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First published online 8 October 2003
doi: 10.1242/dev.00791

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C. elegans ankyrin repeat protein VAB-19 is a component of epidermal attachment structures and is essential for epidermal morphogenesis

Mei Ding1, Alexandr Goncharov2, Yishi Jin2 and Andrew D. Chisholm1,*

1 Sinsheimer Laboratories, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Cruz, CA 95064, USA
2 Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Cruz, CA 95064, USA



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  		Fig. 3. VAB-19::GFP localization to epidermal attachment structures. Because the endogenous VAB-19::GFP fluorescence signal is weak, we used anti-GFP antibodies to visualize VAB-19::GFP expression. (A) At comma stage, VAB-19::GFP was diffusely expressed in dorsal epidermal cells; the uneven expression might reflect differential onset of VAB-19 expression in different cells. (B) During early elongation (1.5-fold), the VAB-19::GFP signal began to accumulate in the regions within dorsal epidermal cells that contact body muscles (arrow). (C) During the intermediate stage of elongation (1.75-fold), VAB-19::GFP mostly localized to epidermal regions adjacent to body wall muscle. (D) During later elongation (threefold stage), VAB-19::GFP was organized in circumferential bands in muscle-adjacent epidermis. Inset (E), higher magnification of the VAB-19::GFP pattern at the threefold stage. (F) In adult epidermal cells, the full length VAB-19::GFP protein is localized to attachment structures. GFP fusions to VAB-19 N-terminal fragments containing residues 1-684 (not shown) or 1-294 (G) display subcellular localization identical to that of the full-length protein. (H) GFP fusions to the VAB-19 ankyrin repeat-containing domain (residues 1-43 and 697-1040) were not localized within epidermal cells. None of the truncated protein constructs rescued vab-19 mutant phenotypes; transgenes containing VAB-19(1-294)::GFP conferred a weak Vab phenotype in a wild-type background. Scale bars, 10 µm.

 


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  		Fig. 4. VAB-19 localizes to epidermal attachment structures. (A-I) VAB-19::GFP progressively co-localizes with intermediate filaments. Confocal images of VAB-19::GFP expression (juIs167) visualized using anti-GFP immunostaining (green); intermediate filaments were visualized using the MH4 monoclonal antibody (red). At the comma stage (A-C), VAB-19::GFP partly colocalized with intermediate filaments. At the intermediate elongation stage (D-F), VAB-19::GFP and intermediate filaments both localized to muscle-adjacent regions of the dorsal and ventral epidermis. During later embryogenesis and larval stages until adulthood, staining of anti-GFP and MH4 was coincident. (G-I) L1 stage epidermis in z-axis section. (J-L) Lateral view of adult epidermis. (M-R) Colocalization of VAB-19::GFP and Myotactin (MH46 antigen). At the intermediate elongation stage (M-O), VAB-19::GFP and Myotactin are both localized to epidermal cell regions adjacent to muscle. In adults (P-R), the bands of Myotactin and VAB-19::GFP are interrupted by gaps corresponding to the positions of neuronal processes (arrows), at which positions MH4 staining is usually enhanced (K, arrow). Like Myotactin, but unlike MH4 staining, VAB-19::GFP is absent from such process gaps (compare J and P). VAB-19::GFP was also expressed in pharyngeal marginal cells (S); unlike Myotactin (T), VAB-19::GFP was localized throughout the apical basal axis of these cells and was more concentrated at the apical and basal surfaces. Scale bars: 5 µm (G-I); 10 µm (all others).

 


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  		Fig. 5. Attachment structures and attachment structure components in vab-19 mutants. (A-F) Localization of intermediate filaments (MH4 immunostaining) in the wild type and in vab-19 mutants. (A) During early elongation in the wild type, IFs accumulate in each epidermal cell adjacent to one muscle quadrant. By the 1.5-fold stage, IFs were organized in circumferential bands in regions of epidermis adjacent to muscle, although these bands are not as regular as in later stages. (C) After the twofold stage, IFs localize to regularly spaced bands. (D-F) In vab-19 mutants, IF staining appears normal until after the twofold stage, when it expands into regions of epidermal cells that do not overlap muscle, compared with the wild type (brackets). (G-M) Myotactin expression (visualized using the MH46 antibody) in wild-type and vab-19 embryos. During early (comma to 1.5-fold; G,K) and intermediate (twofold; H,L) elongation stages, Myotactin appears normal in vab-19 mutants. In wild-type threefold-stage embryos, Myotactin localizes to circumferential bands in muscle-adjacent regions of the epidermis (I, inset). In vab-19 mutants, Myotactin is still localized to muscle-adjacent regions but remains in longitudinal rows rather than circumferential bands (M, inset). Scale bars, 10 µm. Electron microscopy (longitudinal sections) of attachment structures in wild-type L1 larva and vab-19(e1036) arrested larvae. In the wild type (N), attachment structures are confined to epidermis in muscle-adjacent regions, which is ~200 nm thick. In vab-19 mutants (O), attachment structures are present and in phase with cuticle annuli but can be longer than normal. Scale bar, 500 nm (N), 150 nm (insets).

