulated in both funiculi and dorsal midline (data not shown). When El5 spinal cord was analyzed at higher magnification, the fibrous and diffuse expression patterns of EAP-300 and claustrin, respectively, were again evi-

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Fig. 2. EAP-300 and claustrin are both expressed in the roof plate of the late developing spinal cord (A,B). At El3, EAP-300 was expressed only in the roof plate (R), while claustrin was also present in the funiculi (F) (100x). C,D: at E15, EAP-300 and claustrin's expression patterns remained unchanged (F, funiculi; M, muscle; S, spinal nerve; V, vertebrae; 50x 1. E,F: at higher magnification, the fibrous and diffuse patterns of EAP-300 and claustrin expression, respectively, were detected (1,500 x, 950 ×). G,H: in the caudal medulla, the transition between spinal cord roof plate (arrowheads) and ventral midline staining (arrowheads) was detected (*, central canal; 100 × ).

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Fig. 4, EAP-300 and claustrin are expressed in the midline of the mesenc~phalon. A,B: at E12 of development, EAP-300 and claustrin were expressed in the midline (M) of the mesencephalon; however, claustrin was also present in two areas symmetrical to the midline (150 x ). C,D: at higher magnification, EAP-300 and claustrin were detected on radial astrocyte fibers (arrowheads) and in the ECM of the midline, respectively (950 x ).

dent (Fig. 2E,F). These roof plate expression patterns resemble those seen in the marginal layer of the early spinal cord (Fig. 1E,F), lending credence to the suggestion that EAP-300 is expressed by radial astrocytes and that claustrin is secreted by astrocytes 9 into the ECM. Thus, the expression of EAP-300 and claustrin in the spinal cord appeared to be associated with astrocytes and their ECM, respectively, in the marginal layer of the early developing cord and in the roof plate of the late developing cord. At the transition from spinal cord to brainstem a change in staining patterns was observed (Fig. 2G,H). Moderate expression of both molecules was detected dorsally and ventrally on each side of the midline; however, no detectable levels were present in the area surrounding the central canal. In addition, funiculus staining with claustrin MAb was absent at this level of the neuraxis.

EAP.300 and claustrin in the rhombencephalon Similar expression patterns for EAP-300 .~nd claustrin, as seen during spinal cord development, were detected during hindbrain development (Fig. 3). For example, at ES, both MAbs stained cranial nerves and the marginal zone of the hindbrain, with the exception of the floor plate (Fig. 3A,B). Two notable exceptions were A2BI~ MAb staining the early roof plate and AH10 MAb staining small groups of cells within the dorsal mantle layer, but not the roof plate or dorsal midline. At E9, the ventral midline of the rostral medulla expressed both molecules (Fig. 3C,D). The A2B~I MAb also stained the mantle layer with a multifocal pattern. In contrast, the AH10 MAb bilaterally stained an area in the dorsal mantle layer, which might have been derived from the dorsal areas stained in Fig. 3B. With high magnification, the fibers of midline

Fig. 3. EAP-300 and claustrin are present in or near the ventral midline of the developing hindbrain. A,B: at E5 of development, EAP-300 and claustrin were expressed in the marginal layer of the developing hindbrain, except at the ventral midline (*) (CN, cranial nerve; R, roof plate: 4V, fourth ventricle; 125 ×). EAP-300 and claustrin were also present radially throughout the tissue and in a dorsal bilateral group, respectively• C,D: at E9 at the level of the rostral medulla, EAP-300 and claustrin were detected in the ventral midline (arrowhead) (4V, fourth ventricle: 60 x ). E,F: in the ventral midline, EAP-300 was localized to radial astrocyte fibers (arrowheads), and not their cell bodies (arrow), while claustrin was localized diffusely in the ECM (950x). G,H: at E12 in the rostral medulla, EAP-300 was present solely in the ventral midline, whereas claustrin was also expressed in a circular grouping on each side of the midline (150 x ).

16 radial astrocytes that expressed EAP-300 were seen (Fig. 3E). Claustrin expression in the ECM of the ventral midline of the rhombencephalon (Fig. 3F) was similar to that detected in the spinal cord, being diffuse and resembling an extracellular distribution. At El2, EAP-300 expression was restricted solely to the ventral midline (Fig. 3G). Claustrin expression was also localized to the ventral midline, but was also expressed by a bilateral grouping near the ventral midline (Fig. 3F). Thus, during rhombencephalon development, EAP-300 and claustrin were first expressed in the marginal zone, but with development became restricted to the ventral midline. The localization of claustrin to the ventral midline of the hindbrain agrees with previous studies that examined KSPG expression in developing rat and hamster (J. Silver, personal communication).

