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First published online 23 February 2005
doi: 10.1242/dev.01697

Development 132, 1525-1537 (2005)
Published by The Company of Biologists 2005
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Roles of cell-adhesion molecules nectin 1 and nectin 3 in ciliary body development

Maiko Inagaki1, Kenji Irie1, Hiroyoshi Ishizaki2,3, Miki Tanaka-Okamoto3, Koji Morimoto2, Eiji Inoue2, Toshihisa Ohtsuka2, Jun Miyoshi3 and Yoshimi Takai1,*

1 Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan
2 KAN Research Institute, 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815, Japan
3 Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan



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  		Fig. 1. Targeted disruption of the nectin 1 and nectin 3 genes. (A) Targeted disruption of the nectin 1 gene. (A) (a) The structure of the mouse nectin 1 gene with coding exons 2-5 is shown at the top. A targeting vector was designed to remove the exon 2. The construct contained 2.8 kb 5' flanking sequence and 4.0 kb 3' flanking sequence. (b) Southern blotting of ES clones. HindIII-digested DNAs derived from ES cells were hybridized with the 5'- or 3'-probe. WT, wild-type; +/–, nectin 1+/–. (c) Genotyping by PCR of genomic DNA extracted from littermate mice at 21 days of age. Wild-type (WT) and targeted alleles give the bands of 639 bp and 459 bp, respectively. +/–, nectin 1+/–; –/–, nectin 1–/–. (d) Western blotting of the extracts from the brains of wild-type and nectin 1–/– mice with the anti-nectin 1 and anti-actin antibodies. (B) Targeted disruption of the nectin 3 gene. (a) The structure of the mouse nectin 3 gene with coding exon 1 is shown at the top. A targeting vector was designed to remove the exon 1. The construct contained 6.5 kb 5' flanking sequence and 4.2 kb 3' flanking sequence. (b) Southern blotting of ES clones. BamHI-digested DNAs derived from ES cells were hybridized with the 5'- or 3'-probe. WT, wild-type; +/–, nectin 3+/–. (c) Genotyping by PCR of genomic DNA extracted from littermate mice at 21 days of age. Wild-type and targeted alleles give the bands of 379 bp and 814 bp, respectively. WT, wild-type; +/–, nectin 3+/–; –/–, nectin 3–/–. (d) Western blotting of the extracts from the brains of wild-type and nectin 3–/– mice with the anti-nectin 3 and anti-actin antibodies.

 


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  		Fig. 2. Microphthalmia in both nectin 1–/– and nectin 3–/– mice. (A) View of adult wild-type (WT) and nectin 1–/– (–/–) mice. (B) View of adult wild-type and nectin 3–/– (–/–) mice. The results shown are representative of nectin 1–/– and nectin 3–/– mice.

 


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  		Fig. 3. Histological analysis of the eyes of adult nectin 1–/– and nectin 3–/– mice. (A-C, part a) Sections of the eyes of adult wild-type, nectin 1–/– and nectin 3–/– mice. (A-C, part b) Higher magnifications. Arrows, ciliary processes; L, lens; V, vitreous body. Scale bars: 1 mm in a; 100 µm in b.

 


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  		Fig. 4. Developmental analysis of the eyes of nectin 1–/– and nectin 3–/– mice. (A-D) Sections of the eyes of E15.5 (A), E16.5 (B) and P0 (C,D) mice. (A-D, part a) Wild-type embryos. (A-C, part b) A nectin 1–/– embryo. (D, part b) A nectin 3–/– mouse. Scale bars: 500 µm.

 


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  		Fig. 5. Developmental analysis of the ciliary bodies of nectin 1–/– and nectin 3–/– mice. (A-C) Wild-type (A), nectin 1–/– (B) and nectin 3–/– (C) mice. E15.5 (a), E16.5 (b) and P0 (c) mice. PE, pigment epithelia; NPE, non-pigment epithelia; L, lens; R, retina. Scale bars: 50 µm.

 


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  		Fig. 8. Developmental analysis of the localization of nectin 1, nectin 2, nectin 3 and afadin at the apex-apex junctions between the pigment and non-pigment cell layers of the ciliary epithelia. E15.5 (left), E16.5 (middle) and P0 (right) mice. The bottom row shows DIC microscopic images. PE, pigment epithelia; NPE, non-pigment epithelia; L, lens; R, retina. Insets show higher magnifications of the boxed areas. Scale bars, 50 µm.

 


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  		Fig. 6. The expression of collagen IX (top row) and cadherin 11 (middle row) in the ciliary body of nectin 1–/– and nectin 3–/– mice. Wild-type (left), nectin 1–/– (middle) and nectin 3–/– (right) P0 mice are shown. The bottom row shows DIC microscopic images. PE, pigment epithelia; NPE, non-pigment epithelia; L, lens. Scale bars: 50 µm.

 


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  		Fig. 7. Localization of nectins at the apex-apex junctions between the pigment and non-pigment cell layers of the ciliary epithelia in a wild-type adult albino mouse. (A,B, part a) Nectin-1. (C, part a) Nectin-2. (D, part a) Nectin-3. (Ea, part) P-Cadherin. (A, part b) ZO-1. (B-E, part b) Afadin. (A-E, part c) Merge. (A-E, part d) DIC image. Insets show higher magnifications of the boxed areas. Scale bars: 50 µm.

 


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  		Fig. 9. Localization of nectin 1 and nectin 3 at the puncta adherentia junctions between the pigment and non-pigment epithelia of the ciliary body. (A,B) Localization of nectin 1 (A) and nectin 3 (B) at the ciliary body of the adult wild-type eye in immunoelectron microscopy. PE, pigment epithelium; NPE, non-pigment epithelium; M, melanin; N, nucleus. Arrows indicate immunogold particles; arrowhead indicates a gap junction. Scale bars: 500 nm. (C,D) The expression patterns of nectin 1 (C) and nectin 3 (D) mRNAs at the ciliary body of the adult wild-type eye were analyzed by in situ hybridization. Nectin 1 (C, part a) and nectin 3 (D, part a) mRNA. (c) Sense controls for the nectin 1 and nectin 3 mRNAs. (b,d) Corresponding DIC images. Insets show higher magnifications of the boxed areas. Scale bars: 50 µm.

 


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  		Fig. 10. Localization of nectins at the apicolateral boundaries of the pigment and non-pigment epithelia. (A) Localization of nectin 1, nectin 2, nectin 3, afadin, P-cadherin and ZO-1 at the apicolateral boundaries of the pigment and non-pigment epithelia. Wild-type (left,) nectin 1–/– (middle) and nectin 3–/– (right) mice. (B) Localization of nectin 1 at the apicolateral boundaries of the pigment epithelia, but not at those of the non-pigment epithelia in nectin 3–/– mice. (C) Localization of nectin 3 at the apicolateral boundaries of both the pigment and non-pigment epithelia in nectin 1–/– mice.. PE, pigment epithelia; NPE, non-pigment epithelia; R, retina. Insets show higher magnifications of the boxed areas. Scale bars: 50 µm.

 


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  		Fig. 11. CAMs and their associated proteins at the apex-apex junctions between the pigment and non-pigment epithelia, the apicolateral junctions between the pigment epithelia and the apicolateral junctions between the non-pigment epithelia in the ciliary body. (A) Schematic diagram of the eye. The area surrounded by the broken red line is shown in B. PE, pigment epithelia; NPE, non-pigment epithelia. (B) The localization of CAMs and their associated proteins in the ciliary body. Nectin 1, nectin 3, P-cadherin, afadin and ZO-1 localize at the puncta adherentia junctions between the pigment and non-pigment cell layers of the ciliary epithelia. Connexins localizes at the gap junctions between the pigment and non-pigment cell layers of the ciliary epithelia. Nectin 1, nectin 2 and nectin 3, afadin, ZO-1 and P-cadherin localize at the AJs between the pigment epithelia. Nectin 2, nectin 3 and afadin localize at the AJs between the non-pigment epithelia. Cadherin 11 and/or other unknown cadherins, different from P-, E- and N-cadherins, associated with {alpha}- and ß-catenins are likely to localize at these AJs, as cadherin 11 and {alpha}- and ß-catenins localize there. Claudins, occludin and ZO-1 localize at the TJs between the non-pigment epithelia. PE, pigment epithelia; NPE, non-pigment epithelia.

 

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© The Company of Biologists Ltd 2005