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Cell-type-specific expression of protein tyrosine kinase-related receptor RYK in the central nervous system of the rat
Molecular Brain Research 104 (2002) 255–266 www.bres-interactive.com
Interactive report
Cell-type-specific expression of protein tyrosine kinase-related receptor RYK in the central nervous system of the rat Kazuyo Kamitori a , Mitsuru Machide a , Kazuhiko Tomita a,b , Masato Nakafuku c , Shinichi Kohsaka a,b , * a
Department of Neurochemistry, National Institute of Neuroscience, 4 -1 -1 Ogawa-higashi, Kodaira, Tokyo 187 -8502, Japan b Graduate School of Lifescience, Tokyo University of Pharmacy and Lifescience, Hachiouji, Tokyo, Japan c Division of Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Accepted 12 November 2001
Abstract The mammalian RYK is an orphan receptor that contains a catalytically inactive tyrosine-kinase-related domain. Its Drosophila homolog, Lio / Drl, is required for axon pathfinding in developing brain. Our previous study suggested that RYK mRNA is expressed in nestin-positive progenitor cells and neurons. In the present study, immunohistochemistry has been used to further localize RYK in the central nervous system of rats to identify the lineage of the RYK-expressing cells. In the embryonic forebrain, RYK colocalized with nestin in the ventricular zone and with MAP2 in the cortical plate, suggesting that RYK is expressed in neural progenitor cells and neurons. Localization of RYK in embryonic spinal cord also suggested its expression in both cell types. In primary cultures of rat cerebrum, RYK expression was observed in all neurons, as well as in a significant population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. However, no RYK expression was detected in type-1 astrocytes or microglia. Multipotent neural stem cell line MNS-70 was also analyzed for expression of RYK, and most of the cells were positive for both RYK and nestin in the undifferentiated stage. In the differentiated stage, expression of RYK was detected in the neurons, but not in type-1 astrocytes. In conclusion, RYK is expressed in nestin-positive progenitor cells and neurons, and in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes in developing CNS. These findings show that expression of RYK in rat CNS is tightly regulated in a cell-type-specific manner. 2002 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Neuroglia and myelin Keywords: Central nervous system; Neural progenitor cells; Receptor; Differentiation; Immunohistochemistry
1. Introduction During the development of the mammalian central nervous system (CNS), multipotent neural stem cells proliferate to generate mature cells: neurons, astrocytes, and oligodendrocytes. This cell differentiation is regulated by various transcription factors, including Mash-1 [10], neurogenin [16], and olig1 / 2 [15,40], and signaling pathways through membrane receptors are hypothesized to control this event, as exemplified by Notch [8,36], PDGF *Corresponding author. Tel.: 181-42-346-1721; fax: 181-42-3461751. E-mail address: [email protected] (S. Kohsaka).
[22,27], and CNTF [4]. In addition, an increasing number of reports suggest a contributory role of signaling pathways through membrane receptors in the migration and layer formation of postmitotic neurons [1,9,28,30]. Numerous unidentified signaling molecules surely play important roles in the developing CNS by modulating key events, such as cell differentiation or cell migration. The tyrosine-kinase-related protein RYK is an orphan receptor that has been isolated from mammalian species [12,33,34,37,39]. It is structurally distinct from other members of the receptor tyrosine kinase family in two regards. Firstly, its tyrosine-kinase-like domain possesses amino acid substitutions that may lead to impaired ATP binding. Secondly, its extracellular domain is smaller than
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00358-3
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that of other receptor tyrosine kinases, and contains only two leucine-rich motifs (LRMs). Although the tyrosine kinase activity of RYK is thought to have been lost as a result of the amino acid substitutions in the catalytic domain, a chimeric trkA and human RYK receptor have been shown to have the ability to activate the MAP kinase pathway upon stimulation by NGF, a ligand for the trk receptor [14]. Recently, gene targeting analysis has shown that RYK is essential for normal morphogenesis, possibly through association with Eph family receptors, which are involved in the pattern formation of the nervous system [11]. Halford et al. have also demonstrated that RYK binds to AF-6, a Ras effector cell–cell junction protein. Independently, Lio / Drl (also called linotte or derailed), the Drosophila homolog of RYK, has been reported to play key roles in the development and maintenance of the fruitfly brain by controlling axonal pathfinding [2,3,6,18,32]. All these findings imply significant functions of mammalian RYK during development of the CNS through signaling events. In spite of the importance of its Drosophila homolog in the CNS, as described above, the function of RYK in the mammalian CNS has not been fully analyzed, possibly because Northern blot analysis of mammalian tissues has shown relatively low levels of RYK mRNA in the adult brain. However, our previous characterization of the spatial and temporal distribution of RYK mRNA in the rat CNS implied that RYK is expressed in nestin-positive cells (neural progenitor cells) and neurons during development [13]. Here we describe the results of localization of RYK by double-immunostaining for various marker proteins that show its expression in nestin-positive neural progenitor cells and neurons, as well as in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. This information should lead to further understanding of the physiological function of RYK in the mammalian CNS.
2. Materials and methods
2.1. Animals Wistar rats were obtained from CLEA JAPAN. The dams were ether-anesthetized while the embryos were removed from the uterus.
centration was determined using a BCA protein assay kit (PIERCE). Twenty micrograms of the protein were separated by SDS–PAGE, and transferred to PROTRAN nitrocellulose transfer membranes (Schleicher & Schuell) followed by immunoblotting with the affinity-purified antiRYK rabbit antiserum [13] at a concentration of 2 mg / ml. Then the membrane was incubated in horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham Pharmacia) diluted to 1:2000, and the reaction was visualized with an ECL chemiluminescence detection system (Amersham Pharmacia).
2.3. Primary cultured cells The cerebral cortex was isolated from embryonic day 21 (E21) Wistar rats. After removing the meninges, pieces of the tissue were minced and incubated in 0.25% trypsin solution for 10 min to digest the extracellular matrix. Fetal calf serum was added to the cell suspension to inactivate the trypsin, and the suspension was subsequently incubated in 500 mg / ml DNase I solution. The cells were then collected by centrifugation and resuspended in D-MEM medium containing 10% fetal calf serum. After subsequent filtration through a Cell Strainer (70 mm pore size, Falcon), the cells were seeded at a density of 1310 5 / cm 2 onto culture dishes coated with the 50 mg / ml poly-L-lysine, and were incubated at 37 8C under 10% CO 2 for 7 days.
2.4. Neural stem cell line MNS-70 Neural stem cell line MNS-70, isolated from E12.5 rat brain, was maintained as described previously [20]. Briefly, the monolayer culture was maintained in D-MEM / F-12 (1:1) containing 10% fetal calf serum and 5% horse serum (growth medium) on dishes coated with 10 mg / ml poly-Dlysine. To form aggregates of progenitor cells, the monolayer culture cells were trypsinized and resuspended in the growth medium. They were then incubated for 5 days in the presence of 20 ng / ml bFGF and 1 mM b-estradiol on bacterial dishes coated with poly[2-hydroxyethyl methacrylate] to inhibit attachment of the cells to the surface of the dishes. The aggregates were transferred onto dishes coated with 100 mg / ml poly-D-lysine and incubated in D-MEM / F-12 (1:1) medium containing 10% fetal calf serum (differentiation medium).
