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Differential Expression of Cadherin Adhesion Receptors in Neural Retina of the Postnatal Mouse Megumi Honjo,1 Hidenobu Tanihara,1 Sachihiro Suzuki,2 Taro Tanaka,2 Yoshihito Honda,1 and Masatoshi Takeichi2 PURPOSE. To determine the expression pattern of multiple subtypes of cadherin adhesion receptor in postnatal mouse neural retina. METHODS. The expression of N-cadherin, R-cadherin, cadherin-6, cadherin-8, and cadherin-11 in retinas at postnatal days 0 to 42 was analyzed by in situ hybridization of mRNA as well as by immunohistochemistry. RESULTS. Each cadherin was expressed by different cell populations of the retina, and the following expression patterns were established by postnatal day 14: in the ganglion cell layer, all these molecules were expressed, but each occurred only in a subset of the cells. Likewise, in the inner nuclear layer, R-cadherin and cadherin-6 and -8 were expressed by a restricted population of amacrine cells, and cadherin-8 also by a subpopulation of bipolar cells. All horizontal cells expressed R-cadherin, and Mu ¨ ller cells expressed N-cadherin and cadherin-11. Proteins of Rcadherin and cadherin-6 were concentrated in neuropil layers. CONCLUSIONS. The pattern of differential expression of the five cadherins supports the idea that these molecules may play a role in selective cell interactions within the heterogeneous cell pool of the neural retina. (Invest Ophthalmol Vis Sci. 2000;41:546 –551)

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adherins are a family of homophilic adhesion receptors, and play a crucial role in cell-cell adhesion by most cell types. It has been established that a given type of cadherin preferentially binds to its own type via homophilic interaction, although cross-interaction can occur between different cadherin types, depending on their combinations.1 This binding property of cadherins was implicated in selective cell adhesion processes.1 Recent studies demonstrated that cadherins and associated proteins, known as catenins, are localized in synaptic junctions, suggesting that they may play a role in the connection of pre- and postsynaptic membranes.2 In the brain, multiple cadherin subtypes are differentially expressed, each being detected in restricted brain nuclei or cortical sublayers.3 Anal-

From the 1Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Shogoin, Sakyo-ku, Kyoto; and 2Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, Japan. Supported by the programs Grants-in-Aid for Specially Promoted Research (M.T.) and Grants-in-Aid for Scientific Research (H.T.) of the Ministry of Education, Science, Sports, and Culture of Japan. M.H. and S.S. are recipients of Fellowships of the Japan Society for the Promotion of Sciences for Young Scientists. Submitted for publication June 3, 1999; revised September 3, 1999; accepted September 29, 1999. Commercial relationships policy: N. Corresponding author: Masatoshi Takeichi, Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. [email protected]

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ysis of these patterns revealed that the expression of each cadherin was correlated with neural connectivity. From these observations, each cadherin was proposed to take part in connecting neurons to their specific partners.3 It would be interesting to know if the same can be said for retinal circuits. Cadherin expression in the visual system was recently studied in chicken neural retina4 as well as in the optic tectum.5,6 Because the retinal circuits are better understood for mammalian species, we opted to map cadherin distribution in postnatal mouse retina. We studied five cadherin subtypes, that is, N-cadherin (N-cad), R-cadherin (R-cad), cadherin-6 (cad6), cadherin-8 (cad8), and cadherin-11 (cad11) and found that each cadherin subtype is expressed in a restricted population of retinal cells, supporting the view that cadherin adhesion receptors may play a role in selective cell associations in the retina, as they do in the brain.

MATERIALS

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METHODS

Animals Experiments were performed with ICR mice. The day on which the animals were born was regarded as postnatal day 0 (P0). All studies were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

In Situ Hybridization of mRNA Retinas were fixed, and isolated as described below, except that the enucleated eyes were postfixed for 12 hours. In situ Investigative Ophthalmology & Visual Science, February 2000, Vol. 41, No. 2 Copyright © Association for Research in Vision and Ophthalmology

IOVS, February 2000, Vol. 41, No. 2

Cadherin Expression in the Neural Retina

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hybridization methods and the probes used for detection of N-cad, R-cad, cad6, cad8, and cad11 were previously described.3,7 Hybridized signals were visualized by the digoxigenin method, using BM purple (Boehringer Mannheim, Mannheim, Germany).