EAP-300 and claustrin in the mesencephalon The exgression patterns of EAP-300 and claustrin that were observed in the developing hindbrain continued linearly and dorsally into the developing mesencephalon (Fig. 4). After 12 days of development, EAP300 was strongly expressed by midline fibers (Fig. 4A,C). Since this protein was not detected in this region on the astrocytes forming the ventral mkliine or external limiting membrane, EAP-300 appears to be restricted to a unique population of radial astrocytes present only in the midline of the mesencephalon.

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Fig. 5. Claustrin and EAP-300 are differentially expressed in the developing retina and cerebellum. E9 retina (lanes 2 and 4) and El5 cerebellum (lanes 3 and 6) were immunoblotted using AHI0 (lanes 1-3) and A2Bll (lanes 4-6) MAbs. Age-matched brain tissues w e r e used as positive controls (lanes 1 and 4). At E9 of retina development, claustrin levels (faint band, lane 2) were barely detectable by Western blotting, in contrast to EAP-300 levels which were easily detected. In El5 cerebellum, both claustrin (lane 3) and EAP-300 (lane 6) were expressed at high levels (40/zg total protein/lane). The bars at the left indicate molecular weight standards (myosin, 205; /3-galactosidase, 116; and bovine serum albumin, 77 kDa).

Similar to claustrin's expression in the rhombencephalon, KSPG was present in the midline, as well as a nearby bilaterally symmetrical group (Fig. 4B). Like claustrin's expression in spinal cord and hindbrain, claustrin appeared to be localized to the ECM in the midbrain (Fig. 4D). In conclusion, from the mesencephalon through the cervical spinal cord, EAP-300 and claustrin coexpression was associated with midline barrier structures.

Differences in EAP-300 and claustrin expression in retina and cerebellum We have previously reported EAP-300 expression in the E21 cerebellum, where it was expressed along radial astrocyte fibers a6. Since claustrin expression is associated with EAP-300 in many other areas, we analyzed the expression of these molecules in the cerebellum during granule cell migration, and in the retina during neuronal migration. We first confirmed the presence of these molecules in these tissues by Western blot analysis (Fig. 5). Using E9 total brain homogenates as positive controls (Fig. 5, lanes 1 and 4), it was shown that both claustrin and EAP-300 were expressed in El5 cerebellum (lanes 3 and 6, resp.). In E9 retinal tissue, EAP-300 was expressed at similar levels as observed in cerebellum; however, claustrin was present only at very low levels in E9 retina (lanes 5 and 2, resp.). This suggests that claustrin and EAP-300 expression is not coordinately regulated in all neuronal tissues that express EAP-300. EAP.300 and claustrin in the developing retina In earlier studies from our laboratory, immunohistochemical analysis indicated that EAP-300 was selectively expressed in the ganglion cell and optic fiber layers of the embryonic chick retinas, which led us to suggest that ganglion cells were responsible for EAP300 expression. However, 'the possibility that Miiller cell processes and endfeet were expressing EAP-300, and the lack of suitable markers for this cell type, did not allow us to determine expression by this cell type. Thus, the study of EAP-300 expression in retina, and its comparison to claustrin expression, was aimed at identifying the cell types that express these proteins during retinal development. Since claustrin was weakly detected in retina by Western blotting, immunohistochemistry was performed to determine its presence in the developing retina, and to compare its distribution to EAP-300. After 5 days of development, EAP-300 was expressed by the radial glia of retina, Miiller cells, throughout the non-stratified retina (Fig. 6A). Miiller cell expression of EAP-300 was also confirmed using confocal microscopy (data not shown). This expression

I7 of EAP-300 coincides with the period of ganglion cell migration in chick retina 32'33. Claustrin was faintly present as individual groups of punctate staining in the optic fiber layer (OFL) of the peripheral retina (Fig. 6B), which may represent staining of Miiller cell endfeet. After stratification of the retina at E9, EAP-300 was expressed on radial fibers extending from the OFL to the pigment epithelium (PE), whereas claustrin was restricted to the OFL (Fig. 6C,D). C!austrin's faint

expression in the OFL at this age corresponds well with the very weak levels detected by Western blot analysis. After 12 days of development, EAP-300 was readily detected along radial astrocytes, but with a diminished expression, when compared to E9, that no longer extended to the outer nuclear layer (ONL) (Fig. 6A). Claustrin levels had also decreased from the levels present in E9 retina. Furthermore, claustrin was down-regulated to undetectable levels in the retina by