2.5. Immunohistochemistry 2.2. Western blot analysis Brains of E13 Wistar rats were isolated and rinsed in ice-cold PBS. They were homogenized in the tissue lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and Complete EDTA-free protease inhibitor cocktail (Roche)). The cell suspension was centrifuged at 14,0003g for 30 min, and the supernatant was used as the total cell lysate. The protein con-
Embryos of Wistar rats were fixed in 4% paraformaldehyde / PBS and then successively soaked in 10% and 20% sucrose / PBS. Embryos were embedded in OCT compound, and frozen sections were prepared at 16 mm. Primary cultured cells and MNS-70 cells were seeded onto Sonicseal Slides (Nulge Nunc). To stain cell surface antigens, sections or cell cultures were fixed in 4% paraformaldehyde / PBS, incubated in the first antibody
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diluted in 3% BSA and 3% goat serum / PBS for 1 h, and washed in PBS. They were then incubated in the diluted secondary antibody for 1 h, washed in PBS, and mounted in PermaFluor Aqueous Mountant (Immunon). To stain the cytoplasmic antigens, the cells were permeabilized in 99.5% ice-cold ethanol before adding the first antibody. Samples were examined with a confocal laser scanning microscope (Molecular Dynamics). Whole-mount immunohistochemistry was performed as described, with some modifications [16]. Briefly, embryos were fixed in 4% paraformaldehyde / PBS, dehydrated and bleached in 6% H 2 O 2 / methanol, and rehydrated in PBS. Blocking was carried out in a solution containing 2% skim milk and 0.3% Tween 20 / PBS (PBS / MTw), and samples were incubated for 24 h in the first antibody diluted in PBS / MT. They were then washed in PBS / MTw, incubated for 24 h in the HRP-conjugated secondary antibody diluted in PBS / MTw, and washed in PBS. Color development was performed in 0.3 mg / ml diaminobenzidine (DAB) and 0.0003% H 2 O 2 / PBS. The affinity-purified anti-RYK rabbit antiserum [13] was used at a concentration of 5 mg / ml. Antibodies for marker proteins were: anti-nestin mouse monoclonal antibody (clone Rat 401, PharMingen) diluted to 1:500; anti-MAP2 mouse monoclonal antibody (clone HM-2, Sigma) diluted to 1:200; anti-GFAP mouse monoclonal antibody (clone G-A-5, Sigma) diluted to 1:400; anti-galactocerebroside
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(GalC) mouse monoclonal antibody (clone mGalC, RocheBoehringer) diluted to 1:50; and mouse monoclonal antibody A2B5 (conditioned medium of hybridoma, no. CRL1520, ATCC) diluted to 1:5. The secondary antibodies were: FITC-conjugated antirabbit IgG (Jackson Lab) diluted to 1:200; Cy3-conjugated anti-mouse IgG (Amersham Pharmacia) 1:200; Cy3-conjugated anti-mouse IgM (Jackson Lab) diluted to 1:200; and HRP-conjugated anti-rabbit IgG (Amersham Pharmacia) diluted to 1:200.
3. Results
3.1. Localization of RYK in embryonic brain In our previous study, in situ hybridization demonstrated that RYK mRNA was distributed in the ventricular zone and cortical plate of rat embryonic forebrain. We also detected colocalization of RYK and nestin in the neuroepithelium at E13 [13]. In this report, we first examined the specificity of the anti-RYK antibody using E13 rat brains, where abundant RYK mRNA could be observed [13]. An immunoblot analysis showed that the rabbit antiRYK antibody recognized a protein band at 65–70 kDa in the total cell lysate of E13 rat brains (Fig. 1A). The apparent size of rat RYK protein is larger than the size of
Fig. 1. Expression of RYK protein in early embryonic rat brains. Total cell lysate of E13 rat brain was separated by SDS–PAGE, and subjected to an immunoblot with the rabbit anti-RYK polyclonal antibody (A). Coronal sections of rat forebrain at E15 were labeled with anti-RYK antibody (green) and anti-nestin antibody (red) (B), or with anti-RYK antibody (green) and anti-MAP2 antibody (red) (C). RYK-positive cells are observed between nestin-positive radial fibers in the preplate (open arrows), whereas RYK and nestin are colocalized in the ventricular zone (closed arrows) (B). RYK and MAP2 are colocalized in the preplate at E15 (C, arrows). PP, preplate; VZ, ventricular zone. Scale bars: 20 mm.
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63 kDa calculated based on the amino acid sequence, suggesting that rat RYK is glycosylated as is the case in human [37] and mouse RYK [39]. In the preabsorption test, RYK immunoreactivity was abolished at the peptide antigen concentration of 1 mg / ml (data not shown). Double-immunostaining using the anti-RYK antibody and antibodies for marker proteins was performed on coronal sections of forebrain, to further specify the lineage of the RYK-expressing cells in embryonic brain. At E15, colocalization of RYK and nestin was observed in the ventricular zone, where neural stem cells proliferate and committed progenitor cells are produced, indicating expression of RYK in neural stem cells or neural progenitor cells. At this stage, expression of nestin became prominent in the vertical processes of the radial glia, which guide postmitotic neurons migrating from the ventricular zone toward the pial surface. RYK-positive cells were observed between these radial processes at the pial surface, supporting the notion that RYK is expressed in postmitotic neurons (Fig. 1B). Double-immunostaining for RYK and MAP2 was performed, to confirm that the RYK-positive cells observed between the nestin-positive radial fibers were neurons. The results showed that MAP2-positive postmitotic neurons emerged in the preplate at E15, and these cells were also labeled with anti-RYK antibody,
suggesting expression of RYK in postmitotic neurons (Fig. 1C). At E18, RYK-positive cells between nestin-positive radial fibers accumulated to form a thick layer, but colocalization of RYK and nestin was still observed in the ventricular zone (Fig. 2A and B). At this stage, MAP2positive neurons accumulated to form the cortical plate, and expression of RYK and MAP2 completely overlapped in this area. Cells in the marginal zone and the subplate, both of which originate from the preplate, were also strongly positive for both RYK and MAP2 (Fig. 2C and D). These observations suggest expression of RYK in nestin-positive neural stem cells or progenitor cells, and neurons at this stage.
3.2. Localization of RYK in embryonic spinal cord Besides its expression in the brain, our whole-mount in situ hybridization analysis showed expression of RYK in the spinal cord in early embryos [13]. This observation motivated us to analyze the distribution and lineage of RYK-positive cells in developing spinal cord. Wholemount immunohistochemical analysis at E12 revealed strong expression of RYK in the telencephalon, mesencephalon, spinal cord, and dorsal root ganglia. Apart from the
Fig. 2. Immunostaining for RYK in rat brains at E18. Coronal sections of rat forebrain at E18 were labeled with anti-RYK antibody (green) and anti-nestin antibody (red) (A, B), or with anti-RYK antibody (green) and anti-MAP2 antibody (red) (C, D). RYK-positive cells are detected between nestin-positive radial fibers in the cortical plate, whereas colocalization of RYK and nestin is observed in the ventricular zone (A). B is a high-magnification view of the cortical plate, and RYK-positive cells (open arrows) are observed between nestin-positive radial fibers (arrows). RYK and MAP2 double-positive cells have accumulated to form the cortical plate (C). D is a high-magnification view of the cortical plate. CP, cortical plate; MZ, marginal zone; SP, subplate; VZ, ventricular zone. Scale bars: A, C, 100 mm; B, D, 20 mm.