Antibodies The following antibodies were used: rat monoclonal antibodies to mouse N-cad, MNCD2 and to mouse R-cad, MRCD57; rabbit polyclonal antiserum against mouse cad6; mouse monoclonal anti– calbindin-D antibody (Sigma Chemical Co., St. Louis, MO); rabbit anti-tyrosine hydroxylase antibody (Chemicon International, Temecula, CA); and mouse monoclonal anti-choline acetyltransferase antibody (Boehringer Mannheim). Secondary antibodies labeled with indocarbocyanin dye Cy3 were obtained from Chemicon International and peroxidase-conjugated secondary antibodies from Amersham Pharmacia Biotech (Buckinghamshire, England). Preparation of anti-cad8 antibodies has been described elsewhere (Manabe T, Suzuki SC, Takeichi M, Chisaka O, unpublished results).

Immunohistochemical and Immunoblotting Procedures Immunohistochemistory was performed as described previously.7 Briefly, mice were perfusion-fixed with 4% paraformaldehyde dissolved in HEPES-buffered Hanks’ balanced salt solution (HBSS) before enucleation. Subsequently, the enucleated eyes were postfixed for 2 hours at 4°C in 4% paraformaldehyde/HBSS, washed for 5 minutes in phosphate-buffered saline, and then immersed in a graded series of sucrose solutions (12%–18% sucrose), embedded in Tissue-Tek (Miles, Inc., Elkhart, IN), and frozen in liquid nitrogen. Sections (10 ␮m) were cut on a cryostat. The samples were incubated successively in methanol at ⫺20°C for 20 minutes, in 5% skim milk in TBS-Ca for 30 minutes, and in a solution of primary antibodies for 60 minutes at room temperature. They were then treated for 30 minutes with secondary antibodies. For double-immunostaining, the same procedures were repeated. Fluorescence was visualized under an epifluorescence microscope (Zeiss Axioplan, Oberkochen, Germany) or a confocal laser scanning microscope (Bio-Rad Laboratories, Hercules, CA). For doublestaining by RNA in situ hybridization and immunostaining, the latter was carried out after the completion of the in situ hybridization steps, and the immunostaining signals were visualized by the dimethylaminoazobenzene reaction. Immunoblotting was performed as described.7

RESULTS Immunoblotting Analysis of Retinal Cadherin Expression By means of immunoblotting, we determined the expression of five cadherins, N-cad, R-cad, cad6, cad8, and cad11, and of two catenins, ␣N-catenin and ␤-catenin, during postnatal development (P0 –P42) of the neural retina. N-cad and R-cad were expressed at a constant level throughout the postnatal development (Fig. 1), and cad6 and cad11 showed a similar expression pattern (data not shown). The levels of ␣N- and ␤-catenin

FIGURE 1. Western blotting analysis of cadherin protein expression during postnatal development of the neural retina (P0 –P42). Arrows indicate two isoforms of ␣N-catenin; their relative amount changes during development. The total amount of proteins loaded into each electrophoretic lane was adjusted to be equal. N-cad, N-cadherin; R-cad, R-cadherin; cad8, cadherin-8; ␣N-cat, ␣N-catenin; ␤-cat, ␤-catenin.

were also little changed, although the proportion of the two isoforms of ␣N-catenin was changed. An exception was that the cad8 level tended to decrease at later postnatal stages (Fig. 1).

In Situ Hybridization Analysis of Cadherin mRNA Expression We next determined the distribution of the above five cadherins in the neural retina at P0 to P42 (adult stage) by in situ hybridization for detection of mRNA (Fig. 2). Each cadherin showed a unique expression profile, as described below.

