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Fig. S1. P granules and nucleolar lobes. (A) Example of a P granule in a fourth larval stage (L4) hermaphrodite showing the crest (white arrow) and base (black arrowhead). (B-D) Examples of L4 (B,C) and adult (D) germ nuclei with nucleolar lobes (open arrows) in contact with the nuclear envelope. Note the absence of P granules (small white arrows) at the lobe. Scale bars: 1 µm in A; 2.5 µm in B-D.
Fig. S2. α-amanitin causes the loss of PGL-1 and GLH-2 from pachytene P granules. (A) Dissected gonad stained for PGL-1::GFP, GLH-2 and DNA as indicated, 30 minutes after injection with α-amanitin (asterisk marks the distal end of the gonad). Note the loss of perinuclear PGL-1::GFP and GLH-2 in the pachytene zone but persistence in oogonia and oocytes. (B-G) Examples of P granules after 30 minutes (B-D) or 3 hours (E-G) of α-amanitin. Although the crest (white arrow) remains prominent at 3 hours of treatment, by 30 minutes the electron-dense base is faint or absent in 98.2% of the P granules (n=159) compared with 22% of P granules in mock-injected control gonads (n=127). (H,I) Control gonads showing MEX-3 localization in the mitotic zone (asterisk) and oocytes (H), and PGL-1 on pachytene nuclei (I). (J,K) Gonads 5 hours after cycloheximide injection stained as above. Scale bars: 25 µm in A,H,J; 0.5 µm in B-G; 5 µm in I,K.
Fig. S3. Developmental loss of PGL-1 is associated with decreased transcription. (A,B) Loss of perinuclear PGL-1 during male spermatogenesis. (A) Male gonad stained for PGL-1, the nuclear envelope (NPP-9) and DNA as indicated; note the loss of perinuclear PGL-1 in the bracketed region. (B) Male gonad stained as in A for PGL-1 and DNA and with H14, which recognizes phosphorylated Ser5 on the CTD of active RNA polymerase II. (C-F) Loss of perinuclear PGL-1 during germline apoptosis. (C) ced-1 mutant hermaphrodite gonad stained for PGL-1, the nuclear envelope (NPP-9) and DNA as indicated; the ced-1 mutation prevents removal of apoptotic cells. Note the large number of cells (arrows) that lack perinuclear PGL-1 but that have envelopes. (D) ced-1; ced-3 double mutant stained as in C; the ced-3 mutation suppresses cell death. Note the absence of cells without perinuclear PGL-1. Thus, cells in C that lack perinuclear PGL-1 are presumably apoptotic cells. (E) High magnification of wild-type germ cells showing examples of cells that lack perinuclear PGL-1 but do not have the compacted DNA typical of late stages of apoptosis (compare with arrowhead in F); thus, these cells are presumably in early stages of apoptosis. (F) Germ nuclei showing decreased levels of H14 staining in germ nuclei that have lost perinuclear PGL-1. (G) Electron micrograph of a dying cell with compacted chromatin (arrows); this cell has been engulfed for removal by a gonadal sheath cell (blue). Note that P granules (green) remain visible at this stage. (H) High magnification of G showing P granules (white arrows). Scale bars: 25 µm in A-D; 10 µm in E,F; 1 µm in G,H.
Fig. S4. Specificity of αNPP-9, αDDX-19 and αNXF-1 antibodies. (A,B,D-G). Mitotic and early pachytene zones of young adult hermaphrodite gonads stained as indicated. Each pair of panels, (A,B), (D,E) and (F,G), compares an RNAi-treated gonad with an empty vector-treated control. npp-9(RNAi) and nxf-1(RNAi) gonads first show a loss of staining in the mitotic zone, presumably from new cells born after the RNAi treatment began. For comparison, the arrow in B indicates a non-dividing somatic cell (the distal tip cell) that is adjacent to the mitotic germ cells. (C) Western blot of worm lysate stained with αNPP-9; the two predicted isoforms of NPP-9 are 93 and 96 kDa (http://www.wormbase.org/db/seq/protein). The αDDX-19 and αNXF-1 antibodies do not stain western blots. Scale bars: 25 µm.
Fig. S5. Characterization of the expression zone for high-copy transgenes. (A-E) Experiments on worms carrying either an integrated, high-copy hsp16.2::gfp::unc-54 3′UTR transgene (A-D) or a non-integrated, high-copy hsp16.2::gfp::pie-1 3′UTR transgene (E). Panels A, B, D and E are in situ hybridizations for the transgene-derived mRNA. For heat-shocked animals, the indicated time (t) is the recovery in minutes at 23°C following a 30 minute pulse at 34°C. The distal tip of each gonad is indicated with an asterisk. (A) The induced mRNA in the expression zone (bracket) is (1) largely if not entirely sense mRNA, (2) dependent on heat shock and (3) recognized by both 5′ (coding region specific) and 3′ (3′UTR-specific) probes to GFP mRNA. (B) The expression zone does not occur in adult males or early L4 hermaphrodite larvae, but weak expression is observed in late L4 larvae; hybridization was with the 5′ antisense probe as in A. Arrows show examples of somatic (non-germ cell) gonadal nuclei that have high levels of transgene expression at all stages and in both sexes. (C) Variable levels of GFP protein appear in the cytoplasm and nuclei of oogonia and ooctyes after heat shock. (D) Transgenic mRNA persists in intestinal cell cytoplasm more than 1 hour after heat shock but disappears from germ cells. Some, but not all, of the disappearance is probably from transport to oocytes. (E) High-magnification image of germ nuclei with a non-integrated high-copy transgene showing one spot (arrow) or two unpaired spots (double arrow) of mRNA immediately after heat shock, presumably from the unpaired transgenes. By contrast, strains with integrated arrays show two paired foci (not shown). The dotted line indicates the outline of the germ cell nucleus. Scale bars: 25 µm in A-D; 5 µm in E.