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SynCAM, a member of the immunoglobulin superfamily, in the developing nervous system
Developmental Brain Research 154 (2005) 199 – 209 www.elsevier.com/locate/devbrainres
Research report
Distribution of RA175/TSLC1/SynCAM, a member of the immunoglobulin superfamily, in the developing nervous system Eriko Fujita1, Koko Urase1,2, Akiko Soyama, Yoriko Kouroku, Takashi Momoi* Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan Accepted 20 October 2004 Available online 28 December 2004
Abstract RA175 is a new member of the immunoglobulin superfamily with trans interaction activity, and it plays a role as a tumor suppressor in lung carcinoma (TSLC1) and as a cell adhesion molecule promoting the formation of functional synapses (SynCAM). Little is known about the biological function of RA175/TSLC1/SynCAM neural network formation during neurogenesis. We examined the distribution and colocalization of the RA175/TSLC1/SynCAM protein with other members of the immunoglobulin superfamily such as NCAM, L1, and TAG-1 in the mouse developing nervous system. Consistent with the expression of RA175/TSLC1/SynCAM mRNA, the protein was localized in the brain neuroepithelium at embryonic day (E) 9.5, neural crest at E10.5, motor neurons at E10.5, and olfactory epithelium at E16.5. In contrast with its mRNA, the protein was intensely detected on the fasciculated axons in the floor plates, ventral root, and dorsal funiculus in the E10.5–11.5 spinal cord and colocalized with NCAM and L1 on the ventral root and dorsal funiculus and partly colocalized with TAG-1 on the commissural axons and dorsal funiculus. In the E13.5–15.5 brain, RA175/TSLC1/SynCAM colocalized with NCAM and L1 on the developing thalamocortical fibers from the internal capsule (IC) and partly colocalized with TAG-1 on the cortical efferent axons in the intermediate zone (IZ). RA175/TSLC1/SynCAM was localized on the axons of some of the cortical neurons cultured in vitro. Thus, in addition to cell adhesion activity in the neuroepithelium and the synapses, RA175/TSLC1/SynCAM may be involved in neuronal migration, axon growth, pathfinding, and fasciculation on the axons of differentiating neurons. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Axon guidance mechanisms and pathways Keywords: Cell adhesion molecule; Axon growth; Tangential migration; SynCAM; RA175; TSLC1; TAG-1; L1; NCAM
1. Introduction RA175 is a new member of the immunoglobulin superfamily that is preferentially expressed during retinoic acid (RA)-induced neuronal differentiation of P19 embryonal carcinoma (EC) cells [13,26]. RA175 is a membrane glycoprotein with significant amino acid sequence similarity to neuronal cell adhesion molecule (NCAM) and Nectin * Corresponding author. Fax: +81 42 346 1754. E-mail address: [email protected] (T. Momoi). 1 These two authors equally contributed to this work. 2 Present address: Department of Biology, School of Medicine Tokyo Women’s Medical University, Shinjuku, Tokyo 162-8666, Japan. 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.10.015
[39]. RA175 is a Ca++-independent cell adhesion molecule with homophilic trans-cell adhesion activity [14]. TSLC1, a human tumor suppressor gene located on chromosome 11q23.2, is the human orthologue of RA175 [16,18,22]. Defects in this gene promote the metastasis of lung carcinoma. In the developing lung epithelium, RA175/TSLC1 is preferentially localized in the lateral membrane of the polarized cells lining the lumen [14]. RA175/TSLC1 has been shown to be a cell adhesion molecule promoting the formation of functional synapses (SynCAM) [6]. The intracellular domain of RA175/ TSLC1/SynCAM has sequence similarity to contactinassociated protein 2 (Caspr2), a member of the neurexin family [14,32]. These molecules contain the respective
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short amino acid sequences, EWLT and EYFI, at their Cterminus, which serve as binding sites for the type II PDZ domain [4,5,29]. RA175/TSLC1/SynCAM interacts with calcium/calmodulin-dependent serine protein kinase (CASK) via EYFI at the presynaptic membrane to form functional synapses [6]. In the developing central nervous system, the neuroepithelium exhibits cell polarity, detaches from the basal membrane, and differentiates into neural precursor cells. Upon differentiation into neurons, these cells form axon and dendrite structures for synaptic interaction. In the developing spinal cord, the commissural cell fibers extend toward the ventral midline from the neurons located in the dorsal region of the spinal cord. In the cortex of the developing brain, the cortical neurons, born early to form the preplate, function as a scaffold for the assembly of the cortical architecture [1,9,23,25,27]. Other cortical neurons, born later, migrate radially and insert themselves into the preplate to generate the cortical plate, which splits the preplate into the marginal zone and the subplate [23,25]. Pioneer neurons derived from the preplate guide the thalamocortical afferent axons into the cortex and the cortical neurons of the cortical plates extend efferent axons toward their targets through intermediate zone (IZ) [1,9]. Members of the immunoglobulin superfamily with cell adhesion activity promote neuronal migration, axonal growth, fasciculation, pathfinding, and synaptic formation in the developing nervous system and are involved in the formation of neural networks [11]. NrCAM and TAG-1 (axonin), members of the immunoglobulin superfamily, are expressed on the commissural axons that outgrow ipsilaterally to the floor plate [34,35]. NCAM, SC1, and L1 (NgCAM), other members of the immunoglobulin superfamily, and TAG-1 are localized at the bundled axons in the floor plates and at the dorsal funiculus [7,12,24,36]. L1 and TAG-1 are localized on the axons in the corticofugal fiber system including the IZ and the thalamocortical pathway (TC), and the latter is involved in the tangential migration of interneurons from the medial ganglionic eminence (MGE) [10]. RA175 mRNA is expressed in the neuroepithelium during brain morphogenesis. Its expression increases in various regions of the brain, including the cortex, hippocampus, thalamus, and amygdala during neurogenesis, and then decreases before birth [39]. RA175 mRNA is transiently expressed in the motor neurons, neural crest, and dorsal root ganglia (DRG) in the developing spinal cord. However, little is known about the involvement of RA175/TSLC1/SynCAM in the neuronal migration, axonal growth, fasciculation, and pathfinding during neural network formation. To clarify the biological role of RA175/TSLC1/SynCAM in neural network formation during neurogenesis, we examined the distribution of RA175/TSLC1/SynCAM in the developing spinal cord and in the corticofugal fiber
system including IZ and TC of the developing brain by in situ hybridization (ISH) and immunostaining.
2. Materials and methods 2.1. Immunohistochemical staining Antibodies of RA175/TSLC1/SynCAM, antibodies against a peptide corresponding to the C-terminal region of RA175/TSLC1/SynCAM, were prepared as described previously [14] and used for the immunostaining of the RA175/TSLC1/SynCAM in mouse developing nervous system. The mouse embryos at embryonic day (E)9.5, E10.5, E11.5, E12.5 E14.5, E15.5, and E16.5 were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 8C overnight and then soaked in 30% sucrose/PBS at 4 8C overnight and embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetec. Tokyo) and were frozen. Frozen sections (10 Am thick) of embryos were cut on a cryostat and attached to slides coated with Vectabond reagent (Vector Laboratories, Burlingame, CA) and incubated with rabbit anti-RA175/TSLC1/SynCAM and or rat anti-NCAM and anti-L1 (Chemicon International, Temecula, CA), mouse anti-TAG-1 (4D7, Developmental Studies Hybridoma Bank, Iowa City, IA) in PBS containing 0.1% skim milk and 0.1% Triton X-100 at 4 8C for 2 days as described previously. Subcellular localization of RA175/TSLC1/SynCAM in the neurons was also examined by immunostaining. Neurons and astrocytes were isolated from the brain of E15.5 mouse embryos described previously [3] and cultured in Dulbecco’s Modified Eagle’s Medium (Sigma) containing 4500 mg/l glucose, 10% Fetal Bovine Serum in the presence or absence of 10 AM Ala-C for 10 days, respectively. Neurons and astrocytes were fixed at 4% paraformaldehyde and were immunostained with anti-RA175/TSLC1/SynCAM, mouse Tuj-1 (Tubulin h-III, Neuromics Antibodies Northfield, MN), mouse anti-GFAP, anti-Map-2 (Sigma, ST, Louis MO), and mouse anti-Tau (Upstate Biotechnology Lake Placid, NY). Anti-RA175/TSLC1/SynCAM immunoreactivity was detected by FITC-labeled goat anti-rabbit IgG (Leinco Technologies, St. Louis, MO). Rat anti-NCAM and antiL1 immunoreactivities were detected by goat anti-rat IgG Alexa Fluor 568 (Molecular Probes, Eugene, OR). Mouse anti-TAG-1, anti-Tuj-1, anti-GFAP, anti-Map-2, and antiTau immunoreactivities were detected by goat anti-mouse IgG Alexa Fluor 568 (Molecular Probes). Immunoreactivity was viewed using a confocal laser-scanning microscope (CSU-10, Yokogawa, Tokyo, Japan). 2.2. In situ hybridization (ISH) The EcoRI fragment of RA175/TSLC1/SynCAM in the pGEM-T vector was subcloned into pBluescript SK(+)
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vectors in both the sense and anti-sense orientations as described previously [39]. Digoxigenin (DIG)-labeled RA175/TSLC1/SynCAM mRNA probes were transcribed from XhoI-cut constructs by T3 RNA polymerase (DIG RNA Labeling Kit T3, Boehringer Mannheim, Mannheim, Germany) in vitro according to the manufacturer’s instructions. Frozen sections (10 Am thick) of embryos at various developmental stages were treated with proteinase K (1 Ag/ml) at 37 8C for 10 min, refixed in 4% paraformaldehyde, and hybridized overnight with the DIG-labeled RNA probes. Hybridized RNA was detected using alkaline phosphatase-conjugated anti-DIG according to the procedure described by Wilkinson [40].
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At later stage [i.e., E14.5 and E16.5], RA175/TSLC1/ SynCAM mRNA was decreased (Fig. 1Q), and protein was still detected on the ventral commissural axons in the floor plate and in the ventral fiber tract (Fig. 1R, double asterisks). We compared the distribution of RA175/TSLC1/SynCAM with those of other members of immunoglobulin superfamily such as TAG-1 and L1 at E11.0 (Fig. 2). TAG-1 was expressed on the dorsal funiculus and the commissural cell fiber (Fig. 2B, E) and partly colocalized with RA175/ TSLC1/SynCAM (Fig. 2C, F). L1 was expressed in the dorsal funiculus and ventral fiber tract but slightly in the floor plate (Fig. 2H, K). L1 colocalized with RA175/ TSLC1/SynCAM on the dorsal funiculus and ventral fiber tract but not in the ventral commissural fibers in the floor plate (Fig. 2I, L).
3. Results 3.1. Distribution of RA175/TSLC1/SynCAM in the developing spinal cord We examined the distribution of RA175/TSLC1/ SynCAM in the developing spinal cord (Fig. 1). At E9.5, both RA175/TSLC1/SynCAM mRNA and proteins were expressed mainly in somites (open arrowheads) and weakly in the neural tube (Fig. 1A–D). At E10.5, RA175/TSLC1/SynCAM mRNA was expressed mainly in the motor neurons and in the neural crest (Fig. 1E, closed arrowheads). In contrast, the RA175/TSLC1/ SynCAM protein was localized, by immunostaining, to axons, in addition to the neural crest (closed arrowheads) and motor neurons (Fig. 1F). RA175/TSLC1/SynCAM as well as NCAM was expressed on the commissural axons and prominently enriched in the ventral commissure (Fig. 1F–H, thick arrows), where the commissural axons transverse the floor plate. RA175/TSLC1/SynCAM and NCAM were also strongly expressed on the ventral root (Fig. 1F–H, single asterisk) which fasciculates the axons from motor neurons. There was a strong contrast between RA175/TSLC1/SynCAM and NCAM immunoreactivity in the neural crest at this stage; NCAM was not detected in the neural crest at this stage (Fig. 1G, closed arrowheads). At E11.5–12.5, RA175/TSLC1/SynCAM mRNA was still expressed in the motor neurons and in the DRG neurons developed from the neural crest (Fig. 1I, M). In contrast, RA175/TSLC1/SynCAM immunostaining was at a low level in the cell bodies of motor neurons and DRG neurons but positive in their axons (Fig. 1J, N). Strong RA175/TSLC1/SynCAM immunoreactivity was detected in the dorsal funiculus (Fig. 1J, N, thin arrows), which fasciculates bundles of sensory axons from the DRG within the spinal cord. RA175/TSLC1/SynCAM immunoreactivity was still at a high level on the ventral commissural axons in the floor plate and ventral root and colocalized with NCAM (Fig. 1J–L, N–P, thick arrows).
3.2. Distribution of RA175/TSLC1/SynCAM in the developing brain RA175/TSLC1/SynCAM mRNA expression was sparsely detected in the neuroepithelium of the forebrain and hindbrain at E9.5 (Fig. 3A) [39]. Consistent with RA175/TSLC1/SynCAM mRNA, RA175/TSLC1/SynCAM protein was also sparsely distributed in the neuroepithelium and localized at adherent regions of neuroepithelial cells (Fig. 3B). During brain development (E13.5–16.5), however, there was a striking contrast in expression between RA175/TSLC1/SynCAM mRNA and protein; i.e., RA175/TSLC1/SynCAM mRNA was detected in the cell bodies of neurons in various regions of the developing brain, while the protein was detected in the extending axons. At E13.5–E15.5, the thalamocortical axons extend into the cortex from the internal capsule (IC) and cortical neurons extend the efferent fibers projecting toward their targets through IZ. At E14.5, RA175/TSLC1/SynCAM mRNA was expressed mainly in the thalamus and developing cortex (Fig. 3C), while RA175/TSLC1/SynCAM protein was positive mainly in the extending thalamocortical axons from the IC (Fig. 3D). At E16.5, RA175/TSLC1/SynCAM mRNA was highly expressed in various regions of the brain, including the cortex, thalamus, and habenular nucleus (Fig. 3F). In the cortex, most RA175/TSLC1/SynCAM mRNA was expressed in the cortical plate, whereas RA175/TSLC1/SynCAM protein was localized to the axons in the IZ and the axons in the TC (Fig. 3G). Most RA175/TSLC1/ SynCAM protein colocalized with NCAM on the thalamocortical axons extending into IZ (Fig. 3H–J) from the IC (Fig. 3K–M). Neural adhesion molecules of the immunoglobulin superfamily, TAG-1 and L1, are expressed by cortical efferent and afferent fibers, respectively [17,21,42]; i.e., the TAG-1-positive efferent axons of the IZ are tangentially oriented, projecting towards the IC, while the L1 afferent fibers extend into the IZ through the TC during
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Fig. 1. Distribution of RA175/TSLC1/SynCAM mRNA and protein the developing spinal cord (E9.5–E14.5). Transverse sections of mouse embryos at E9.5 (A– D), E10.5 (E–H), E11.5 (I–L), E12.5 (M–P), and E14.5 (Q–T) were hybridized with antisense probes for RA175/TSLC1/SynCAM mRNA (A, E, I, M, Q) and immunostained with anti-RA175/TSLC1/SynCAM (B, F, J, N, R), anti-NCAM (C, G, K, O, S), and merged (D, H, L, P, T). mo, motor neurons; DRG, dorsal root ganglia neurons. Open arrowheads and closed arrowheads indicate somite and neural crest, respectively, and thick and thin arrows indicate the ventral commissure and the dorsal funiculus, respectively. Single and double asterisks show ventral root and ventral fiber track, respectively. Bars indicate 100 Am. RA175/TSLC1/SynCAM colocalized with NCAM in the dorsal funiculus, axons of the ventral commissure and ventral root with strong intensity.
cortical development [17] (Fig. 4A). As it is difficult to discriminate afferents and efferents intermingled in the IZ at E16.5 (Fig. 3H–J) [10], we examined the immunor-
eactivity of RA175/TSLC1/SynCAM on the TAG-1positive cortical efferent axons outgrowing in the IZ and on the L1-positive thalamocortical afferent axons extend-
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Fig. 2. Colocalization of RA175/TSLC1/SynCAM and TAG-1 or L1 in the neural tube at E11.0. Transverse sections of mouse embryos at E11.0 were immunostained with anti-RA175/TSLC1/SynCAM in the absence (A, D, G, J) or presence (M) of 10 Ag/ml peptide of C-terminal region of RA175, anti-TAG-1 (B and E), and anti-L1 (H and K). Panels (C, F, I, L) are merge. Panels (D, E, F) are rectangular region in panel (C). Panels (J, K, L) are horizontal section cut at broken line in panels (G, H, I). RA175/TSLC1/SynCAM and TAG-1 were partly colocalized at the dorsal funiculus and the commissural fibers (C, F). RA175/ TSLC1/SynCAM and L1 were colocalized at dorsal funiculus and ventral fiber tract but not at the ventral commissure (I, L). Panels (N, O) were immunostaining with goat FITC-labeled rabbit IgG and goat anti-rat IgG Alexa Fluor 568, respectively. Fluorescences in panels (M, N, O) were due to the cells with autofluorescence. Bars indicate 100 Am.
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Fig. 3. Distribution of RA175/TSLC1/SynCAM mRNA and protein in the developing brain at E9.5, E14.5, and E16.5. RA175/TSLC1/SynCAM mRNA expression and protein distribution in the hindbrain at E9.5 (A, B) and in the brain at E14.5 (C, D, E) and E16.5 (F, G) were examined by in situ hybridization (A, C, F) and immunostaining using anti-RA175/TSLC1/SynCAM in the absence (B, D, G) or presence (E) of the peptide of C-terminal region of RA175. Panel (B) is the rectangular region in (A). Bars in panels (A, C, D, E, F, G) indicate 200 Am. Bar in panel (B) indicates 25 Am. Fb, forebrain; Hb, hindbrain; cx, cortex; th, thalamus; hi, hippocampus; hb, habenular nucleus. RA175/TSLC1/SynCAM with NCAM colocalized in the TC and IZ (TC/IZ) (H, I, J) and the IC (K, L, M). (H, K) Anti-RA175/TSLC1/SynCAM immunoreactivity; (I, L) anti-NCAM; (J, M) merge.
ing from IC at E15.5 (Fig. 4). Most RA175/TSLC1/ SynCAM protein colocalized with L1 on the TC and the IC (Fig. 4F–H, L–N). RA175/TSLC1/SynCAM was also partly overlapped with TAG-1 in the IZ (Fig. 4C–E) but not in the IC (Fig. 4I–K).
RA175/TSLC1/SynCAM was clustered and patched on the axons and surface of the cell body (Fig. 6G–I, J–L, arrowheads).
4. Discussion 3.3. Distribution of RA175/TSLC1/SynCAM in the developing epithelia of sensory organs RA175/TSLC1/SynCAM mRNA was highly expressed in the olfactory system and in the retina at E16.5 (Fig. 5A). In the retina, both RA175/TSLC1/SynCAM and NCAM were strongly coexpressed in the nerve fiber layer (Fig. 5B–D, arrowheads), with relatively weak staining in the inner nuclear layer (arrows). RA175/ TSLC1/SynCAM colocalized with NCAM in the nerve fibers of olfactory sensory neurons projecting to the olfactory bulb (Fig. 5E–G). In contrast with NCAM, RA175/TSLC1/SynCAM was detected in the adherent region in the olfactory epithelium. 3.4. Subcellular localization of RA175/TSLC1/SynCAM in the neuron RA175/TSLC1/SynCAM was expressed in the Tuj-1positive cortical neurons cultured in vitro (Fig. 6A–C) and preferentially localized on the axons but not expressed in the GFAP-positive astrocytes (Fig. 6D–F). The subcellular localization of RA175/TSLC1/SynCAM in cortical neurons was examined by immunostaining (Fig. 6G, J). RA175/ TSLC1/SynCAM was preferentially localized on the Map2-positive axons of the differentiated cortical neurons rather than the cell body (Fig. 6G–I). Some of the
4.1. Change in the localization of RA175/TSLC1/SynCAM There was a great contrast between the localization of the RA175/TSLC1/SynCAM protein in the epithelium, including the neuroepithelium and the nervous system after neurogenesis (Figs. 1–6). In the epithelium, the expression of RA175/TSLC1/SynCAM mRNA and protein were detected in the cell bodies, and RA175/TSLC1/SynCAM protein was localized in the adherent region of epithelial cells. In the developing nervous system, regions expressing RA175/TSLC1/SynCAM mRNA and protein were apparently not consistent. RA175/TSLC1/SynCAM was preferentially expressed on the axons of the differentiated neurons. RA175/TSLC1/SynCAM may play different biological roles as cell adhesion molecules at each neuronal differentiation step; that is, RA175/TSLC1/SynCAM may be involved in the cell adhesion processes on the axons of the differentiating neurons in addition to those in the neuroepithelium [14] and at synapses of mature neurons [6]. 4.2. RA175/TSLC1/SynCAM in the developing spinal cord The floor plate can exert strong influences, both attractive and repulsive, over axon projections [8,37]. The commissural axons are attracted by the floor plate cells, and their early projections are directed ventrally
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Fig. 4. Localization of RA175/TSLC1/SynCAM and L1 or TAG-1 in the IZ, TC, and IC. Coronal sections of the E15.5 mouse brain were double immunostained with anti-L1 (green) and TAG-1 (red) (A), anti-RA175/TSLC1/SynCAM (green) and TAG-1 (red) (B, C–E, I–K), and anti-RA175/TSLC1/ SynCAM (green) and L1 (red) (F–H, L–N). (C–E) IZ, (F–H) TC, (I–K, L–N) IC. (C, F, I, L) Anti-RA175/TSLC1/SynCAM (green), panels (D, J) are anti-TAG1 (red), panels (G, M) are anti-L1 (red), panels (E, K) are merge of anti-RA175/TSLC1/SynCAM and anti-TAG-1, and panels (H, N) are merge of anti-RA175/ TSLC1/SynCAM and anti-L1. Bars in panels (B, H, K, N) indicates 100 Am. Bar in panel (H) indicates 50 Am.
toward the floor plate and fasciculated at the ventral commissure, where they transverse the floor plate. RA175/ TSLC1/SynCAM immunoreactivity was strongly localized
on the ventral commissural axons in the floor plate and weakly localized on the commissural axons directed toward the floor plate (Fig. 1).