 


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  		Fig. 6. VAB-19 localization is dependent on VAB-10A/Plectin and Myotactin. VAB-19::GFP localization in attachment structure mutants examined using anti-GFP immunostaining. In let-805 (Myotactin) mutants, VAB-19::GFP localization is normal in early (1.5- to twofold stage) embryos (A); soon after the twofold stage, VAB-19::GFP dispersed into puncta throughout the dorsal and ventral epidermis (B). Live analysis of VAB-19::GFP in let-805 mutants revealed that this delocalization occurs within 15-20 minutes of the twofold stage (not shown). In vab-10A(ju281ts) animals grown at 25°C, VAB-19::GFP localization is normal at the 1.75-fold stage (C) but becomes disorganized after the twofold stage (D). Scale bar, 10 µm.

 


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  		Fig. 1. vab-19 mutants are defective in embryonic morphogenesis and muscle attachment. (A-H) Frames from time-lapse Nomarski movies of wild-type and vab-19(ju406) embryos. vab-19 mutant embryos appear normal at comma stage and twofold stage (E,F). 70 minutes after the twofold stage, the wild-type embryo has elongated to the threefold stage (C), whereas the vab-19 mutants have stopped elongation and show constrictions and lumps in the epidermis (G). vab-19 mutants typically hatch as deformed L1 stage animals with extensive muscle detachment (H, arrowhead; I,J, arrow). (K-N) Epidermal junctions visualized by AJM-1::GFP in wild-type and vab-19 embryos at 1.75-fold (K,M) and post-twofold stages (L,N), showing that epidermal adherens junctions form normally in vab-19 mutants. (O) The vab-19(e1036cs) temperature-sensitive period (TSP) is during early elongation. The x axis represents developmental stages at which embryos were up- or down-shifted. The y axis represents the proportion of embryos that showed vab-19 lethal and Vab phenotypes at 22.5°C. At least 24 embryos were scored for each time point. Up-shift involved transferring embryos raised at 15°C to 22.5°C, whereas down-shift involved transferring embryos raised at 22.5°C to 15°C. The midpoint of the TSP is between the 1.5-fold and twofold stages.

 


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  		Fig. 2. The vab-19 gene encodes a conserved ankyrin repeat-containing protein. (A) Genetic and physical maps of the vab-19 region and structure of the vab-19 gene. All rescue experiments used vab-19(e1036) and the rol-6 (pRF4) co-injection marker. Initial experiments assayed rescue of the morphological defects of e1036 at the semipermissive temperature 22.5°C. Rescue was classified as follows: no rescue (injection of pRF4 alone) means that 20% of Rol animals are Vab; weak rescue (+) means that 5-10% Rol animals are Vab; partial rescue (++) means that 2-5% Rol animals are Vab; full rescue (+++) means that <2% animals are Vab. All rescuing transgenes also rescue the lethal Vab-19 phenotypes. (B) Comparison of C. elegans VAB-19, human Kank/KIAA0172, mouse NG28 and Drosophila CG10249, isoform C. A second murine VAB-19 homolog is represented by an apparent partial sequence (AAH06647, not shown). The predicted effects of vab-19 mutations are indicated. The four ankyrin repeats are shown as striped boxes. Sequence alignments suggest that the four highly conserved repeats might be flanked by partial or divergent ankyrin repeats (not shown). (C) ClustalW alignments of VAB-19 family N-terminal motifs A and B.

 


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  		Fig. 7. Disorganization of actin filaments in vab-19 and genetic suppression in vab-19 sma-1 double mutants. Confocal images of F-actin distribution in wild-type and vab-19 embryo, visualized with fluorescently labeled phalloidin. In all panels, the bright longitudinal strips of phalloidin staining correspond to body wall muscles. (A-D) In the wild type, actin filaments form parallel bundles circumferentially around the embryo. In vab-19 embryos, actin bundles form normally at early elongation stage (E) but are more randomly oriented in later elongation (F-H) or missing from the apical surface of the epidermis (F). The disorganization of actin bundles is more obvious in regions where muscle is detached from epidermal cells (F). In sma-1(e30);vab-19(ju406) double mutants (I-K), actin bundles are less disorganized than in vab-19 single mutants. Scale bar, 10 µm.

 

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© The Company of Biologists Ltd 2003