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nervous system, intense RYK expression was observed in the limb bud, tail, and branchial arches (Fig. 3A and B). This pattern of expression of RYK protein was consistent with the pattern of RYK mRNA expression previously reported [13]. An immunofluorescence study was performed on transverse sections at E15 and E17, when a large number of neurons are generated in rat spinal cord [25]. Staining of the E15 sections revealed intense expression of RYK in the ventral horn, intermediate gray matter, and ventricular zone, but expression of RYK in the dorsal horn was faint. RYK immunoreactivity in the mantle layer colocalized with MAP2, suggesting its expression in the cell body of neurons. The spatial information implies that RYK expression is intense in motoneurons, but is relatively low in interneurons. Although less intense, expression of RYK was also noted in the marginal zone / white matter, especially in the ventral cord, possibly representing its localization in the axons. The floor plate and roof plate were devoid of RYK expression (Fig. 3C and D). Double-
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immunofluorescence for RYK and nestin at E15 showed colocalization of these proteins in the ventricular zone, where neural stem cells proliferate and committed progenitor cells are generated, and thus RYK is expressed in neural stem cells or neural progenitor cells of the developing spinal cord. In the gray matter, RYK was identified between nestin-positive radial fibers (Fig. 3E and F). Localization of RYK remained basically the same at E17, however, there was some discrepancy between the staining pattern of RYK and MAP2, possibly due to the difference in subcellular localization between these proteins (data not shown).
3.3. Expression of RYK in primary cultured cells We have previously observed prominent expression of RYK mRNA in the cerebrum between postnatal day 0 (P0) and postnatal week 1 (P1W). Since a large number of cells differentiate to constitute the nervous system by this stage,
Fig. 3. Immunostaining for RYK in rat whole embryo and spinal cord sections. Lateral view (A) and posterior view (B) of a whole-mount preparation, immunostained for RYK at E12. Transverse sections at E15 were labeled with anti-RYK antibody (green) and anti-MAP2 antibody (red; C, D), or anti-nestin antibody (red; E, F). RYK and MAP2 are colocalized in the ventral horn and intermediate gray matter, and RYK is positive in the marginal zone / white matter, but less intensely (C). The level of expression of RYK in the dorsal horn is relatively low (D). RYK-positive cells in the gray matter are observed between nestin-positive radial fibers (E). RYK and nestin are colocalized in the ventricular zone (arrows, F). BA, branchial arch; DH, dorsal horn; DRG, dorsal root ganglia; FP, floor plate; IG, intermediate gray matter; LB, limb bud; Mes, mesencephalon; MZ, marginal zone / white matter; RP, roof plate; SC, spinal cord; T, tail; Tel, telencephalon; VH, ventral horn; VZ, ventricular zone. Scale bars: A, B, 500 mm; C–E, 100 mm; F, 20 mm.
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identifying the lineage of RYK-expressing cells is important to understanding the function of this molecule, and immunohistochemistry of primary cultures of E21 rat cerebrum was carried out for this purpose. As shown in Fig. 4A, relatively small round cells constituted the largest population of RYK-positive cells. The double-immunolabeling study revealed that these cells were also positive for MAP2, suggesting expression of RYK in neurons. The distribution of RYK was predominantly in the cell body, whereas expression of MAP2 was distributed in both the cell body and on the dendrites of neurons (Fig. 4A). Although more than 90% of the GFAP-positive astrocytes were negative for RYK, a small population of them was clearly labeled with anti-RYK antibody (Fig. 4B). Because the cells were stellate in shape and had multiple long processes, the typical morphology of type-2 astrocytes, double-immunostaining was performed with anti-RYK antibody and A2B5 antibody, a marker for type-2 astrocytes and O-2A progenitor cells [26]. The results showed that a significant population (more than 80%) of A2B5-positive cells also express RYK (Fig. 4C). The A2B5-positive cells that are type-2 astrocytes can be identified by their stellate-shape, whereas the O-2A progenitor cells have small round morphology, with or without short processes. Cells having both of these sets of characteristics were labeled with anti-RYK antibody, suggesting expression of RYK by both type-2 astrocytes and O-2A progenitor cells. RYK localized in the cell body of both these types of cells, and on their processes less intensely. Double-positive cells for RYK and GFAP exclusively exhibited the stellate-shape, and none had large flat morphology, suggesting that there was no expression of RYK in type-1 astrocytes. Double-immunolabeling for RYK and GalC, a marker for oligodendrocytes, showed that a certain population of oligodendrocytes strongly expressed RYK (Fig. 4D). However, some of the GalC-positive cells extending long processes with branches were negative for RYK, suggesting high expression of RYK in relatively young oligodendrocytes rather than in mature oligodendrocytes. Expression of RYK was prominent not only in the cell body of the oligodendrocytes, but in their processes. This observation was intriguing because of the dominant ex-
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pression of RYK in the cell body of neurons. By contrast, hardly any expression of RYK was detected in ED1-positive microglia in the same culture (data not shown).
3.4. Expression of RYK in MNS-70 cells The results obtained from the immunohistochemical analysis using tissue sections and primary culture cells revealed unique cell-type-specific expression of RYK in the developing forebrains of rats. As the next step, a neural stem cell line with the potential for differentiation would be a useful tool for future analysis. The MNS-70 cell line has such a property [20], and in addition, possibly has characteristics of neural stem cells originating in the forebrain based on the expression pattern of transcription factors [21], therefore we further analyzed RYK expression in MNS-70 by immunostaining. As shown in Fig. 5A, more than 90% of the cells in the monolayer culture of MNS-70 were nestin-positive neural stem cells, as previously described [20,21]. These nestin-positive cells were also intensely labeled with the anti-RYK antibody (Fig. 5A). Although less than 10% of the cells were weakly labeled with anti-GFAP antibody, significant levels of RYK expression were not detected in these cells (data not shown). Under culture conditions for cell differentiation, the cells in the aggregates gradually differentiated after attachment to the culture dish. Cells were immunostained and examined 3 h after attachment of the aggregates to the culture dish. At that point, RYK and MAP2 were already coexpressed in most of the aggregates, mainly in the cells on their surface (Fig. 5B). Expression of GFAP, on the other hand, was detected both in the center and on the surface of the aggregates, and expression of RYK and GFAP hardly overlapped at this stage (Fig. 5C). Coexpression of RYK and nestin was observed in some cells at the edge of spreading aggregates (Fig. 5D). These results suggested that a certain population of cells in the aggregate have already differentiated, with some remaining in the form of nestin-positive progenitor cells at this stage, and that expression of RYK is dominant in nestin-positive progenitor cells and neurons.
Fig. 4. Double-immunostaining of primary cultured cells isolated from E21 rat cerebrum. Cells were labeled with anti-RYK antibody (green; A–D), and anti-MAP2 antibody (red; A), anti-GFAP antibody (red; B), A2B5 antibody (red; C), or anti-GalC antibody (red; D). RYK and MAP2 colocalizes in all neurons (A). A small number of GFAP-positive cells are also RYK-positive (arrows, B). Stellate-shaped type-2 astrocytes (arrows) and small O-2A progenitor cells (open arrow) are observed among the A2B5- and RYK-positive cells (C). A significant population of GalC-positive oligodendrocytes is also positive for RYK (arrows, D). Scale bars represent 50 mm.
Fig. 5. Double-immunostaining of neural stem cell line MNS-70. Monolayer cultured cells (A) or aggregates (B–D) were immunostained for anti-RYK antibody (green) and anti-nestin antibody (red; A, D), anti-MAP2 antibody (red; B), or anti-GFAP antibody (red; C). Most of the monolayer cells are labeled with RYK and nestin (A). Colocalization of RYK and MAP2 is observed in the aggregates (B), but staining for RYK does not overlap staining for GFAP (C). Colocalization of RYK and nestin is visible at the edge of some aggregates (arrows, D). Scale bars represent 50 mm.