N-Cadherin At P0, N-cad mRNA was detected throughout the retinal layers. However, the most intense signals were observed in the middle region of the future inner nuclear layer (INL); at P3 to P7, this expression pattern was maintained. At more advanced stages, as well as in the adult (P42), intense signals remained only in the middle zone of the INL, where the nuclei of Mu ¨ ller cells are located.8

R-Cadherin At P0 to P3, strong signals were detected in a subset of cells in the ganglion cell layer (GCL), and weaker signals were found in the future amacrine layer. At P7, the relative intensity in the amacrine signals increased, and in the deepest area of the inner plexiform layer (IPL), new signals appeared from cells scattered along the outer plexiform layer (OPL), the distribution of which suggested them to be horizontal cells (see below for confirmation). Faint signals were found also in the deep area of the outer nuclear layer (ONL). This expression profile, in principle, continued to the adult

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FIGURE 2. In situ hybridization analysis of cadherin mRNA expression in the neural retina at P0 to P42. Small arrows point to amacrine cells, and large arrows to horizontal cells; arrowheads indicate putative bipolar cells. The differences in the staining intensity among the panels do not quantitatively reflect the real differences in the expression level of the molecules, as staining efficiency varied with the samples.

stage. In the amacrine and ganglion layers, only a subset of cells was positive (see below).

Cadherin-6 At P0 to P3, sparsely distributed positive cells were found in both the GCL and amacrine layer, and this pattern persisted throughout development.

Cadherin-8 As found with R-cad and cad6, cad8 was detected in the GCL as well as in the INL at P0 to P3. At P7, cad8 began to be expressed in the deeper zone of the INL, and by P14, the signals in this zone had become most prominent, whereas those in the GCL were diminished. In the adult, a similar



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Cadherin-11 This cadherin was also detected in a subpopulation of cells in the GCL and INL at P0. At P3 to P7, cad11 signals became intense at the middle zone of the INL, similar to that of N-cad. At P14, the cad11 signals became restricted to this zone, and this pattern was maintained up to the adult stage. As noted for N-cad, the cad11-positive zone corresponded to where Mu ¨ ller cell bodies were located.

Further Identification of Cadherin mRNA-Positive Cells

FIGURE 3. Identification of cadherin-positive cells. (A through C) Double-staining for cadherin mRNA (blue) and calbindin-D proteins (brown) in P14 neural retina. Large arrows point to examples of amacrine cells having both in situ hybridization and calbindin-D staining signals, and small arrows to horizontal cells having both signals. Large dark and white arrowheads point out examples of amacrine and bipolar cells, respectively, that showed only in situ hybridization signals. Small dark and white arrowheads indicate examples of amacrine and horizontal cells, respectively, having only calbindin-D staining. In (A), the calbindin-D staining was reduced to show clearly the double-stained status in horizontal cells. (D) Double-staining for cad6 mRNA (6) and TH protein. Arrow points to a cell having both in situ hybridization signals and TH staining. (E, F) Whole-mount in situ hybridization of P14 neural retina for cad6 and N-cad. The retina is flat mounted and focused on the GCL. Note that only a subpopulation of cells expresses each cadherin.

expression pattern was observed, although the western blot analysis results indicated that the total cad8 protein level was reduced by the adult stage.

FIGURE 4. Immunofluorescence staining of N- and R-cad expression in the neural retina at P0 to P42. Sections were stained with antibodies against Ncad (N) or R-cad (R). Confocal images are shown.