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Fig. 6. EAP-300 is expressed by radial astrocytes, and claustrin by the optic fiber layer, in the developing retina. A: at E5 of development, EAP-300 was expressed by radial astrocytes (arrow) and their fibers (small arrowhea-.as~ throughout the gan~lio, cell laser (ut:L) of the incompletely differentiated retina (950 x). B: in a sister section, claustrin was expressed at low levels m a punctate pattern in the optic fiber layer (OFL) of peripheral retina (PE, pigment epithelium). C: at E9, EAP-300 was expressed by radial astrocytes (arrow) and their fibers (small arrowheads) from the outer nuclear layer (ONL) to the OFL of the differentiating retina. D: at E9, claustrin was expressed diffusely along the OFL (INL, inner nuclear layer; IPL, inner plexiform layer; 750x). E,F: at El2, EAP-300 continued to be expressed by radial astrocyte fibers (small arrowheads), with their endfeet (large arrowhead) anchored in the OFL, whereas claustrin levels were down-regulated (700 x ).

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Fig. 7. EAP-300 expression is down-regulated in retina. A: by El5 of retina development, EAP-300 levels were dramatically down-regulated from El2 levels (compare with Fig. 6E). B: at El7, EAP-300 was barely detectable on Miiller cells (PE, pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; and OFL, optic fiber layer; 600x ).

EAP-300 expression by retinal astrocytes Since EAP-300 was restricted to radial-like fibers in the developing retina, it was of interest to confirm if this pattern was owed to EAP-300 expression by radial astrocytes. To test this possibility, mixed neuronal and glial cultures from E6 chick retinas were double-labeled with A~Bt~ and R5 MAbs (Fig. 9). R5 MAb is a specific filament marker for radial astrocytes in the chick 5°. EAP-300 was expressed only by a subpopulation of cultured retinal radial astrocytes in a punctate pattern along their processes. InterestinglY, A2BI~ MAb staining did not require the cell permeabilization step that was needed for R5 MAb staining, suggesting that EAP-300 is expressed on the cell surface of retinal astrocytes. These results agree with our previously reported expression of EAP-300 by cerebellar radial astrocytes ~6. Thus, EAP-300 is expressed by radial astrocytes in different CNS regions, accounting for the radial astrocyte fiber-like staining detected in midline

El5 (not shown)• EAP-300 expression was also downregulated between El2 and El5 (compare Fig. 6E with Fig. 7A). At El5, EAP-300 could be detected only faintly in the ganglion cell layer (GCL). By El7 of retina development, EAP-300 was barely detectable, with only a few MLiller fibers exhibiting low levels of staining (Fig. 7B). Thus, our immunohistochemistry analysis supports our previously reported Western blot data showing that both EAP-300 (data not shown) and claustrin '~ are down-regulated in the chick retina by El7. Hence, EAP-300 expression in the developing retina was restricted to developing Mi~ller cells, whereas claustrin was expressed at lower levels and only in the OFL. Based on previous analyses '~, the expression of claustrin in the optic fiber layer probably results from astrocyte secretion of the molecule into the ECM in this region of the retina, although it remains possible that the OFL expression of claustrin is associated with ganglion cell axons.

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Fig. 8. EAP-300 and claustrin are expressed in the optic stalk. A: at E9 of development, EAP-300 was expressed throughout the retina (R) on Miiller cells, and continued into the beginning of the optic nerve, the optic stalk (OS). EAP-300 was present in skeletal muscle (M), but not the hyaline cartilage (C) of developing bone (150x). B: claustrin expression was present in the OFL of E9 retina, and the intensity of staining increased as the OFL approached the optic stalk. Nerves (N) also expressed claustrin, but hyaline cartilage and muscle did not.

19 structures of the mesencephalon, brainstem and spinal cord. EAP-300 and claustrin are expressed in the optic stalk To complete the study of EAP-300 and claustrin in the developing retina, we analyzed their expression as the OFL entered the optic nerve (Fig. 8). At low magnification, EAP-300 expression was detected across the E9 retina, continuing into the optic stalk (OS) of the optic nerve (Fig. 8A). In addition, EAP-300 was ..