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Aggregates of MNS-70 cells were also incubated in differentiation medium for 5 days and immunostained. At this stage, staining for RYK and MAP2 overlapped mainly in the center of flattened aggregates, which showed expression of RYK in neurons (Fig. 6A). Both RYK and MAP2 predominantly localized in the cell body, suggesting that the cells were relatively immature neurons without dendrites. Some nestin-positive cells were noted at the edge of the aggregates, and a certain population of them was also positive for RYK (Fig. 6C). However, the levels of expression of both nestin and RYK in these cells were not significant compared with their levels in the monolayer culture. At the same stage, hardly any RYK- or GFAP-double-positive cells were detected, suggesting that the astrocytes did not express RYK (Fig. 6B). By contrast, small numbers of cells with or without short processes were positive for both RYK and A2B5 (Fig. 6D). This result suggests expression of RYK in O-2A progenitor cells. As previously described, no GalC-positive oligodendrocytes were observed among the MNS-70 cells [21]. Taken together, these findings indicate that RYK expressed in nestin-positive neural stem cells or progenitor cells, neurons, and O-2A progenitor cells among MNS-70 cells, which is consistent with the results observed in tissue sections and primary cultured cells.
4. Discussion In this study, we analyzed the distribution of RYK protein in developing rat CNS, and the results showed that the localization of RYK protein in embryonic forebrain was quite similar to that of RYK mRNA described previously [13]. Double-immunostaining for marker proteins revealed expression of RYK protein in nestin-positive cells and neurons in embryonic forebrain and spinal cord. RYK expression was basically prominent in all of the neurons in forebrain sections and in cell cultures isolated from embryonic forebrain (primary cultures and a cell line). By contrast, the level of expression of RYK in the spinal cord seemed to depend on the subtype of the neurons, for example, there was high expression in putative motoneurons. These findings imply a different mechanism of regulation of RYK expression in forebrain and spinal cord. Although Northern blot analysis has revealed widespread distribution of RYK mRNA in mammalian tissues, stage- or lineage-specific expression of RYK has been reported by several groups. Based on these reports taken together with our own present results in this study showing cell-type-specific expression of RYK in the CNS, expression of RYK is tightly regulated, however, the regulation process seems to depend on the type of tissue or cell. For example, during hematopoiesis, RYK mRNA was detected in T cells in both the early and differentiated stages, but only in mature B cells, not in early B cells [33]. It was also observed in differentiated cell lines, but not in
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multipotent cell lines [39]. These findings suggest that RYK is expressed in differentiated populations rather than in proliferating cells in the hematopoietic system. By contrast, a high level of RYK mRNA was detected in embryonic stem (ES) cells [39]. In epithelial cells, transcription of RYK is specifically up-regulated in proliferating cells in the intestine and uterus [31] and in malignant ovarian tumors [37], suggesting dominant expression of RYK in replicating populations. Compared with hematopoietic or epithelial tissues, the pattern of expression of RYK in the CNS observed in the present study is unique, since it was detected in both immature cells, such as nestin-positive cells and O-2A progenitor cells, and in mature cells, such as neurons and oligodendrocytes. The most interesting issue concerns the function of RYK, however, we were unable to simply relate this expression pattern to a single phenomenon, such as replication or migration. Instead, the expression of RYK in various cell types suggests multiple functions of this molecule during the formation of the CNS. The discovery of expression of RYK in oligodendrocytes was unexpected, since we hardly observed any RYK mRNA in the myelin of postnatal cerebrum. The mRNA signals in the white matter of the late embryonic cerebrum [13], however, may represent expression of RYK in oligodendrocytes or their progenitors. Detection of RYK in the ventral spinal cord at around E15, the site and time oligodendrocyte progenitors first arise in the rat CNS [38], may also represent RYK expression in these cells. Expression in both neurons and oligodendrocytes or their progenitors may suggest a function of RYK during myelin formation. However, it is difficult to discuss the significance of RYK in type-2 astrocytes, because the properties of type-2 astrocytes in vivo are controversial [23]. The high expression in type-2 astrocytes, but not in type-1 astrocytes, however, may be related to biochemical differences between these cell types, such as in surface molecules and ion channels [19]. By contrast, the failure to detect RYK in microglia was unexpected, since we observed RYK mRNA in a rat microglia culture in a preliminary study (unpublished). High expression of RYK in hematopoietic cells, which may share the same origin as microglia, also suggested the possibility of expression in microglia. In view of its stage-specific expression in hematopoietic cells, expression of RYK in microglia may be highly regulated, and we did not detect it under the culture conditions described. In Drosophila, expression of the RYK homolog Lio / Drl has been independently identified in neurons [3,18] and glia [32], and Lio / Drl in both types of cells are associated with the control of axon outgrowth. Expression of RYK in both neurons and glia may reflect a role of RYK analogous to that of Lio / Drl during mammalian CNS development. Although the predominant expression of RYK in the cell body of neurons conflicts with this hypothesis, our preliminary observations suggest that it is expressed on
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Fig. 6. Double-immunostaining of neural stem cell line MNS-70 at the differentiated stage. Cells incubated in the differentiation culture for 5 days were immunostained for anti-RYK antibody (green) and anti-MAP2 antibody (red; A), anti GFAP-antibody (red; B), anti-nestin antibody (red; C), or A2B5 antibody (red; D). Colocalization of RYK and MAP2 can be observed (A), but staining for RYK and GFAP is segregated (B). Arrows in C indicate colocalization of RYK and nestin. Some A2B5-positive cells are detected, and some of them are also RYK-positive (arrows, D). Scale bars represent 50 mm.
neurites depending on the culture conditions, and expression in the white matter of developing spinal cord may represent expression in growing axons. Association of RYK with Eph receptors [11], which play a role in axon pathfinding [24,35], may mean that RYK cooperates with Eph receptors in this event, although the physiological significance of this binding has to be further elucidated. Since AF-6 localizes on synapses [5], binding of RYK with AF-6 [11] also suggests an attractive model in which RYK plays a role in axon pathfinding. It is also intriguing that Lio / Drl may be functionally associated with Dek [29] or Canoe [17], Drosophila counterparts of the Eph receptor and AF-6, respectively. The RYK expressed in nestin-positive cells may have as yet unidentified functions, since expression of Lio / Drl has never been reported in neural stem cell or progenitor cells. Expression of this signaling molecule in replicating cells, such as neural stem cells and ES cells, would suggest a function in proliferation. A recent report shows a role of Eph / ephrin signaling in the migration and proliferation of adult neural stem cells [7], and the expression of RYK, a possible partner of Eph receptors, in neural stem cells or neural progenitor cells implies a cooperative function of the RYK and Eph pathway during these events. However, possible roles of RYK through association with other membrane-bound or cytoplasmic proteins cannot be ruled out, and RYK may participate in other key steps in CNS development. The tight regulation of RYK expression, in particular, suggests that it may be involved in cell differentiation. In conclusion, RYK is expressed in neural stem cells or neural progenitor cells, and neurons of the embryonic forebrain and spinal cord. Analysis of primary cultures from forebrain showed its expression in all neurons, and some O-2A progenitor cells, type-2 astrocytes, and oligodendrocytes, but not in type-1 astrocytes or microglia. Neural stem cell line MNS-70 showed a similar profile of expression. The distribution of expression of RYK in both immature and mature cells is quite intriguing, but confusing in terms of identifying its role. Although the function of RYK in mammalian CNS is still unclear, the results of the present study suggest some possible roles of this molecule. Further analysis of loss or gain of expression would supply information about the signaling mechanisms associated with it. We are also interested in learning how RYK expression is regulated in a cell-type-dependent manner, and finding the answer should help clarify its function.
Acknowledgements We are grateful to Dr. Shun Nakamura for kindly providing the MNS-70 cells. This work was supported by the Ministry of Health, Labour and Welfare of Japan and the Japan Health Sciences Foundation.