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Fig. 5. Localization of RA175/TSLC1/SynCAM and NCAM in the retina and olfactory organs at E16.5. Coronal section of mouse head at E16.5 was hybridized with antisense probes for RA175/TSLC1/SynCAM mRNA (A) and immunostained with anti-RA175/TSLC1/SynCAM (B, E) and anti-NCAM (C, F). Panels (D, G) are merge. RA175/TSLC1/SynCAM mRNA was highly expressed in the retina and olfactory epithelia. (B, C, D) Double immunostaining of the retina (coronal section) with anti-RA175/TSLC1/SynCAM (B; green) and anti-NCAM (C; red). Arrowheads indicate the nerve fibers and arrows indicate the inner nuclear layer. (E, F, G) Double immunostaining of the olfactory epithelium (sagittal section) with anti-RA175/TSLC1/SynCAM (E; green) and antiNCAM (F; red). Bars in panels A and E indicate 1 mm and 100 Am, respectively. ob, olfactory bulb; oe, olfactory epithelium.
Cell adhesion molecules (CAMs) have important roles in axon outgrowth and their fasciculation via homophilic or heterophilic interactions [11,19,41]. TAG-1 is expressed on the commissural axons, in particular, on the ventral commissural axons in the floor plate [24] and NCAM, SC1, L1, and TAG-1 are also detected on the fasciculated axons at the dorsal funiculus and the ventral root [7,24,36]. RA175/TSLC1/SynCAM was also prominently expressed on the ventral commissural axons in the floor plate and the fasciculated axons at the dorsal funiculus (Fig. 1). Thus, it is likely that RA175/TSLC1/SynCAM, as well as other CAM proteins, is involved in the outgrowth of the commissural axons attracted by the floor plate and may also play a role in the fasciculation of the axons at the floor plate, dorsal funiculus, and ventral root in the developing spinal cord. 4.3. RA175/TSLC1/SynCAM in the developing brain Two types of cortical migration of neurons have been shown [30]; one is a radial migration by which postmitotic neurons migrate from the ventricular zone and form the preplate. The other is nonradial migration of the interneurons [1,9,23,25,27]. Axons can provide a substratum for nonradial neuronal migration in the developing central nervous system, including the cerebral cortex [20,33]. The developing axons of the IZ and TC have been shown to be the substratum for the GABAergic interneurons tangentially migrating from the MGE to the cortex [2,28,31]. RA175/TSLC1/SynCAM was localized not only on the axons of the TC but also in the IZ. Most of the RA175/TSLC1/SynCAM colocalized with L1 on the TC and at the IC (Fig. 4F–H, L–N), suggesting that RA175/TSLC1/SynCAM is expressed on
the cortical afferent fibers extending from the IC into the cortex. Some of the RA175/TSLC1/SynCAM immunoreactivity colocalized with TAG-1 on the cortical efferent axons outgrowing in the IZ (Fig. 4B–E), suggesting that RA175/ TSLC1/SynCAM is also expressed on the developing cortical efferent axons in the IZ. This was supported by the localization of RA175/TSLC1/SynCAM on the axons of the cortical neurons (Fig. 6A–C). Furthermore, RA175/ TSLC1/SynCAM mRNA and proteins were expressed in the various regions of the brain (Fig. 3), also suggesting that RA175/TSLC1/SynCAM is localized on the axons of the neurons including the efferents and afferents of the cortical neurons and play a role in the pathfinding and migration in the cooperation with other cell adhesion molecules such as NCAM, L1, and TAG-1. TAG-1 is involved as a cellular substrate in the pathfinding of the GABAergic interneurons in the tangential migration to the neocortex [10]. However, TAG-1deficient mice do not show inhibition of GABAergic interneuron migration histologically [15], suggesting that other cell adhesion molecules also play as their cellular substrates. RA175/TSLC1/SynCAM located on the axons of the IZ and TC may be one of the candidate adhesion molecules involved in the pathfinding of the GABAergic interneurons as a substratum. This possibility remains to be clarified by experiments with RA175/TSLC1/SynCAMdeficient mice. 4.4. Subcellular localization of RA175/TSLC1/SynCAM in the neuron In differentiated cortical neurons cultured in vitro, RA175/TSLC1/SynCAM was localized in the cluster or
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Fig. 6. Subcellular localization of RA175/TSLC1/SynCAM in the cortical neurons. Localization of RA175/TSLC1/SynCAM in the neurons was examined by immunostaining using anti-RA175/TSLC1/SynCAM (A, D, G, J; green) and anti-Tuj-1 (B; red), anti-GFAP (E; red), anti-Map-2 (H; red) or anti-Tau (K; red), respectively. Panels (C, F, I, L) are merge. GFAP-positive astrocytes are RA175/TSLC1/SynCAM-negative (rectangular in panels D, E, F). Arrowheads indicate RA175/TSLC1/SynCAM immunoreactivity in the cluster or patched form on the axons. Bars (I, C) indicate 20 Am. Bars (C, F) indicate 100 Am.
patched form on the axons and dendrites and also on the surface of the cell body (Fig. 6). At present, the molecular mechanism of the localization of RA175/TSLC1/SynCAM on axons is not clear. CAMs have biological functions and distributions distinct from other cell adhesion molecules via association with cytoplasmic proteins at the peripheral membrane of the neurons. The intracellular domain of RA175/TSLC1/SynCAM has the putative binding domain of ERM (ezrin, radixin, and moesin), a member of the band 4.1 superfamily, shared with Caspr2 [14,32]. Caspr2 associates with the cytoskeleton via the band 4.1-binding protein and localizes on the axons [38]. Thus, this putative band 4.1-binding region may contribute to the stabilization of the localization of RA175/ TSLC1/SynCAM on the axons. In addition, RA175/ TSLC1/SynCAM interacts with CASK at the presynaptic membrane via EYFI, the binding site for the type II PDZ domain at the C-terminus [29]. RA175/TSLC1/SynCAM
may also be associated with proteins with PDZ domain on the axons or dendrites and clustered to form functional synapses. In conclusion, the RA175/TSLC1/SynCAM protein is localized on axons in many fiber systems. RA175/TSLC1/ SynCAM, as well as TAG-1, may be involved in the guidance of commissural axons in the floor plate, the fasciculation of axons, axonal pathfinding, and the tangiential migration of neuronal cells.
Acknowledgments This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and by Research Grant 14A-1 for Nervous and Mental Disorders and Research on Brain Science from the Ministry of Health and Welfare of Japan, the Human Science Foundation. E.F.
