First published online 28 September 2004
doi: 10.1242/jcs.01403
Journal of Cell Science 117, 5209-5220 (2004)
Published by The Company of Biologists 2004
Tetraspanin protein (TSP-15) is required for epidermal integrity in Caenorhabditis elegans
Hiroki Moribe1,*,
John Yochem2,*,
Hiromi Yamada1,
Yo Tabuse3,
Toyoshi Fujimoto4 and
Eisuke Mekada1,
1 Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
2 Department of Genetics, Cell Biology, and Development, University of Minnesota, 420 Washington Avenue, SE Minneapolis, MN 55455, USA
3 Fundamental Research Laboratories, NEC Corporation, 34 Miyukigaoka, Tsukuba, Ibaraki 305-8501, Japan
4 Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
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Fig. 1. The tsp-15 gene of Caenorhabditis elegans and its predicted product. (A) Schematic diagram of the tsp-15 genomic region, transgene, the relative position of the sv15 mutation, and results of rescue analyses. +, rescue; –, no rescue. (B) ClustalW comparison of TSP-15 (Q9XVM9) with human CD151 (P48509), mouse CD151 (Q35566) and fruit fly Tsp74F (Q9VVM5), which are the tetraspanins from other species that most closely resemble TSP-15. Identical and similar amino acids are indicated. The identities of the sequences between human and worm or fly and worm are both  24%. (C) Representation of the position of the sv15 mutation (a G-to-T transversion), and the effect of the mutation on splicing of intron 4. The mutation alters the splice donor site, and a cryptic splice donor in the middle of intron 4 is often used (upper versus lower case). (D) Schematic representation of the putative structure of TSP-15 and the relative position of the sv15 mutation. Well-conserved amino acid residues among the tetraspanin family are indicated. (E) Expression of the wild-type transcript is reduced in tsp-15(sv15) mutants and a cryptic donor site is often used. RT-PCR and subsequent Southern blotting of tsp-15 transcripts obtained from single mutant larvae. The region of tsp-15 examined by the RT-PCR is indicated with a double arrow in A. tsp-15(sv15) mutants produced a PCR product of the same length as that of the wild type and the ratio of correct and irregular transcripts differs for each individual mutant.
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Fig. 2. Abnormal epidermal morphology caused by loss or reduction of tsp-15 in C. elegans. (A-I) Nomarski images of N2 L4 larva (A,D), tsp-15(RNAi) L4 larva (B,E), bli-2(e768) adult (C,F), tsp-15(sv15) L3 (G) and L4 larva (H), and Ex[Pdpy-7::tsp-15His]; tsp-15(sv15) L4 larva (I) are shown. tsp-15(RNAi) animals (B,E) show numerous epidermal blisters and a swollen body morphology. Unlike bli-2(e768) mutants (C,F), unidentified material accumulates within the blisters in tsp-15(RNAi) animals (arrows in B and E). tsp-15(sv15) mutants typically have milder defects than tsp-15(RNAi) animals (compare G with B and E) and sometimes only exhibit a dumpy shape (H). (I) Rescue of sv15 by expression of the wild-type tsp-15 gene in the hypodermis. Bars, 50 µm.
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Fig. 3. Protrusions in C. elegans associated with tsp-15(sv15). (A) An L4 larva from the SP2275 strain with small protrusions of the hypodermis. A Nomarski image of the cluster of protrusions is shown at higher magnification in the inset. Nuclei can be seen in several of them, and the posterior-most protrusion is connected to the main body by a narrow stalk (arrow), a common feature of small protrusions. (B) An L4 larva from SP2275 with two large protrusions. The dorsal protrusion is connected to the main body in the region delineated by the arrowheads. The protrusion clearly contains part of the intestine. Bars, 50 µm.
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Fig. 4. Expression pattern of TSP-15::GFP in C. elegans. (A-E) Confocal images of the expression pattern of TSP-15::GFP in transgenic animals. Expression begins in 1.5-fold embryos (A) and continues into adulthood, where it is seen in the pharynx, vulva and body wall (B). (C,D) A cryosectioned Ex[tsp-15::gfp] adult worm is shown. TSP-15::GFP is not seen in the body muscle quadrants, which are visualized by rhodamine-phalloidin staining (C), but is present in the hypodermis, as confirmed by colocalization with MH5 antigen (D, arrows), a component of the FOs. (E) A punctate localization of TSP-15::GFP (arrows) in thick region of hypodermis. Bars, 25 µm.
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Fig. 5. Genetic mosaics of C. elegans implicate hyp7 as the focus of tsp-15 activity. A total of 28 mosaics were identified that had had early losses of mnEx140 but displayed no aspects of the sv15 mutant phenotype, indicating where in the early cell lineage inheritance of wild-type tsp-15 DNA is sufficient to prevent the mutant phenotype. Based on expression of sur-5::gfp in descendents of the early cells of an embryo (Sulston et al., 1983 ), cells establishing a positive clone in an individual mosaic are indicated with a square next to the name of the cell. For two mosaics, one depicted with circles and the other with diamonds, two cells of the embryo independently established positive clones. The early cells of an embryo that contribute to the hyp7 syncytium are indicated.
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Fig. 6. Cuticular and hypodermal abnormalities in tsp-15(RNAi) C. elegans. (A-D) Transmission electron micrographs of the epidermis of N2 and tsp-15(RNAi) animals. C and D are higher magnification images of tsp-15(RNAi) animals. In tsp-15(RNAi) animals, the cuticle is dissociated in the cortical layer (c). Unidentified material accumulates within the cortical and medial layers (m) of the cuticle (black arrows in B and C; compare with A), but the fiber layer (f) remains intact. There are some abnormal gaps within the hypodermis (asterisks in B and D). Note that the hypodermis and basement membrane are severely distorted, and the basement membrane has inconsistent thickness (C,D). The FOs, represented as electron-dense structures are indicated by white arrows. Cu, cuticle; Hy, hypodermis; BM, basement membrane; Mu, muscle. Bars, 200 nm.
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Fig. 7. Functional deficiency of epidermal membrane integrity in tsp-15 hypomorph C. elegans. (A,B) Pdpy-7::gfp expression patterns merged with the Nomarski images in the tsp-15(sv15) and bli-2(e768) backgrounds. In tsp-15(sv15) mutants, but not in bli-2(e768) mutants, GFP fluorescence is observed within blisters (arrows), suggesting that cellular components have leaked into the blisters in tsp-15 mutants. Small arrowheads indicate the edge of the blisters in bli-2(e768) mutants. Bars, 50 µm.
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Fig. 8. Impairment of the membrane barrier function owing to reduction of tsp-15. (A-F) Living animals were incubated for 15 minutes with the membrane-impermeable fluorescent dye Hoechst 33258. N2, tsp-15(RNAi), and bli-2(e768) adult animals (A-C) and tsp-15(RNAi), tsp-15(sv15) and dpy-5(e61) larvae (D-F) are shown. Staining of nuclei by Hoechst 33258 is indicated with arrows. Although autofluorescence of gut granules is apparent, nuclear fluorescence is usually not observed within living N2, bli-2, or dpy-5 animals (A,C,F). In contrast, nuclear staining is frequently seen in the tsp-15(RNAi) and tsp-15(sv15) animals (B,D,E). Moreover, nuclear staining can be seen in tsp-15(RNAi) larvae that do not yet exhibit blisters (D). (G) The ratio of larvae that showed Hoechst-positive nuclei was determined for each genotype (n>100). Bars, 50 µm.
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© The Company of Biologists Ltd 2004