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Interactive report
Cell-type-specific expression of protein tyrosine kinase-related receptor RYK in the central nervous system of the rat Kazuyo Kamitori a , Mitsuru Machide a , Kazuhiko Tomita a,b , Masato Nakafuku c , Shinichi Kohsaka a,b , * a
Department of Neurochemistry, National Institute of Neuroscience, 4 -1 -1 Ogawa-higashi, Kodaira, Tokyo 187 -8502, Japan b Graduate School of Lifescience, Tokyo University of Pharmacy and Lifescience, Hachiouji, Tokyo, Japan c Division of Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Accepted 12 November 2001
Abstract The mammalian RYK is an orphan receptor that contains a catalytically inactive tyrosine-kinase-related domain. Its Drosophila homolog, Lio / Drl, is required for axon pathfinding in developing brain. Our previous study suggested that RYK mRNA is expressed in nestin-positive progenitor cells and neurons. In the present study, immunohistochemistry has been used to further localize RYK in the central nervous system of rats to identify the lineage of the RYK-expressing cells. In the embryonic forebrain, RYK colocalized with nestin in the ventricular zone and with MAP2 in the cortical plate, suggesting that RYK is expressed in neural progenitor cells and neurons. Localization of RYK in embryonic spinal cord also suggested its expression in both cell types. In primary cultures of rat cerebrum, RYK expression was observed in all neurons, as well as in a significant population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. However, no RYK expression was detected in type-1 astrocytes or microglia. Multipotent neural stem cell line MNS-70 was also analyzed for expression of RYK, and most of the cells were positive for both RYK and nestin in the undifferentiated stage. In the differentiated stage, expression of RYK was detected in the neurons, but not in type-1 astrocytes. In conclusion, RYK is expressed in nestin-positive progenitor cells and neurons, and in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes in developing CNS. These findings show that expression of RYK in rat CNS is tightly regulated in a cell-type-specific manner. 2002 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Neuroglia and myelin Keywords: Central nervous system; Neural progenitor cells; Receptor; Differentiation; Immunohistochemistry
1. Introduction During the development of the mammalian central nervous system (CNS), multipotent neural stem cells proliferate to generate mature cells: neurons, astrocytes, and oligodendrocytes. This cell differentiation is regulated by various transcription factors, including Mash-1 [10], neurogenin [16], and olig1 / 2 [15,40], and signaling pathways through membrane receptors are hypothesized to control this event, as exemplified by Notch [8,36], PDGF *Corresponding author. Tel.: 181-42-346-1721; fax: 181-42-3461751. E-mail address: [email protected] (S. Kohsaka).
[22,27], and CNTF [4]. In addition, an increasing number of reports suggest a contributory role of signaling pathways through membrane receptors in the migration and layer formation of postmitotic neurons [1,9,28,30]. Numerous unidentified signaling molecules surely play important roles in the developing CNS by modulating key events, such as cell differentiation or cell migration. The tyrosine-kinase-related protein RYK is an orphan receptor that has been isolated from mammalian species [12,33,34,37,39]. It is structurally distinct from other members of the receptor tyrosine kinase family in two regards. Firstly, its tyrosine-kinase-like domain possesses amino acid substitutions that may lead to impaired ATP binding. Secondly, its extracellular domain is smaller than
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00358-3
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that of other receptor tyrosine kinases, and contains only two leucine-rich motifs (LRMs). Although the tyrosine kinase activity of RYK is thought to have been lost as a result of the amino acid substitutions in the catalytic domain, a chimeric trkA and human RYK receptor have been shown to have the ability to activate the MAP kinase pathway upon stimulation by NGF, a ligand for the trk receptor [14]. Recently, gene targeting analysis has shown that RYK is essential for normal morphogenesis, possibly through association with Eph family receptors, which are involved in the pattern formation of the nervous system [11]. Halford et al. have also demonstrated that RYK binds to AF-6, a Ras effector cell–cell junction protein. Independently, Lio / Drl (also called linotte or derailed), the Drosophila homolog of RYK, has been reported to play key roles in the development and maintenance of the fruitfly brain by controlling axonal pathfinding [2,3,6,18,32]. All these findings imply significant functions of mammalian RYK during development of the CNS through signaling events. In spite of the importance of its Drosophila homolog in the CNS, as described above, the function of RYK in the mammalian CNS has not been fully analyzed, possibly because Northern blot analysis of mammalian tissues has shown relatively low levels of RYK mRNA in the adult brain. However, our previous characterization of the spatial and temporal distribution of RYK mRNA in the rat CNS implied that RYK is expressed in nestin-positive cells (neural progenitor cells) and neurons during development [13]. Here we describe the results of localization of RYK by double-immunostaining for various marker proteins that show its expression in nestin-positive neural progenitor cells and neurons, as well as in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. This information should lead to further understanding of the physiological function of RYK in the mammalian CNS.
2. Materials and methods
2.1. Animals Wistar rats were obtained from CLEA JAPAN. The dams were ether-anesthetized while the embryos were removed from the uterus.
centration was determined using a BCA protein assay kit (PIERCE). Twenty micrograms of the protein were separated by SDS–PAGE, and transferred to PROTRAN nitrocellulose transfer membranes (Schleicher & Schuell) followed by immunoblotting with the affinity-purified antiRYK rabbit antiserum [13] at a concentration of 2 mg / ml. Then the membrane was incubated in horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham Pharmacia) diluted to 1:2000, and the reaction was visualized with an ECL chemiluminescence detection system (Amersham Pharmacia).
2.3. Primary cultured cells The cerebral cortex was isolated from embryonic day 21 (E21) Wistar rats. After removing the meninges, pieces of the tissue were minced and incubated in 0.25% trypsin solution for 10 min to digest the extracellular matrix. Fetal calf serum was added to the cell suspension to inactivate the trypsin, and the suspension was subsequently incubated in 500 mg / ml DNase I solution. The cells were then collected by centrifugation and resuspended in D-MEM medium containing 10% fetal calf serum. After subsequent filtration through a Cell Strainer (70 mm pore size, Falcon), the cells were seeded at a density of 1310 5 / cm 2 onto culture dishes coated with the 50 mg / ml poly-L-lysine, and were incubated at 37 8C under 10% CO 2 for 7 days.
2.4. Neural stem cell line MNS-70 Neural stem cell line MNS-70, isolated from E12.5 rat brain, was maintained as described previously [20]. Briefly, the monolayer culture was maintained in D-MEM / F-12 (1:1) containing 10% fetal calf serum and 5% horse serum (growth medium) on dishes coated with 10 mg / ml poly-Dlysine. To form aggregates of progenitor cells, the monolayer culture cells were trypsinized and resuspended in the growth medium. They were then incubated for 5 days in the presence of 20 ng / ml bFGF and 1 mM b-estradiol on bacterial dishes coated with poly[2-hydroxyethyl methacrylate] to inhibit attachment of the cells to the surface of the dishes. The aggregates were transferred onto dishes coated with 100 mg / ml poly-D-lysine and incubated in D-MEM / F-12 (1:1) medium containing 10% fetal calf serum (differentiation medium).