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and Y.K. are Research Fellows of the Japan Society for the Promotion of Science. [19]
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Research report
Distribution of RA175/TSLC1/SynCAM, a member of the immunoglobulin superfamily, in the developing nervous system Eriko Fujita1, Koko Urase1,2, Akiko Soyama, Yoriko Kouroku, Takashi Momoi* Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan Accepted 20 October 2004 Available online 28 December 2004
Abstract RA175 is a new member of the immunoglobulin superfamily with trans interaction activity, and it plays a role as a tumor suppressor in lung carcinoma (TSLC1) and as a cell adhesion molecule promoting the formation of functional synapses (SynCAM). Little is known about the biological function of RA175/TSLC1/SynCAM neural network formation during neurogenesis. We examined the distribution and colocalization of the RA175/TSLC1/SynCAM protein with other members of the immunoglobulin superfamily such as NCAM, L1, and TAG-1 in the mouse developing nervous system. Consistent with the expression of RA175/TSLC1/SynCAM mRNA, the protein was localized in the brain neuroepithelium at embryonic day (E) 9.5, neural crest at E10.5, motor neurons at E10.5, and olfactory epithelium at E16.5. In contrast with its mRNA, the protein was intensely detected on the fasciculated axons in the floor plates, ventral root, and dorsal funiculus in the E10.5–11.5 spinal cord and colocalized with NCAM and L1 on the ventral root and dorsal funiculus and partly colocalized with TAG-1 on the commissural axons and dorsal funiculus. In the E13.5–15.5 brain, RA175/TSLC1/SynCAM colocalized with NCAM and L1 on the developing thalamocortical fibers from the internal capsule (IC) and partly colocalized with TAG-1 on the cortical efferent axons in the intermediate zone (IZ). RA175/TSLC1/SynCAM was localized on the axons of some of the cortical neurons cultured in vitro. Thus, in addition to cell adhesion activity in the neuroepithelium and the synapses, RA175/TSLC1/SynCAM may be involved in neuronal migration, axon growth, pathfinding, and fasciculation on the axons of differentiating neurons. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Axon guidance mechanisms and pathways Keywords: Cell adhesion molecule; Axon growth; Tangential migration; SynCAM; RA175; TSLC1; TAG-1; L1; NCAM
1. Introduction RA175 is a new member of the immunoglobulin superfamily that is preferentially expressed during retinoic acid (RA)-induced neuronal differentiation of P19 embryonal carcinoma (EC) cells [13,26]. RA175 is a membrane glycoprotein with significant amino acid sequence similarity to neuronal cell adhesion molecule (NCAM) and Nectin * Corresponding author. Fax: +81 42 346 1754. E-mail address: [email protected] (T. Momoi). 1 These two authors equally contributed to this work. 2 Present address: Department of Biology, School of Medicine Tokyo Women’s Medical University, Shinjuku, Tokyo 162-8666, Japan. 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.10.015
[39]. RA175 is a Ca++-independent cell adhesion molecule with homophilic trans-cell adhesion activity [14]. TSLC1, a human tumor suppressor gene located on chromosome 11q23.2, is the human orthologue of RA175 [16,18,22]. Defects in this gene promote the metastasis of lung carcinoma. In the developing lung epithelium, RA175/TSLC1 is preferentially localized in the lateral membrane of the polarized cells lining the lumen [14]. RA175/TSLC1 has been shown to be a cell adhesion molecule promoting the formation of functional synapses (SynCAM) [6]. The intracellular domain of RA175/ TSLC1/SynCAM has sequence similarity to contactinassociated protein 2 (Caspr2), a member of the neurexin family [14,32]. These molecules contain the respective
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short amino acid sequences, EWLT and EYFI, at their Cterminus, which serve as binding sites for the type II PDZ domain [4,5,29]. RA175/TSLC1/SynCAM interacts with calcium/calmodulin-dependent serine protein kinase (CASK) via EYFI at the presynaptic membrane to form functional synapses [6]. In the developing central nervous system, the neuroepithelium exhibits cell polarity, detaches from the basal membrane, and differentiates into neural precursor cells. Upon differentiation into neurons, these cells form axon and dendrite structures for synaptic interaction. In the developing spinal cord, the commissural cell fibers extend toward the ventral midline from the neurons located in the dorsal region of the spinal cord. In the cortex of the developing brain, the cortical neurons, born early to form the preplate, function as a scaffold for the assembly of the cortical architecture [1,9,23,25,27]. Other cortical neurons, born later, migrate radially and insert themselves into the preplate to generate the cortical plate, which splits the preplate into the marginal zone and the subplate [23,25]. Pioneer neurons derived from the preplate guide the thalamocortical afferent axons into the cortex and the cortical neurons of the cortical plates extend efferent axons toward their targets through intermediate zone (IZ) [1,9]. Members of the immunoglobulin superfamily with cell adhesion activity promote neuronal migration, axonal growth, fasciculation, pathfinding, and synaptic formation in the developing nervous system and are involved in the formation of neural networks [11]. NrCAM and TAG-1 (axonin), members of the immunoglobulin superfamily, are expressed on the commissural axons that outgrow ipsilaterally to the floor plate [34,35]. NCAM, SC1, and L1 (NgCAM), other members of the immunoglobulin superfamily, and TAG-1 are localized at the bundled axons in the floor plates and at the dorsal funiculus [7,12,24,36]. L1 and TAG-1 are localized on the axons in the corticofugal fiber system including the IZ and the thalamocortical pathway (TC), and the latter is involved in the tangential migration of interneurons from the medial ganglionic eminence (MGE) [10]. RA175 mRNA is expressed in the neuroepithelium during brain morphogenesis. Its expression increases in various regions of the brain, including the cortex, hippocampus, thalamus, and amygdala during neurogenesis, and then decreases before birth [39]. RA175 mRNA is transiently expressed in the motor neurons, neural crest, and dorsal root ganglia (DRG) in the developing spinal cord. However, little is known about the involvement of RA175/TSLC1/SynCAM in the neuronal migration, axonal growth, fasciculation, and pathfinding during neural network formation. To clarify the biological role of RA175/TSLC1/SynCAM in neural network formation during neurogenesis, we examined the distribution of RA175/TSLC1/SynCAM in the developing spinal cord and in the corticofugal fiber
system including IZ and TC of the developing brain by in situ hybridization (ISH) and immunostaining.