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expressed by surrounding non-neuronal tissues, inclutaing muscle. Claustrin was detected weakly in the optic fiber layer near the eye's posterior pole (see Fig. 6D) where the OFL approaches the OS, but was absent in the OFL at the anterior pole of the eye (data not shown). However, near the optic fissure where ganglion cell fibers gather to travel withii, the optic nerve, claustrin levels increased, with higher amounts present throughout the OS. Within the OS, EAP-300 and claustrin were expressed in a rectalinear pattern, which raises the possibility that they may play a role in the guidance of optic nerve axons. These data may also indicate that claustrin is associated with retinal ganglion cell axons, although electron microscopic and in situ hybridization analyses will be required to confirm the cell type responsible for synthesis of this proteoglycan. EAP-300 and claustrin expression in developing cerebellum We previously reported EAP-300 expression in E21 cerebellum and by cerebellar radial astrocytes in culture 36. Since our Western blotting analysis indicated that claustrin is also present in embryonic cerebellum, it was of interest to compare the expression patterns of EAP-300 and claustrin at earlier cerebellar developmental stages during periods of cell migration (Fig. 10). At El2 of development, prior to the major migration of granule cell neurons from the external granule cell layer (EGL) into the internal granule cell layer (IGL) '2, the cerebellum expressed EAP-300, but not claustrin (Fig. 10A,B). Radial astrocytes expressed EAP-300 on their fibers which extend from the IGL into the EGL in a spoke-like pattern. Radial astrocyte fibers arc used by migrating neurons as guides during migration to the IGL ~s. At El5, when the granule ~eurons have begun their migration22, EAP-300 continued to be expressed by radial astrocytes, while claustrin levels were dramatically increased (Fig. 10C,D). Induction of claustrin expression occurred in the EGL, ML and fiber tracts (FT); however, the IGL expressed notably lower levels of claustrin. Thus, these data indicate that EAP-300 and claustrin appear to be differentially expressed at different developmental times during cerebellum development and granule cell migration.

DISCUSSION Fig. 9. Cultured retinal radial astroeytes express EAP-300. Dissociated E6 retina cells were grown for 5-7 days as described. A: phase contrast image of two glia displaying the typical long bipolar morpholo~ of immature radial astrocytes (600×). B: EAP-300 was expressed in a punctate pattern (arrows) along these processes. C: the glia were confirmed to be radial astrocytes by their expression of the radial astroeyte filament protein, R5.

Immunohistochemistry, immunocytochemistry and Western blotting were used to compare the expression patterns of EAP-300 and claustrin in the developing chick CNS. The histological distribution of EAP-300 showed the protein to be restricted to radial astrocyte-

2O like cells throughout the CNS. Claustrin, a KSPO of the ECM, was detected in many CNS regions that also expressed EAP-300. Immunocytochemistry confirmed the expression of EAP-300 to radial astrocytes, especially their fibers. Western blotting detected EAP-300 in both retina and cerebellum, although claustrin was present in higher levels in the cerebellum than in the retina. The expression patterns of EAP-300 and claustrin frequently were related to putative barriers to migrating neurons and growing axons. In a recent study 36, we showed that both EAP-300 and claustrin were localized to the glial knot, a putative bar,'ier that segregates the olfactory and optic projections 52. In this study, we have extended our analysis of EAP-300 and claustrin to additional putative barriers, as well as other CNS regions. In the early developing spinal cord, MAbs to both proteins intensely stained the marginal layer of the spinal cord. EAP-300 has been shown to be present in the marginal layer of the cord as early as E23~. During this early developmental period in the spinal cord, neurons are migrating from the germinal epithe-

lium into the mantle layer, b u t d o not enter the marginal layer of the cord nT'a°. In addition, pioneer axons of commissural neurons begin to grow ventrally to cross the floor plate 2s'61, and do not enter the marginal layer until they have crossed the midline. It is therefore possible that the presence of claustrin plays a role in regulating the extent of neuron migration, as well as the guidance of commissural axons, by acting as a barrier. Previous studies in our laboratory have shown that claustrin acts as an inhibitory substrate for the extension of axons in vitro 9. Thus, the expression of claustrin and/or EAP-300 may also create an inhibitory environment for the migration of neurons in vivo. In the late developing spinal cord, both EAP-300 and claustrin were expressed by the roof plate. The roof plate, or dorsal septum of the spinal cord, is composed of astrocytes and their ECM 37'54'56. This midline area may function as a physical a n d / o r chemical barrier to growing somatoscnsory axons attempting to cross the roof plate 37'54. Ventral commissural axons also avoid the roof plate and cross the ventral floor plate to reach their contralateral targets 42'56'6°. Both