2.5. Immunohistochemistry 2.2. Western blot analysis Brains of E13 Wistar rats were isolated and rinsed in ice-cold PBS. They were homogenized in the tissue lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and Complete EDTA-free protease inhibitor cocktail (Roche)). The cell suspension was centrifuged at 14,0003g for 30 min, and the supernatant was used as the total cell lysate. The protein con-
Embryos of Wistar rats were fixed in 4% paraformaldehyde / PBS and then successively soaked in 10% and 20% sucrose / PBS. Embryos were embedded in OCT compound, and frozen sections were prepared at 16 mm. Primary cultured cells and MNS-70 cells were seeded onto Sonicseal Slides (Nulge Nunc). To stain cell surface antigens, sections or cell cultures were fixed in 4% paraformaldehyde / PBS, incubated in the first antibody
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diluted in 3% BSA and 3% goat serum / PBS for 1 h, and washed in PBS. They were then incubated in the diluted secondary antibody for 1 h, washed in PBS, and mounted in PermaFluor Aqueous Mountant (Immunon). To stain the cytoplasmic antigens, the cells were permeabilized in 99.5% ice-cold ethanol before adding the first antibody. Samples were examined with a confocal laser scanning microscope (Molecular Dynamics). Whole-mount immunohistochemistry was performed as described, with some modifications [16]. Briefly, embryos were fixed in 4% paraformaldehyde / PBS, dehydrated and bleached in 6% H 2 O 2 / methanol, and rehydrated in PBS. Blocking was carried out in a solution containing 2% skim milk and 0.3% Tween 20 / PBS (PBS / MTw), and samples were incubated for 24 h in the first antibody diluted in PBS / MT. They were then washed in PBS / MTw, incubated for 24 h in the HRP-conjugated secondary antibody diluted in PBS / MTw, and washed in PBS. Color development was performed in 0.3 mg / ml diaminobenzidine (DAB) and 0.0003% H 2 O 2 / PBS. The affinity-purified anti-RYK rabbit antiserum [13] was used at a concentration of 5 mg / ml. Antibodies for marker proteins were: anti-nestin mouse monoclonal antibody (clone Rat 401, PharMingen) diluted to 1:500; anti-MAP2 mouse monoclonal antibody (clone HM-2, Sigma) diluted to 1:200; anti-GFAP mouse monoclonal antibody (clone G-A-5, Sigma) diluted to 1:400; anti-galactocerebroside
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(GalC) mouse monoclonal antibody (clone mGalC, RocheBoehringer) diluted to 1:50; and mouse monoclonal antibody A2B5 (conditioned medium of hybridoma, no. CRL1520, ATCC) diluted to 1:5. The secondary antibodies were: FITC-conjugated antirabbit IgG (Jackson Lab) diluted to 1:200; Cy3-conjugated anti-mouse IgG (Amersham Pharmacia) 1:200; Cy3-conjugated anti-mouse IgM (Jackson Lab) diluted to 1:200; and HRP-conjugated anti-rabbit IgG (Amersham Pharmacia) diluted to 1:200.
3. Results
3.1. Localization of RYK in embryonic brain In our previous study, in situ hybridization demonstrated that RYK mRNA was distributed in the ventricular zone and cortical plate of rat embryonic forebrain. We also detected colocalization of RYK and nestin in the neuroepithelium at E13 [13]. In this report, we first examined the specificity of the anti-RYK antibody using E13 rat brains, where abundant RYK mRNA could be observed [13]. An immunoblot analysis showed that the rabbit antiRYK antibody recognized a protein band at 65–70 kDa in the total cell lysate of E13 rat brains (Fig. 1A). The apparent size of rat RYK protein is larger than the size of
Fig. 1. Expression of RYK protein in early embryonic rat brains. Total cell lysate of E13 rat brain was separated by SDS–PAGE, and subjected to an immunoblot with the rabbit anti-RYK polyclonal antibody (A). Coronal sections of rat forebrain at E15 were labeled with anti-RYK antibody (green) and anti-nestin antibody (red) (B), or with anti-RYK antibody (green) and anti-MAP2 antibody (red) (C). RYK-positive cells are observed between nestin-positive radial fibers in the preplate (open arrows), whereas RYK and nestin are colocalized in the ventricular zone (closed arrows) (B). RYK and MAP2 are colocalized in the preplate at E15 (C, arrows). PP, preplate; VZ, ventricular zone. Scale bars: 20 mm.
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63 kDa calculated based on the amino acid sequence, suggesting that rat RYK is glycosylated as is the case in human [37] and mouse RYK [39]. In the preabsorption test, RYK immunoreactivity was abolished at the peptide antigen concentration of 1 mg / ml (data not shown). Double-immunostaining using the anti-RYK antibody and antibodies for marker proteins was performed on coronal sections of forebrain, to further specify the lineage of the RYK-expressing cells in embryonic brain. At E15, colocalization of RYK and nestin was observed in the ventricular zone, where neural stem cells proliferate and committed progenitor cells are produced, indicating expression of RYK in neural stem cells or neural progenitor cells. At this stage, expression of nestin became prominent in the vertical processes of the radial glia, which guide postmitotic neurons migrating from the ventricular zone toward the pial surface. RYK-positive cells were observed between these radial processes at the pial surface, supporting the notion that RYK is expressed in postmitotic neurons (Fig. 1B). Double-immunostaining for RYK and MAP2 was performed, to confirm that the RYK-positive cells observed between the nestin-positive radial fibers were neurons. The results showed that MAP2-positive postmitotic neurons emerged in the preplate at E15, and these cells were also labeled with anti-RYK antibody,
suggesting expression of RYK in postmitotic neurons (Fig. 1C). At E18, RYK-positive cells between nestin-positive radial fibers accumulated to form a thick layer, but colocalization of RYK and nestin was still observed in the ventricular zone (Fig. 2A and B). At this stage, MAP2positive neurons accumulated to form the cortical plate, and expression of RYK and MAP2 completely overlapped in this area. Cells in the marginal zone and the subplate, both of which originate from the preplate, were also strongly positive for both RYK and MAP2 (Fig. 2C and D). These observations suggest expression of RYK in nestin-positive neural stem cells or progenitor cells, and neurons at this stage.
3.2. Localization of RYK in embryonic spinal cord Besides its expression in the brain, our whole-mount in situ hybridization analysis showed expression of RYK in the spinal cord in early embryos [13]. This observation motivated us to analyze the distribution and lineage of RYK-positive cells in developing spinal cord. Wholemount immunohistochemical analysis at E12 revealed strong expression of RYK in the telencephalon, mesencephalon, spinal cord, and dorsal root ganglia. Apart from the
Fig. 2. Immunostaining for RYK in rat brains at E18. Coronal sections of rat forebrain at E18 were labeled with anti-RYK antibody (green) and anti-nestin antibody (red) (A, B), or with anti-RYK antibody (green) and anti-MAP2 antibody (red) (C, D). RYK-positive cells are detected between nestin-positive radial fibers in the cortical plate, whereas colocalization of RYK and nestin is observed in the ventricular zone (A). B is a high-magnification view of the cortical plate, and RYK-positive cells (open arrows) are observed between nestin-positive radial fibers (arrows). RYK and MAP2 double-positive cells have accumulated to form the cortical plate (C). D is a high-magnification view of the cortical plate. CP, cortical plate; MZ, marginal zone; SP, subplate; VZ, ventricular zone. Scale bars: A, C, 100 mm; B, D, 20 mm.