We analyzed cadherin-positive cells by use of cell type–specific markers, focusing on the INL of the P14 retina. In the INL, subtypes of amacrine cells can be immunostained with antibodies to calbindin-D.9 To examine whether the calbindin-D expression overlaps cadherin expression, we double-stained retinas for calbindin-D with antibodies, and R-cad, cad6, or cad8 by in situ hybridization. The results showed that some of R-cad– and cad6-positive cells also reacted with anti– calbindin-D antibody (Figs. 3A, 3B), but many were calbindin-D– negative, indicating that the cell population that expressed R-cad or cad6 was heterogeneous in terms of calbindin-D expression. Most of the cad8-positive cells did not react with the anti– calbindin-D antibody (Fig. 3C). When we stained for tyrosine hydroxylase (TH), a marker for dopaminergic amacrine cells,10 its immunostaining signals coincided with weak cad6 in situ hybridization signals (Fig. 3D). In this case, therefore, all TH-positive cells seem to express cad6. Calbindin-D also can be used as a marker of horizontal cells.9 In contrast to the case of amacrine cells, all calbindinD–positive horizontal cells were positive for R-cadherin expression (Fig. 3A). In this deep area of the INL, cad8-positive cells also were present, but none of the cad8 signals overlapped with the calbindin-D staining (Fig. 3C). The cad8-positive cells located in this area are, therefore, most likely bipolar cells. In the GCL, all the cadherins studied were expressed, but each appeared to occur only in a subpopulation of cells present in this layer. This was confirmed by whole-mount in situ hybridization of P14 retinas (see Figs. 3E, 3F for examples

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of cad6 and N-cad, respectively). The size of the cadherinpositive cells in the GCL varied from small to large.

Immunostaining Analysis of Cadherin Expression The in situ hybridization analysis provided information about which cells expressed a given cadherin gene. We then determined the distribution of proteins encoded by each cadherin gene. However, for immunohistologic staining, the antibodies only to N-cad, R-cad, and cad6 were available; accordingly, our analysis was restricted to these three cadherins. N-cad proteins were rather ubiquitously detected in the neural retina throughout development (Fig. 4), despite the enrichment of its mRNA in putative Mu ¨ ller cells. In contrast, R-cad protein distribution was regional, and correlated with its mRNA expression profile (Fig. 4). At PO to P3, strong immunostaining signals were found in the IPL as well as in some cells localized in the GCL. At P7, the IPL signals split into two zones, and this staining profile persisted to the adult stage. It should be noted that, after P7, a particular population of amacrine cells located deeper in the INL accumulated R-cad proteins in the cytoplasm of their soma (Figs. 5A, 5B). For further identification of the R-cad–positive areas in the IPL, we double-stained P14 retinas for R-cad and syntaxin, a synaptic marker for the entire IPL. The results indicated that the R-cad–positive zone in the IPL corresponded to the deeper half (likely the sublamina a) of the IPL (Fig. 5A). Horizontal cells and OPL became positive for R-cad protein expression after P7. This cadherin was also detected in the outer limiting membrane (OLM), as was N-cad (Fig. 4). Cad6 protein was confined to two narrow strata in the IPL (Figs. 5B right, 5C middle). Because this pattern was reminiscent of that of choline acetyltransferase (ChAT) (Fig. 5C left), known to be expressed by cholinergic amacrine cells,11 we double-stained for cad6 and ChAT and found that their expressions coincided (Fig. 5C right). We could not detect conspicuous immunostaining signals from TH-positive neurons, probably because the cad6 expression level in these cells was low. We also compared the staining results for R-cad and cad6 and found that their expressions did not overlap (Fig. 5B), being complementary, indicating that they are accumulated in different zones of the IPL.

DISCUSSION In this study, we analyzed the expression of five cadherins in postnatal neural retina. As previously found in chicken retina,4 every cadherin was expressed by a restricted population of cells in mouse retina. The expression patterns changed during development, but were basically established by P14. The established patterns differed with the various cadherins. N-cad and cad11 appeared to be expressed predominantly by a single cell type, that is, Mu ¨ ller cells, as assessed by their mRNA distribution. Although N-cadherin proteins were detected in many regions of the retina, this protein localization pattern probably represents the distribution of Mu ¨ ller cell processes, which form an extensive scaffolding across the retina.12 On the other hand, R-cad, cad6, and cad8 were expressed by multiple cell types, but, in general, these molecules occurred in a limited subpopulation of cells for most cell types. This restricted distribution likely reflects the heterogeneity of

FIGURE 5. Identification of cadherin-positive regions in P14 retina. (A) Double-immunostaining for R-cad and syntaxin. R-cad signals in the IPL are confined to its deeper half, displaying double stripes. (B) Immunostaining for R-cad and cad6 in adjacent sections. Note that major cad6 signals are restricted to double narrow zones in the IPL, whose locations are separate from the R-cadherin–negative zones. Staining in the inner-half of the IPL, shown in the left panel, is not reproducible. (C) Double-immunostaining for ChAT (red) and cad6 (green) visualized with confocal laser scanning microscopy. These signals are merged in the right-most panel. The cad6 signals coincide with the ChAT signals.

each cell group. It should be noted that, in the amacrine layer, certain identified cell groups expressed a particular cadherin; for example, all TH-positive amacrine neurons expressed cad6 mRNA, and ChAT colocalized with cad6 proteins. These observations suggest that cells expressing a particular cadherin may represent a specific functional group, not only in the amacrine but also in other layers. Consistently, in the case of horizontal cells, which are less heterogeneous than are amacrine cells, they evenly expressed R-cad. Interestingly, however, the cadherin expression pattern did not always correlate with that of known markers, such as calbindin-D. Thus, cadherin expression may provide novel information for grouping of highly heterogeneous retinal cells. Immunostaining for R-cad and cad6 proteins provides additional important information. Unlike the mRNA staining patterns, these proteins were concentrated in synapse-enriched zones, as previously found in chicken retina.4 Most likely,

IOVS, February 2000, Vol. 41, No. 2 R-cad and cad6 proteins, once synthesized, are transported to synaptic areas, and are used for interneuronal connections at these sites. The differential distribution of R-cad and cad6 in the IPL implies that each may serve as connectors between restricted pairs of neurons. To summarize, our findings support the hypothesis that each cadherin plays a role in grouping of selected cells in the heterogeneous retinal cell pool. This idea will be tested by future analyses, including gene knockout, electron microscopy, and electrophysiology studies.

Acknowledgments The authors thank Ryuichiro Kageyama (Kyoto University) and Yutaka Fukuda (Osaka University) for critical suggestions on cell type markers.

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4. Wo ¨ hrn JC, Puelles L, Nakagawa S, Takeichi M, Redies C. Cadherin expression in the retina and retinofugal pathways of the chicken embryo. J Comp Neurol. 1998;396:20 –38. 5. Miskevich F, Zhu Y, Ranscht B, Sanes JR. Expression of multiple cadherins and catenins in the chick optic tectum. Mol Cell Neurosci. 1998;12:240 –255. 6. Wo ¨ hrn JC, Nakagawa S, Ast M, Takeichi M, Redies C. Combinatorial expression of cadherins in the tectum and the sorting of neurites in the tectofugal pathways of the chicken embryo. Neuroscience. 1999;90:985–1000. 7. Matsunami H, Takeichi M. Fetal brain subdivisions defined by Rand E-cadherin expressions: evidence for the role of cadherin activity in region-specific, cell-cell adhesion. Dev Biol. 1995;172: 466 – 478. 8. Jeon CJ, Strettoi E, Masland RH. The major cell populations of the mouse retina. J Neurosci. 1998;18:8936 – 8946. 9. Hamano K, Kiyama H, Emson PC, Manabe R, Nakauchi M, Tohyama M. Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina. J Comp Neurol. 1990;302:417– 424. 10. Voigt T, Wa¨ssle H. Dopaminergic innervation of AII amacrine cells in mammalian retina. J Neurosci. 1987;7:4115– 4128. 11. Voigt T. Cholinergic amacrine cells in the rat retina. J Comp Neurol. 1986;248:19 –35. 12. Gabriel R, Wilhelm M, Straznicky C. Morphology and distribution of Mu ¨ ller cells in the retina of the toad Bufo marinus. Cell Tissue Res. 1993;272:183–192.