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Fig. 10. EAP-300 and claustrin are differentially expressed in the developing cerebellum. A: at El2 of development, EAP-300 was expressed by radial astrocytes (arrow) and their fibers (arrowheads). The radial astrocytes present in the internal granule cell layer (IGL) send their fibers up through the Purkinje cell layer (P), molecular layer (ML) and into the external granule cell layer (EC;L) (600x). B: claustrin was not detected at El2 of cerebellar development. C: at El5 of development, EAP-300 continued to be expressed by radial astrocytes (arrow), their fibers (arrowheads) and fiber tracts (FT) (375 x ). D: claustrin expression was strongly induced by El5 in the EGL, ML and FT; however, only low levels of claustrin could be detected in the P and IGL.

21 KSPG 9'54 and chondroitin sulfate proteoglycan 54 (CSPG) are known to be present in the roof plate, and thus, claustrin may be the physiologically relevant KSPG of CNS barriers. Similar to the expression observed in the developing spinal cord, both EAP-300 and claustrin were expressed in the marginal layer and midline region of the developing brainstem. Early in rhombencephalon development, both EAP-300 and claustrin were found in the marginal layer, with a striking absence in the ventral midline. A few days later at E9, these molecules were no longer expressed in the marginal layer, but were expressed in the ventral midline. Here again, EAP-300 and claustrin appeared to be present on radial astrocyte fibers and in the ECM, respectively. The ventral midline expression pattern observed in the developing rhombencephalon appears to be a continuation of the dorsal midline expression pattern detected in the developing spinal cord. A transition from spinal cord roof plate to rostral medulla ventral midline expression of these molecules was detected in the caudal medulla, where their expression pattern split onto the sides of the dorsal and ventral midline. The caudal medulla is a region of nerve tract decussation, where a meshwork of radial astrocytes bulges out from the midline to allow the crossing of pyramidal tract axons aa. Thus, CNS barriers may serve a guidance role by inhibiting axon growth in inappropriate directions and by channeling nerve fiber tracts. It is difficult to delineate a dorsal septum in the rostral medulla, because the roof plate is transformed into a thin sheet of cells. This change in morphology results from the great expansion of the fourth ventricle from the central canal. Thus, the ventral floor plate is given the role of separating left and right sides of the hindbrain, and can be considered to assume the function of the dorsal midline at this level of the neuraxis. Therefore, the ventral midline of the developing hindbrain separates almost all left and right side nerve tracts traveling through this part of the CNS. In a previous study, we demonstrated that EAP-300 was expressed by the midline of the developing mesencephalon a6. The midline of the mescncephalon contains radial astrocytes 3s which have been reported to act as a barrier to growing axons, in order to maintain the ipsilateral separation of afferent fibers in the optic tectum 43. Interestingly, the transient expression of EAP-300 in the mesencephalon midline coincides with the formation of retinotectal connections 44. The midline expression patterns of EAP-300 and claustrin have also been shown to continue into the diencephalon, where they again appear to be present on radial astrocyte fibers and in the ECM, respectively, of

the glial knot 36. In the present study, we have shown that both EAP-300 and claustrin were expressed in the midline, and not in the peripheral gliai limiting membrane, of the midbrain. Thus, EAP-300 and claustrin are unlike other markers of the midline which are also expressed in the limiting membrane or enriched in non-nervous tissues 19,3a,56. These data, therefore, suggest that EAP-300 and claustrin may be specific markers for the radial gila and ECM of CNS barrier structures during development of the nervous system, since all known putative barriers in the CNS express these two proteins. In the studies described here, we also extended our analyses to other regions of the CNS that have not yet been shown to contain barriers, but express EAP-300 and claustrin. In the developing retina, EAP-300 and claustrin were not expressed in the same cellular regions. Throughout the stratification of the developing retina, only radial astrocyte fibers and the optic fiber layer were positive for EAP-300 and claustrin, respectively. Both EAP-300 and claustrin were transiently expressed from E5 to E17. Since retinal ganglion cells migrate from the neuroepithelium to the ganglion cell layer during this time period 32'33, one can speculate that EAP-300 may be involved in cell migration, and that claustrin may act as a barrier to further cell migration and/or to channel groups of retinal ganglion cell axons into the optic nerve. Interestingly, both molecules were expressed in the stalk of the optic nerve, with claustrin exhibiting intense staining in the optic stalk. In addition, the OFL staining with antibodies to claustrin spread peripherally from the optic stalk as development progressed. Hence, a greater proportion of the OFL expressed claustrin at E9, when compared to E5 (data not shown). Since intercellular spaces are present in the marginal layer of the developing retina that continue through the optic stalk, it has been suggested that these spaces channel growing axons into the optic nerve 53. Thus, EAP-300 and claustrin seem to be likely candidates for axon channeling through rectalinear tracts, such as the optic nerve. In addition, recent studies have suggested that CSPG may channel ganglion cell axons to the optic fissure 6. The apparent expression of EAP-300 by radial astrocytes of the developing retina was confirmed by immunocytochemical studies. Retinal cell cultures were double labeled with A2BIi MAb and R5 MAb, a gliai filament marker specific for radial astrocytcs 59. Furthermore, EAP-300 appears to be a cell surface marker of radial astrocytes, since A2BI~ immunostaining of the astrocytes did not require permeabilization, as did R5 staining. Thig confirms our previous report that radial astrocytcs express the EAP-300 protein 36. Thus,

22 the radial fiber-like staining detected in the developing spinal cord and hindbrain is probably also resulting from radial astrocyte expression of EAP-300. In the developing cerebellum, EAP-300 and claustrin expression was not synchronous. During early cerebellum development, EAP-300 was expressed by radial astrocytes, but claustrin expression could not be detected. At this stage of cerebellum development, granule cells are rapidly dividing and preparing to load onto radial astrocyte fibers ~. Three days later at El5, claustrin was dramatically up-regulated in the EGL, ML and FT regions, it is during this developmental stage that granule cell neurons are rapidly migrating toward their permanent residence in the IGL layer ~'22 Claustrin expression was noticeably lacking in this layer. Interestingly, these barrier-associated proteins are also expressed in cerebellum, which raises the intriguing possibility that inhibitory molecules may have diverse functions based on their level and site of expression. For example, gradients of inhibitory CSPG have been shown to regulate the rate of axonal growth "~5.Thus, in regions such as retina and cerebellum, barrier-associated molecules could regulate processes as diverse as neuron migration and axon pathway tbrmation, possibly controlling the rate of these migratory processes. For example, it is of interest to speculate that claustrin expression surrounding the IGL could further prevent the migration of granule cells out of the IGL. Of course, the regulation of adhesion processes by inhibitory barrier molecules will also depend on the expression of cell receptors for barrier molecules, which introduces an additional level of regulation by moleeu. lar barriers, Several relationships of EAP-300 and claustrin expression during CNS development deserve special mention. First, EAP-300 expression in the absence of claustrin expression has only been observed on radial astrocytes during periods of cell migration, as detected in the early developing spinal cord during neuron migration, the retina during ganglion cell migration and the cerebellum during granule cell migration. Furthermore, EAP-300 levels are strongly down-regulated after cell migration has been completed 36. Second, EAP30{) and claustrin coexpression was associated with putative barriers to neuron migration, such as the marginal layer of the spinal cord, the subplate of the telencephalon and the fiber tracts of the cerebellum, or with barriers to axon extension, such as the dorsal midline of the spinal cord, the midline of the mesencephalon or the ventral midline structures of the rostral medulla and gliai knot of the diencephalon 36. Expression of both molecules, as determined by Western blotting, was down-regulated after the neurons or ax-

ons of interest had reached their targets. Thus, it seems reasonable to speculate that the expression of EAP-300 on immature radial astrocytes may serve to regulate early neuron migration, and that mature radial astrocytes expressing EAP-300, as well as secreting claustrin 36, could create a barrier to neuron migration or axon extension. Since immunopurification schemes using AEBzz MAb under non-dissociating conditions will coprecipitate both EAP-300 and claustrin 9, the secreted claustrin may bind to the EAP-300 expressed on the plasma membrane of the radial astrocyte, suggesting a role for both molecules in the regulation of neuron migration and axonal growth. However, experiments to test this interesting hypothesis have been hampered because of difficulties in obtaining purified EAP-300. In summary, we have mapped the expression of two developmentally regulated barrier-associated molecules, EAP-300 and claustrin, in the developing CNS. EAP-300 is a glycoprotein that appears to be expressed on the cell surface of radial astrocytes during periods of neuron migration. Claustrin is an ECM KSPG that colocalizes with EAP-300 in several barrier structures that impede neuron migration and axon growth. The transient expression of these proteins in several putative barriers, and on radial gila during periods of neuron migration, suggests that these molecules may play an integral role in the regulation of neuronal migration and axon pathway formation during development of the CNS. Acknowledgements. The authors thank Deborah Turner for technical

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