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nervous system, intense RYK expression was observed in the limb bud, tail, and branchial arches (Fig. 3A and B). This pattern of expression of RYK protein was consistent with the pattern of RYK mRNA expression previously reported [13]. An immunofluorescence study was performed on transverse sections at E15 and E17, when a large number of neurons are generated in rat spinal cord [25]. Staining of the E15 sections revealed intense expression of RYK in the ventral horn, intermediate gray matter, and ventricular zone, but expression of RYK in the dorsal horn was faint. RYK immunoreactivity in the mantle layer colocalized with MAP2, suggesting its expression in the cell body of neurons. The spatial information implies that RYK expression is intense in motoneurons, but is relatively low in interneurons. Although less intense, expression of RYK was also noted in the marginal zone / white matter, especially in the ventral cord, possibly representing its localization in the axons. The floor plate and roof plate were devoid of RYK expression (Fig. 3C and D). Double-
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immunofluorescence for RYK and nestin at E15 showed colocalization of these proteins in the ventricular zone, where neural stem cells proliferate and committed progenitor cells are generated, and thus RYK is expressed in neural stem cells or neural progenitor cells of the developing spinal cord. In the gray matter, RYK was identified between nestin-positive radial fibers (Fig. 3E and F). Localization of RYK remained basically the same at E17, however, there was some discrepancy between the staining pattern of RYK and MAP2, possibly due to the difference in subcellular localization between these proteins (data not shown).
3.3. Expression of RYK in primary cultured cells We have previously observed prominent expression of RYK mRNA in the cerebrum between postnatal day 0 (P0) and postnatal week 1 (P1W). Since a large number of cells differentiate to constitute the nervous system by this stage,
Fig. 3. Immunostaining for RYK in rat whole embryo and spinal cord sections. Lateral view (A) and posterior view (B) of a whole-mount preparation, immunostained for RYK at E12. Transverse sections at E15 were labeled with anti-RYK antibody (green) and anti-MAP2 antibody (red; C, D), or anti-nestin antibody (red; E, F). RYK and MAP2 are colocalized in the ventral horn and intermediate gray matter, and RYK is positive in the marginal zone / white matter, but less intensely (C). The level of expression of RYK in the dorsal horn is relatively low (D). RYK-positive cells in the gray matter are observed between nestin-positive radial fibers (E). RYK and nestin are colocalized in the ventricular zone (arrows, F). BA, branchial arch; DH, dorsal horn; DRG, dorsal root ganglia; FP, floor plate; IG, intermediate gray matter; LB, limb bud; Mes, mesencephalon; MZ, marginal zone / white matter; RP, roof plate; SC, spinal cord; T, tail; Tel, telencephalon; VH, ventral horn; VZ, ventricular zone. Scale bars: A, B, 500 mm; C–E, 100 mm; F, 20 mm.
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identifying the lineage of RYK-expressing cells is important to understanding the function of this molecule, and immunohistochemistry of primary cultures of E21 rat cerebrum was carried out for this purpose. As shown in Fig. 4A, relatively small round cells constituted the largest population of RYK-positive cells. The double-immunolabeling study revealed that these cells were also positive for MAP2, suggesting expression of RYK in neurons. The distribution of RYK was predominantly in the cell body, whereas expression of MAP2 was distributed in both the cell body and on the dendrites of neurons (Fig. 4A). Although more than 90% of the GFAP-positive astrocytes were negative for RYK, a small population of them was clearly labeled with anti-RYK antibody (Fig. 4B). Because the cells were stellate in shape and had multiple long processes, the typical morphology of type-2 astrocytes, double-immunostaining was performed with anti-RYK antibody and A2B5 antibody, a marker for type-2 astrocytes and O-2A progenitor cells [26]. The results showed that a significant population (more than 80%) of A2B5-positive cells also express RYK (Fig. 4C). The A2B5-positive cells that are type-2 astrocytes can be identified by their stellate-shape, whereas the O-2A progenitor cells have small round morphology, with or without short processes. Cells having both of these sets of characteristics were labeled with anti-RYK antibody, suggesting expression of RYK by both type-2 astrocytes and O-2A progenitor cells. RYK localized in the cell body of both these types of cells, and on their processes less intensely. Double-positive cells for RYK and GFAP exclusively exhibited the stellate-shape, and none had large flat morphology, suggesting that there was no expression of RYK in type-1 astrocytes. Double-immunolabeling for RYK and GalC, a marker for oligodendrocytes, showed that a certain population of oligodendrocytes strongly expressed RYK (Fig. 4D). However, some of the GalC-positive cells extending long processes with branches were negative for RYK, suggesting high expression of RYK in relatively young oligodendrocytes rather than in mature oligodendrocytes. Expression of RYK was prominent not only in the cell body of the oligodendrocytes, but in their processes. This observation was intriguing because of the dominant ex-
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pression of RYK in the cell body of neurons. By contrast, hardly any expression of RYK was detected in ED1-positive microglia in the same culture (data not shown).
3.4. Expression of RYK in MNS-70 cells The results obtained from the immunohistochemical analysis using tissue sections and primary culture cells revealed unique cell-type-specific expression of RYK in the developing forebrains of rats. As the next step, a neural stem cell line with the potential for differentiation would be a useful tool for future analysis. The MNS-70 cell line has such a property [20], and in addition, possibly has characteristics of neural stem cells originating in the forebrain based on the expression pattern of transcription factors [21], therefore we further analyzed RYK expression in MNS-70 by immunostaining. As shown in Fig. 5A, more than 90% of the cells in the monolayer culture of MNS-70 were nestin-positive neural stem cells, as previously described [20,21]. These nestin-positive cells were also intensely labeled with the anti-RYK antibody (Fig. 5A). Although less than 10% of the cells were weakly labeled with anti-GFAP antibody, significant levels of RYK expression were not detected in these cells (data not shown). Under culture conditions for cell differentiation, the cells in the aggregates gradually differentiated after attachment to the culture dish. Cells were immunostained and examined 3 h after attachment of the aggregates to the culture dish. At that point, RYK and MAP2 were already coexpressed in most of the aggregates, mainly in the cells on their surface (Fig. 5B). Expression of GFAP, on the other hand, was detected both in the center and on the surface of the aggregates, and expression of RYK and GFAP hardly overlapped at this stage (Fig. 5C). Coexpression of RYK and nestin was observed in some cells at the edge of spreading aggregates (Fig. 5D). These results suggested that a certain population of cells in the aggregate have already differentiated, with some remaining in the form of nestin-positive progenitor cells at this stage, and that expression of RYK is dominant in nestin-positive progenitor cells and neurons.
Fig. 4. Double-immunostaining of primary cultured cells isolated from E21 rat cerebrum. Cells were labeled with anti-RYK antibody (green; A–D), and anti-MAP2 antibody (red; A), anti-GFAP antibody (red; B), A2B5 antibody (red; C), or anti-GalC antibody (red; D). RYK and MAP2 colocalizes in all neurons (A). A small number of GFAP-positive cells are also RYK-positive (arrows, B). Stellate-shaped type-2 astrocytes (arrows) and small O-2A progenitor cells (open arrow) are observed among the A2B5- and RYK-positive cells (C). A significant population of GalC-positive oligodendrocytes is also positive for RYK (arrows, D). Scale bars represent 50 mm.
Fig. 5. Double-immunostaining of neural stem cell line MNS-70. Monolayer cultured cells (A) or aggregates (B–D) were immunostained for anti-RYK antibody (green) and anti-nestin antibody (red; A, D), anti-MAP2 antibody (red; B), or anti-GFAP antibody (red; C). Most of the monolayer cells are labeled with RYK and nestin (A). Colocalization of RYK and MAP2 is observed in the aggregates (B), but staining for RYK does not overlap staining for GFAP (C). Colocalization of RYK and nestin is visible at the edge of some aggregates (arrows, D). Scale bars represent 50 mm.