2. Materials and methods 2.1. Immunohistochemical staining Antibodies of RA175/TSLC1/SynCAM, antibodies against a peptide corresponding to the C-terminal region of RA175/TSLC1/SynCAM, were prepared as described previously [14] and used for the immunostaining of the RA175/TSLC1/SynCAM in mouse developing nervous system. The mouse embryos at embryonic day (E)9.5, E10.5, E11.5, E12.5 E14.5, E15.5, and E16.5 were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 8C overnight and then soaked in 30% sucrose/PBS at 4 8C overnight and embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetec. Tokyo) and were frozen. Frozen sections (10 Am thick) of embryos were cut on a cryostat and attached to slides coated with Vectabond reagent (Vector Laboratories, Burlingame, CA) and incubated with rabbit anti-RA175/TSLC1/SynCAM and or rat anti-NCAM and anti-L1 (Chemicon International, Temecula, CA), mouse anti-TAG-1 (4D7, Developmental Studies Hybridoma Bank, Iowa City, IA) in PBS containing 0.1% skim milk and 0.1% Triton X-100 at 4 8C for 2 days as described previously. Subcellular localization of RA175/TSLC1/SynCAM in the neurons was also examined by immunostaining. Neurons and astrocytes were isolated from the brain of E15.5 mouse embryos described previously [3] and cultured in Dulbecco’s Modified Eagle’s Medium (Sigma) containing 4500 mg/l glucose, 10% Fetal Bovine Serum in the presence or absence of 10 AM Ala-C for 10 days, respectively. Neurons and astrocytes were fixed at 4% paraformaldehyde and were immunostained with anti-RA175/TSLC1/SynCAM, mouse Tuj-1 (Tubulin h-III, Neuromics Antibodies Northfield, MN), mouse anti-GFAP, anti-Map-2 (Sigma, ST, Louis MO), and mouse anti-Tau (Upstate Biotechnology Lake Placid, NY). Anti-RA175/TSLC1/SynCAM immunoreactivity was detected by FITC-labeled goat anti-rabbit IgG (Leinco Technologies, St. Louis, MO). Rat anti-NCAM and antiL1 immunoreactivities were detected by goat anti-rat IgG Alexa Fluor 568 (Molecular Probes, Eugene, OR). Mouse anti-TAG-1, anti-Tuj-1, anti-GFAP, anti-Map-2, and antiTau immunoreactivities were detected by goat anti-mouse IgG Alexa Fluor 568 (Molecular Probes). Immunoreactivity was viewed using a confocal laser-scanning microscope (CSU-10, Yokogawa, Tokyo, Japan). 2.2. In situ hybridization (ISH) The EcoRI fragment of RA175/TSLC1/SynCAM in the pGEM-T vector was subcloned into pBluescript SK(+)
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vectors in both the sense and anti-sense orientations as described previously [39]. Digoxigenin (DIG)-labeled RA175/TSLC1/SynCAM mRNA probes were transcribed from XhoI-cut constructs by T3 RNA polymerase (DIG RNA Labeling Kit T3, Boehringer Mannheim, Mannheim, Germany) in vitro according to the manufacturer’s instructions. Frozen sections (10 Am thick) of embryos at various developmental stages were treated with proteinase K (1 Ag/ml) at 37 8C for 10 min, refixed in 4% paraformaldehyde, and hybridized overnight with the DIG-labeled RNA probes. Hybridized RNA was detected using alkaline phosphatase-conjugated anti-DIG according to the procedure described by Wilkinson [40].
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At later stage [i.e., E14.5 and E16.5], RA175/TSLC1/ SynCAM mRNA was decreased (Fig. 1Q), and protein was still detected on the ventral commissural axons in the floor plate and in the ventral fiber tract (Fig. 1R, double asterisks). We compared the distribution of RA175/TSLC1/SynCAM with those of other members of immunoglobulin superfamily such as TAG-1 and L1 at E11.0 (Fig. 2). TAG-1 was expressed on the dorsal funiculus and the commissural cell fiber (Fig. 2B, E) and partly colocalized with RA175/ TSLC1/SynCAM (Fig. 2C, F). L1 was expressed in the dorsal funiculus and ventral fiber tract but slightly in the floor plate (Fig. 2H, K). L1 colocalized with RA175/ TSLC1/SynCAM on the dorsal funiculus and ventral fiber tract but not in the ventral commissural fibers in the floor plate (Fig. 2I, L).
3. Results 3.1. Distribution of RA175/TSLC1/SynCAM in the developing spinal cord We examined the distribution of RA175/TSLC1/ SynCAM in the developing spinal cord (Fig. 1). At E9.5, both RA175/TSLC1/SynCAM mRNA and proteins were expressed mainly in somites (open arrowheads) and weakly in the neural tube (Fig. 1A–D). At E10.5, RA175/TSLC1/SynCAM mRNA was expressed mainly in the motor neurons and in the neural crest (Fig. 1E, closed arrowheads). In contrast, the RA175/TSLC1/ SynCAM protein was localized, by immunostaining, to axons, in addition to the neural crest (closed arrowheads) and motor neurons (Fig. 1F). RA175/TSLC1/SynCAM as well as NCAM was expressed on the commissural axons and prominently enriched in the ventral commissure (Fig. 1F–H, thick arrows), where the commissural axons transverse the floor plate. RA175/TSLC1/SynCAM and NCAM were also strongly expressed on the ventral root (Fig. 1F–H, single asterisk) which fasciculates the axons from motor neurons. There was a strong contrast between RA175/TSLC1/SynCAM and NCAM immunoreactivity in the neural crest at this stage; NCAM was not detected in the neural crest at this stage (Fig. 1G, closed arrowheads). At E11.5–12.5, RA175/TSLC1/SynCAM mRNA was still expressed in the motor neurons and in the DRG neurons developed from the neural crest (Fig. 1I, M). In contrast, RA175/TSLC1/SynCAM immunostaining was at a low level in the cell bodies of motor neurons and DRG neurons but positive in their axons (Fig. 1J, N). Strong RA175/TSLC1/SynCAM immunoreactivity was detected in the dorsal funiculus (Fig. 1J, N, thin arrows), which fasciculates bundles of sensory axons from the DRG within the spinal cord. RA175/TSLC1/SynCAM immunoreactivity was still at a high level on the ventral commissural axons in the floor plate and ventral root and colocalized with NCAM (Fig. 1J–L, N–P, thick arrows).
3.2. Distribution of RA175/TSLC1/SynCAM in the developing brain RA175/TSLC1/SynCAM mRNA expression was sparsely detected in the neuroepithelium of the forebrain and hindbrain at E9.5 (Fig. 3A) [39]. Consistent with RA175/TSLC1/SynCAM mRNA, RA175/TSLC1/SynCAM protein was also sparsely distributed in the neuroepithelium and localized at adherent regions of neuroepithelial cells (Fig. 3B). During brain development (E13.5–16.5), however, there was a striking contrast in expression between RA175/TSLC1/SynCAM mRNA and protein; i.e., RA175/TSLC1/SynCAM mRNA was detected in the cell bodies of neurons in various regions of the developing brain, while the protein was detected in the extending axons. At E13.5–E15.5, the thalamocortical axons extend into the cortex from the internal capsule (IC) and cortical neurons extend the efferent fibers projecting toward their targets through IZ. At E14.5, RA175/TSLC1/SynCAM mRNA was expressed mainly in the thalamus and developing cortex (Fig. 3C), while RA175/TSLC1/SynCAM protein was positive mainly in the extending thalamocortical axons from the IC (Fig. 3D). At E16.5, RA175/TSLC1/SynCAM mRNA was highly expressed in various regions of the brain, including the cortex, thalamus, and habenular nucleus (Fig. 3F). In the cortex, most RA175/TSLC1/SynCAM mRNA was expressed in the cortical plate, whereas RA175/TSLC1/SynCAM protein was localized to the axons in the IZ and the axons in the TC (Fig. 3G). Most RA175/TSLC1/ SynCAM protein colocalized with NCAM on the thalamocortical axons extending into IZ (Fig. 3H–J) from the IC (Fig. 3K–M). Neural adhesion molecules of the immunoglobulin superfamily, TAG-1 and L1, are expressed by cortical efferent and afferent fibers, respectively [17,21,42]; i.e., the TAG-1-positive efferent axons of the IZ are tangentially oriented, projecting towards the IC, while the L1 afferent fibers extend into the IZ through the TC during
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Fig. 1. Distribution of RA175/TSLC1/SynCAM mRNA and protein the developing spinal cord (E9.5–E14.5). Transverse sections of mouse embryos at E9.5 (A– D), E10.5 (E–H), E11.5 (I–L), E12.5 (M–P), and E14.5 (Q–T) were hybridized with antisense probes for RA175/TSLC1/SynCAM mRNA (A, E, I, M, Q) and immunostained with anti-RA175/TSLC1/SynCAM (B, F, J, N, R), anti-NCAM (C, G, K, O, S), and merged (D, H, L, P, T). mo, motor neurons; DRG, dorsal root ganglia neurons. Open arrowheads and closed arrowheads indicate somite and neural crest, respectively, and thick and thin arrows indicate the ventral commissure and the dorsal funiculus, respectively. Single and double asterisks show ventral root and ventral fiber track, respectively. Bars indicate 100 Am. RA175/TSLC1/SynCAM colocalized with NCAM in the dorsal funiculus, axons of the ventral commissure and ventral root with strong intensity.
cortical development [17] (Fig. 4A). As it is difficult to discriminate afferents and efferents intermingled in the IZ at E16.5 (Fig. 3H–J) [10], we examined the immunor-
eactivity of RA175/TSLC1/SynCAM on the TAG-1positive cortical efferent axons outgrowing in the IZ and on the L1-positive thalamocortical afferent axons extend-
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Fig. 2. Colocalization of RA175/TSLC1/SynCAM and TAG-1 or L1 in the neural tube at E11.0. Transverse sections of mouse embryos at E11.0 were immunostained with anti-RA175/TSLC1/SynCAM in the absence (A, D, G, J) or presence (M) of 10 Ag/ml peptide of C-terminal region of RA175, anti-TAG-1 (B and E), and anti-L1 (H and K). Panels (C, F, I, L) are merge. Panels (D, E, F) are rectangular region in panel (C). Panels (J, K, L) are horizontal section cut at broken line in panels (G, H, I). RA175/TSLC1/SynCAM and TAG-1 were partly colocalized at the dorsal funiculus and the commissural fibers (C, F). RA175/ TSLC1/SynCAM and L1 were colocalized at dorsal funiculus and ventral fiber tract but not at the ventral commissure (I, L). Panels (N, O) were immunostaining with goat FITC-labeled rabbit IgG and goat anti-rat IgG Alexa Fluor 568, respectively. Fluorescences in panels (M, N, O) were due to the cells with autofluorescence. Bars indicate 100 Am.
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Fig. 3. Distribution of RA175/TSLC1/SynCAM mRNA and protein in the developing brain at E9.5, E14.5, and E16.5. RA175/TSLC1/SynCAM mRNA expression and protein distribution in the hindbrain at E9.5 (A, B) and in the brain at E14.5 (C, D, E) and E16.5 (F, G) were examined by in situ hybridization (A, C, F) and immunostaining using anti-RA175/TSLC1/SynCAM in the absence (B, D, G) or presence (E) of the peptide of C-terminal region of RA175. Panel (B) is the rectangular region in (A). Bars in panels (A, C, D, E, F, G) indicate 200 Am. Bar in panel (B) indicates 25 Am. Fb, forebrain; Hb, hindbrain; cx, cortex; th, thalamus; hi, hippocampus; hb, habenular nucleus. RA175/TSLC1/SynCAM with NCAM colocalized in the TC and IZ (TC/IZ) (H, I, J) and the IC (K, L, M). (H, K) Anti-RA175/TSLC1/SynCAM immunoreactivity; (I, L) anti-NCAM; (J, M) merge.
ing from IC at E15.5 (Fig. 4). Most RA175/TSLC1/ SynCAM protein colocalized with L1 on the TC and the IC (Fig. 4F–H, L–N). RA175/TSLC1/SynCAM was also partly overlapped with TAG-1 in the IZ (Fig. 4C–E) but not in the IC (Fig. 4I–K).
RA175/TSLC1/SynCAM was clustered and patched on the axons and surface of the cell body (Fig. 6G–I, J–L, arrowheads).
4. Discussion 3.3. Distribution of RA175/TSLC1/SynCAM in the developing epithelia of sensory organs RA175/TSLC1/SynCAM mRNA was highly expressed in the olfactory system and in the retina at E16.5 (Fig. 5A). In the retina, both RA175/TSLC1/SynCAM and NCAM were strongly coexpressed in the nerve fiber layer (Fig. 5B–D, arrowheads), with relatively weak staining in the inner nuclear layer (arrows). RA175/ TSLC1/SynCAM colocalized with NCAM in the nerve fibers of olfactory sensory neurons projecting to the olfactory bulb (Fig. 5E–G). In contrast with NCAM, RA175/TSLC1/SynCAM was detected in the adherent region in the olfactory epithelium. 3.4. Subcellular localization of RA175/TSLC1/SynCAM in the neuron RA175/TSLC1/SynCAM was expressed in the Tuj-1positive cortical neurons cultured in vitro (Fig. 6A–C) and preferentially localized on the axons but not expressed in the GFAP-positive astrocytes (Fig. 6D–F). The subcellular localization of RA175/TSLC1/SynCAM in cortical neurons was examined by immunostaining (Fig. 6G, J). RA175/ TSLC1/SynCAM was preferentially localized on the Map2-positive axons of the differentiated cortical neurons rather than the cell body (Fig. 6G–I). Some of the
4.1. Change in the localization of RA175/TSLC1/SynCAM There was a great contrast between the localization of the RA175/TSLC1/SynCAM protein in the epithelium, including the neuroepithelium and the nervous system after neurogenesis (Figs. 1–6). In the epithelium, the expression of RA175/TSLC1/SynCAM mRNA and protein were detected in the cell bodies, and RA175/TSLC1/SynCAM protein was localized in the adherent region of epithelial cells. In the developing nervous system, regions expressing RA175/TSLC1/SynCAM mRNA and protein were apparently not consistent. RA175/TSLC1/SynCAM was preferentially expressed on the axons of the differentiated neurons. RA175/TSLC1/SynCAM may play different biological roles as cell adhesion molecules at each neuronal differentiation step; that is, RA175/TSLC1/SynCAM may be involved in the cell adhesion processes on the axons of the differentiating neurons in addition to those in the neuroepithelium [14] and at synapses of mature neurons [6]. 4.2. RA175/TSLC1/SynCAM in the developing spinal cord The floor plate can exert strong influences, both attractive and repulsive, over axon projections [8,37]. The commissural axons are attracted by the floor plate cells, and their early projections are directed ventrally
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Fig. 4. Localization of RA175/TSLC1/SynCAM and L1 or TAG-1 in the IZ, TC, and IC. Coronal sections of the E15.5 mouse brain were double immunostained with anti-L1 (green) and TAG-1 (red) (A), anti-RA175/TSLC1/SynCAM (green) and TAG-1 (red) (B, C–E, I–K), and anti-RA175/TSLC1/ SynCAM (green) and L1 (red) (F–H, L–N). (C–E) IZ, (F–H) TC, (I–K, L–N) IC. (C, F, I, L) Anti-RA175/TSLC1/SynCAM (green), panels (D, J) are anti-TAG1 (red), panels (G, M) are anti-L1 (red), panels (E, K) are merge of anti-RA175/TSLC1/SynCAM and anti-TAG-1, and panels (H, N) are merge of anti-RA175/ TSLC1/SynCAM and anti-L1. Bars in panels (B, H, K, N) indicates 100 Am. Bar in panel (H) indicates 50 Am.
toward the floor plate and fasciculated at the ventral commissure, where they transverse the floor plate. RA175/ TSLC1/SynCAM immunoreactivity was strongly localized
on the ventral commissural axons in the floor plate and weakly localized on the commissural axons directed toward the floor plate (Fig. 1).
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Fig. 5. Localization of RA175/TSLC1/SynCAM and NCAM in the retina and olfactory organs at E16.5. Coronal section of mouse head at E16.5 was hybridized with antisense probes for RA175/TSLC1/SynCAM mRNA (A) and immunostained with anti-RA175/TSLC1/SynCAM (B, E) and anti-NCAM (C, F). Panels (D, G) are merge. RA175/TSLC1/SynCAM mRNA was highly expressed in the retina and olfactory epithelia. (B, C, D) Double immunostaining of the retina (coronal section) with anti-RA175/TSLC1/SynCAM (B; green) and anti-NCAM (C; red). Arrowheads indicate the nerve fibers and arrows indicate the inner nuclear layer. (E, F, G) Double immunostaining of the olfactory epithelium (sagittal section) with anti-RA175/TSLC1/SynCAM (E; green) and antiNCAM (F; red). Bars in panels A and E indicate 1 mm and 100 Am, respectively. ob, olfactory bulb; oe, olfactory epithelium.
Cell adhesion molecules (CAMs) have important roles in axon outgrowth and their fasciculation via homophilic or heterophilic interactions [11,19,41]. TAG-1 is expressed on the commissural axons, in particular, on the ventral commissural axons in the floor plate [24] and NCAM, SC1, L1, and TAG-1 are also detected on the fasciculated axons at the dorsal funiculus and the ventral root [7,24,36]. RA175/TSLC1/SynCAM was also prominently expressed on the ventral commissural axons in the floor plate and the fasciculated axons at the dorsal funiculus (Fig. 1). Thus, it is likely that RA175/TSLC1/SynCAM, as well as other CAM proteins, is involved in the outgrowth of the commissural axons attracted by the floor plate and may also play a role in the fasciculation of the axons at the floor plate, dorsal funiculus, and ventral root in the developing spinal cord. 4.3. RA175/TSLC1/SynCAM in the developing brain Two types of cortical migration of neurons have been shown [30]; one is a radial migration by which postmitotic neurons migrate from the ventricular zone and form the preplate. The other is nonradial migration of the interneurons [1,9,23,25,27]. Axons can provide a substratum for nonradial neuronal migration in the developing central nervous system, including the cerebral cortex [20,33]. The developing axons of the IZ and TC have been shown to be the substratum for the GABAergic interneurons tangentially migrating from the MGE to the cortex [2,28,31]. RA175/TSLC1/SynCAM was localized not only on the axons of the TC but also in the IZ. Most of the RA175/TSLC1/SynCAM colocalized with L1 on the TC and at the IC (Fig. 4F–H, L–N), suggesting that RA175/TSLC1/SynCAM is expressed on
the cortical afferent fibers extending from the IC into the cortex. Some of the RA175/TSLC1/SynCAM immunoreactivity colocalized with TAG-1 on the cortical efferent axons outgrowing in the IZ (Fig. 4B–E), suggesting that RA175/ TSLC1/SynCAM is also expressed on the developing cortical efferent axons in the IZ. This was supported by the localization of RA175/TSLC1/SynCAM on the axons of the cortical neurons (Fig. 6A–C). Furthermore, RA175/ TSLC1/SynCAM mRNA and proteins were expressed in the various regions of the brain (Fig. 3), also suggesting that RA175/TSLC1/SynCAM is localized on the axons of the neurons including the efferents and afferents of the cortical neurons and play a role in the pathfinding and migration in the cooperation with other cell adhesion molecules such as NCAM, L1, and TAG-1. TAG-1 is involved as a cellular substrate in the pathfinding of the GABAergic interneurons in the tangential migration to the neocortex [10]. However, TAG-1deficient mice do not show inhibition of GABAergic interneuron migration histologically [15], suggesting that other cell adhesion molecules also play as their cellular substrates. RA175/TSLC1/SynCAM located on the axons of the IZ and TC may be one of the candidate adhesion molecules involved in the pathfinding of the GABAergic interneurons as a substratum. This possibility remains to be clarified by experiments with RA175/TSLC1/SynCAMdeficient mice. 4.4. Subcellular localization of RA175/TSLC1/SynCAM in the neuron In differentiated cortical neurons cultured in vitro, RA175/TSLC1/SynCAM was localized in the cluster or
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Fig. 6. Subcellular localization of RA175/TSLC1/SynCAM in the cortical neurons. Localization of RA175/TSLC1/SynCAM in the neurons was examined by immunostaining using anti-RA175/TSLC1/SynCAM (A, D, G, J; green) and anti-Tuj-1 (B; red), anti-GFAP (E; red), anti-Map-2 (H; red) or anti-Tau (K; red), respectively. Panels (C, F, I, L) are merge. GFAP-positive astrocytes are RA175/TSLC1/SynCAM-negative (rectangular in panels D, E, F). Arrowheads indicate RA175/TSLC1/SynCAM immunoreactivity in the cluster or patched form on the axons. Bars (I, C) indicate 20 Am. Bars (C, F) indicate 100 Am.
patched form on the axons and dendrites and also on the surface of the cell body (Fig. 6). At present, the molecular mechanism of the localization of RA175/TSLC1/SynCAM on axons is not clear. CAMs have biological functions and distributions distinct from other cell adhesion molecules via association with cytoplasmic proteins at the peripheral membrane of the neurons. The intracellular domain of RA175/TSLC1/SynCAM has the putative binding domain of ERM (ezrin, radixin, and moesin), a member of the band 4.1 superfamily, shared with Caspr2 [14,32]. Caspr2 associates with the cytoskeleton via the band 4.1-binding protein and localizes on the axons [38]. Thus, this putative band 4.1-binding region may contribute to the stabilization of the localization of RA175/ TSLC1/SynCAM on the axons. In addition, RA175/ TSLC1/SynCAM interacts with CASK at the presynaptic membrane via EYFI, the binding site for the type II PDZ domain at the C-terminus [29]. RA175/TSLC1/SynCAM
may also be associated with proteins with PDZ domain on the axons or dendrites and clustered to form functional synapses. In conclusion, the RA175/TSLC1/SynCAM protein is localized on axons in many fiber systems. RA175/TSLC1/ SynCAM, as well as TAG-1, may be involved in the guidance of commissural axons in the floor plate, the fasciculation of axons, axonal pathfinding, and the tangiential migration of neuronal cells.
Acknowledgments This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and by Research Grant 14A-1 for Nervous and Mental Disorders and Research on Brain Science from the Ministry of Health and Welfare of Japan, the Human Science Foundation. E.F.
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and Y.K. are Research Fellows of the Japan Society for the Promotion of Science. [19]
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