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MEC-8 regulates alternative splicing of unc-52 transcripts in C. elegans hypodermal cells

Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA

View larger version (140K): [in a new window]   Fig. 1. MEC-8 expression pattern. (A,C) Early embryos with anti-MEC-8 staining in all nuclei. (B) DAPI staining of the embryo shown in A. (D,E) Older embryos with anti-MEC-8 staining in hypodermal nuclei. (F) A lower focal plane of the same embryo as in E. This embryo was transgenic for pPD37.48, an hlh-1::lacZ fusion construct (Krause et al., 1994); muscle cells stained with anti-ß-galactosidase and not with anti-MEC-8. (G) MEC-8::GFP in a 1.5-fold embryo. (H) Anti-LIN-26 hypodermal staining of the embryo shown in G, showing overlapping staining with anti-MEC-8. (I) A mec-8(u391) embryo lacking anti-MEC-8 nuclear stain. (J) The mid-body region of an L1 larva in which hyp7 nuclei were stained with anti-MEC-8. (K) DAPI stain of the larva shown in J. (L) The mid-body region of an adult stained with anti-MEC-8. The white arrow indicates ALML (touch neuron), and the black arrow indicates the vulva. Scale bar: 10 µm.

View larger version (19K): [in a new window]   Fig. 2. Exon-intron structures of six unc-52 minigenes. Exons are represented by boxes, introns by horizontal lines and splices by angled lines that join exons together. unc-52 exons are filled with black; the position of a nuclear localization signal (NLS) is marked by a white line. The positions of nonsense codons found in unc-52(e669) and unc-52(e444) are marked. Known mec-8-dependent splices of transcripts from the complete unc-52 gene (Lundquist et al., 1996) are shown below the unc-52(+) minigene; an expected mec-8-independent splice form (Rogalski et al., 1995) that includes all of the minigene exons is shown above the unc-52(+) minigene.

View larger version (84K): [in a new window]   Fig. 3. Embryos expressing unc-52::gfp minigenes driven by hypodermis-specific or by muscle-specific promoters. (A,B) 0.5 second exposures of wild-type (A) and mec-8 (B) embryos carrying the hypodermis-expressing unc-52(+)::gfp minigene on mnIs61. (C,D) 2 second exposures of wild-type (C) and mec-8 embryos (D) carrying the muscle-expressing unc-52(+)::gfp minigene on mnIs64. (E) 0.75 second exposure of a wild-type embryo carrying the hypodermis-expressing unc-52(e669)::gfp mini-gene on integrated array mnIs63. (F) 6 second exposure of a mec-8 embryo carrying mnIs63. (G,H) 6 second exposures of wild-type (G) and mec-8 (H) embryos carrying the muscle-specific unc-52(e669)::gfp mini-gene on mnIs27. Scale bar: 10 µm.

View larger version (43K): [in a new window]   Fig. 4. Tissue-specific expression of mec-8(+) transgenes in embryos. (A) A 2.5-fold mec-8(u314) embryo carrying Phlh-1::mec-8(+) on mnEx113 stained with anti-MEC-8. (B) Embryo shown in A stained with MH2; UNC-52 localizes in stripes over muscle cell quadrants at this stage. (C) Merged images of A and B. (D) mec-8(u314) embryo carrying Pdpy-7::mec-8(+) on mnEx134 stained with anti-MEC-8. (E) Embryo shown in D stained with DM5.6; myosin is diffuse and outlines muscle cell nuclei (arrows) at this stage. (F) Merged images of D and E. (G) 7 second exposure of an embryo carrying muscle-specific unc-52(e444)::gfp minigene mnIs25. (H) 7 second exposure of a mec-8; unc-36; mnIs25; mnEx113 embryo. Scale bar: 10 µm.

View larger version (16K): [in a new window]   Fig. 5. Genetic mosaics in which an unc-52(+)-bearing extrachromosomal array was lost somatically in animals that were otherwise homozygous for unc-52(e669). The early C. elegans cell lineage and the cell types generated by each founder cell are indicated. Points in the lineage at which array losses were identified are shown. One mosaic animal lost the array at both AB and EMS. All other mosaic animals had single array losses in this part of the lineage. White and black circles represent non-Unc-52 and Unc-52 animals, respectively; thus, one P1(-) mosaic was Unc-52 and six were wild type.

View larger version (15K): [in a new window]   Fig. 6. Genetic mosaics in which an unc-52(+)-bearing extrachromosomal array was lost somatically in animals that were otherwise homozygous for unc-52(st549). A wild-type mosaic is represented by an white circle. Black circles represent viable and fertile dumpy rollers. One of the C(-) mosaics also suffered array loss at EMS, and the P2(-) mosaic also suffered array loss at E. The other mosaic animals had single array losses in this part of the lineage.