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Aggregates of MNS-70 cells were also incubated in differentiation medium for 5 days and immunostained. At this stage, staining for RYK and MAP2 overlapped mainly in the center of flattened aggregates, which showed expression of RYK in neurons (Fig. 6A). Both RYK and MAP2 predominantly localized in the cell body, suggesting that the cells were relatively immature neurons without dendrites. Some nestin-positive cells were noted at the edge of the aggregates, and a certain population of them was also positive for RYK (Fig. 6C). However, the levels of expression of both nestin and RYK in these cells were not significant compared with their levels in the monolayer culture. At the same stage, hardly any RYK- or GFAP-double-positive cells were detected, suggesting that the astrocytes did not express RYK (Fig. 6B). By contrast, small numbers of cells with or without short processes were positive for both RYK and A2B5 (Fig. 6D). This result suggests expression of RYK in O-2A progenitor cells. As previously described, no GalC-positive oligodendrocytes were observed among the MNS-70 cells [21]. Taken together, these findings indicate that RYK expressed in nestin-positive neural stem cells or progenitor cells, neurons, and O-2A progenitor cells among MNS-70 cells, which is consistent with the results observed in tissue sections and primary cultured cells.
4. Discussion In this study, we analyzed the distribution of RYK protein in developing rat CNS, and the results showed that the localization of RYK protein in embryonic forebrain was quite similar to that of RYK mRNA described previously [13]. Double-immunostaining for marker proteins revealed expression of RYK protein in nestin-positive cells and neurons in embryonic forebrain and spinal cord. RYK expression was basically prominent in all of the neurons in forebrain sections and in cell cultures isolated from embryonic forebrain (primary cultures and a cell line). By contrast, the level of expression of RYK in the spinal cord seemed to depend on the subtype of the neurons, for example, there was high expression in putative motoneurons. These findings imply a different mechanism of regulation of RYK expression in forebrain and spinal cord. Although Northern blot analysis has revealed widespread distribution of RYK mRNA in mammalian tissues, stage- or lineage-specific expression of RYK has been reported by several groups. Based on these reports taken together with our own present results in this study showing cell-type-specific expression of RYK in the CNS, expression of RYK is tightly regulated, however, the regulation process seems to depend on the type of tissue or cell. For example, during hematopoiesis, RYK mRNA was detected in T cells in both the early and differentiated stages, but only in mature B cells, not in early B cells [33]. It was also observed in differentiated cell lines, but not in
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multipotent cell lines [39]. These findings suggest that RYK is expressed in differentiated populations rather than in proliferating cells in the hematopoietic system. By contrast, a high level of RYK mRNA was detected in embryonic stem (ES) cells [39]. In epithelial cells, transcription of RYK is specifically up-regulated in proliferating cells in the intestine and uterus [31] and in malignant ovarian tumors [37], suggesting dominant expression of RYK in replicating populations. Compared with hematopoietic or epithelial tissues, the pattern of expression of RYK in the CNS observed in the present study is unique, since it was detected in both immature cells, such as nestin-positive cells and O-2A progenitor cells, and in mature cells, such as neurons and oligodendrocytes. The most interesting issue concerns the function of RYK, however, we were unable to simply relate this expression pattern to a single phenomenon, such as replication or migration. Instead, the expression of RYK in various cell types suggests multiple functions of this molecule during the formation of the CNS. The discovery of expression of RYK in oligodendrocytes was unexpected, since we hardly observed any RYK mRNA in the myelin of postnatal cerebrum. The mRNA signals in the white matter of the late embryonic cerebrum [13], however, may represent expression of RYK in oligodendrocytes or their progenitors. Detection of RYK in the ventral spinal cord at around E15, the site and time oligodendrocyte progenitors first arise in the rat CNS [38], may also represent RYK expression in these cells. Expression in both neurons and oligodendrocytes or their progenitors may suggest a function of RYK during myelin formation. However, it is difficult to discuss the significance of RYK in type-2 astrocytes, because the properties of type-2 astrocytes in vivo are controversial [23]. The high expression in type-2 astrocytes, but not in type-1 astrocytes, however, may be related to biochemical differences between these cell types, such as in surface molecules and ion channels [19]. By contrast, the failure to detect RYK in microglia was unexpected, since we observed RYK mRNA in a rat microglia culture in a preliminary study (unpublished). High expression of RYK in hematopoietic cells, which may share the same origin as microglia, also suggested the possibility of expression in microglia. In view of its stage-specific expression in hematopoietic cells, expression of RYK in microglia may be highly regulated, and we did not detect it under the culture conditions described. In Drosophila, expression of the RYK homolog Lio / Drl has been independently identified in neurons [3,18] and glia [32], and Lio / Drl in both types of cells are associated with the control of axon outgrowth. Expression of RYK in both neurons and glia may reflect a role of RYK analogous to that of Lio / Drl during mammalian CNS development. Although the predominant expression of RYK in the cell body of neurons conflicts with this hypothesis, our preliminary observations suggest that it is expressed on
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Fig. 6. Double-immunostaining of neural stem cell line MNS-70 at the differentiated stage. Cells incubated in the differentiation culture for 5 days were immunostained for anti-RYK antibody (green) and anti-MAP2 antibody (red; A), anti GFAP-antibody (red; B), anti-nestin antibody (red; C), or A2B5 antibody (red; D). Colocalization of RYK and MAP2 can be observed (A), but staining for RYK and GFAP is segregated (B). Arrows in C indicate colocalization of RYK and nestin. Some A2B5-positive cells are detected, and some of them are also RYK-positive (arrows, D). Scale bars represent 50 mm.
neurites depending on the culture conditions, and expression in the white matter of developing spinal cord may represent expression in growing axons. Association of RYK with Eph receptors [11], which play a role in axon pathfinding [24,35], may mean that RYK cooperates with Eph receptors in this event, although the physiological significance of this binding has to be further elucidated. Since AF-6 localizes on synapses [5], binding of RYK with AF-6 [11] also suggests an attractive model in which RYK plays a role in axon pathfinding. It is also intriguing that Lio / Drl may be functionally associated with Dek [29] or Canoe [17], Drosophila counterparts of the Eph receptor and AF-6, respectively. The RYK expressed in nestin-positive cells may have as yet unidentified functions, since expression of Lio / Drl has never been reported in neural stem cell or progenitor cells. Expression of this signaling molecule in replicating cells, such as neural stem cells and ES cells, would suggest a function in proliferation. A recent report shows a role of Eph / ephrin signaling in the migration and proliferation of adult neural stem cells [7], and the expression of RYK, a possible partner of Eph receptors, in neural stem cells or neural progenitor cells implies a cooperative function of the RYK and Eph pathway during these events. However, possible roles of RYK through association with other membrane-bound or cytoplasmic proteins cannot be ruled out, and RYK may participate in other key steps in CNS development. The tight regulation of RYK expression, in particular, suggests that it may be involved in cell differentiation. In conclusion, RYK is expressed in neural stem cells or neural progenitor cells, and neurons of the embryonic forebrain and spinal cord. Analysis of primary cultures from forebrain showed its expression in all neurons, and some O-2A progenitor cells, type-2 astrocytes, and oligodendrocytes, but not in type-1 astrocytes or microglia. Neural stem cell line MNS-70 showed a similar profile of expression. The distribution of expression of RYK in both immature and mature cells is quite intriguing, but confusing in terms of identifying its role. Although the function of RYK in mammalian CNS is still unclear, the results of the present study suggest some possible roles of this molecule. Further analysis of loss or gain of expression would supply information about the signaling mechanisms associated with it. We are also interested in learning how RYK expression is regulated in a cell-type-dependent manner, and finding the answer should help clarify its function.
Acknowledgements We are grateful to Dr. Shun Nakamura for kindly providing the MNS-70 cells. This work was supported by the Ministry of Health, Labour and Welfare of Japan and the Japan Health Sciences Foundation.
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