Figure S2. CLC-2 in C. elegans (A) A reporter, which fuses GFP to the predicted last amino acid of the CLC-2 protein (CLC-2-GFP) and is driven by 2.0 kb of the genomic sequence upstream of the endogenous CLC-2 gene, was constructed. In adult stable transformants expressing CLC-2-GFP, in- tense GFP signals were detected in hypodermal seam cells (arrows in [a] and [b]), which fuse to form a single elongated syncytium, although the GFP signal did not appear to be concentrated at cell-cell borders. When adult wild-type worms were whole-mount stained with anti-CLC-2 pAb, the seam cell syncytium appeared to be outlined to give two parallel lines, although these lines were thick and not very sharp (arrow in [c]). The scale bars represent 10 um. (B) The dsRNA of GFP (control) or CLC-2 was injected into one side of the gonads of the worms, and F1 worms were applied to the tracer experiment (see Figure 4). In all of the control F1 worms (n = 97), the outline of the body surface was clearly visualized without any leakage across the hypodermal cell layers (see Figure 4A). By contrast, when dsRNA specific for CLC-2 was injected, 56% of F1 worms exhibited the dye infiltration across the hypodermal cell layers (n = 216). The scale bar represents 7 um.
Figure S1. Additional Information on the Localization of CLC-1-GPF in Worms. CLC-1-GFP was detected in spermatheca (arrows), vulva (arrowhead), and a pore cell (double arrowhead). The scale bars represent 50 um in the upper panel and 20 um in the lower panels.
Figure 2. Localization of CLC-1 in C. elegans. (A) Transformants expressing CLC-1-GFP. CLC-1-GFP was driven by 1.24 kb of the genomic sequence upstream of the endogenous CLC-1 gene. The DIC image represents four portions of the pharyngeal region: procorpus (P), metacorpus (M), isthmus (I), and terminal bulb (T) (left panel). In both the procorpus (upper middle panel) and isthmus (lower middle panel) portions, three parallel GFP-positive lines (arrows) were observed to run longitudinally and to surround the interior lumen, as shown in the merged image (right panel). Occasionally, one or two additional lines were detected in the isthmus portion (arrowhead).(B) Stereo pairs of immunofluorescence micrographs of the isthmus portion. The CLC-1 distribution shown in (A) was confirmed by whole-mount immunofluorescence staining with anti-CLC-1 pAb.(C) Comparison of the distribution of CLC-1 with that of AJM-1, a junctional marker, in the isthmus portion. The DsRed fusion protein with CLC-1 (CLC-1-DsRed) was exogenously expressed in transformants stably expressing AJM-1-GFP. CLC-1 appeared to be colocalized with AJM-1-GFP. I, isthmus; P, procorpus.The scale bars represent 5 μm.
CLC-2 in C. elegans (A) A reporter, which fuses GFP to the predicted last amino acid of the CLC-2 protein (CLC-2-GFP) and is driven by 2.0 kb of the genomic sequence upstream of the endogenous CLC-2 gene, was constructed. In adult stable transformants expressing CLC-2-GFP, intense GFP signals were detected in hypodermal seam cells (arrows in [a] and [b]), which fuse to form a single elongated syncytium, although the GFP signal did not appear to be concentrated at cell-cell borders. When adult wild-type worms were whole-mount stained with anti-CLC-2 pAb, the seam cell syncytium appeared to be outlined to give two parallel lines, although these lines were thick and not very sharp (arrow in [c]). The scale bars represent 10 um.
Fig. 7. Embryonic expression and subnuclear localization of H1.X. (A,B) show H1.X::GFP fluorescence detection in a few cells in the periphery of a >100-cell stage embryos. In (A), a fixed specimen shows a shallow fluorescence of the nucleoplasm and bright fluorescence of the nucleoli, whereas in (B), a live observation shows a shallow fluorescence in the cytoplasm and a bright fluorescence of the nucleoplasm. The brightest spots in the nucleoplasm correspond to the nucleoli. Bars, 20 μm.
(A) CED-9 expression in a WT embryo of ~30 to 50 cells. (B) Mitotracker Red localization in the same embryo as in (A). (C) Merged image of (A) and (B).
(A) A Northern blot of total RNA isolated from synchronized wild-type animals was probed with a gla-3 cDNA as described in Materials and Methods. Beta-Tubulin was used as a loading control.
(A) GLD-4 expression is equal across the distal germ line. GLD-2 intensities increase from low-to-high in a distal-to-proximal manner. Extruded gonads of indicated genotype stained with DAPI, a-GLD-2, a-GLD-4, and a-GLH-2 as a positive tissue penetration control (not shown). Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary.