DEV074047 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1-S9 -
• Supplemental Figure S1 -

  Fig. S1. rsks-1 (S6K) is required for several aspects of reproduction. (A) Number of proliferative zone nuclei in early adult wild type and rsks-1 raised at 20°C or 25°C. (B) Average number of sperm per gonad arm in wild type and rsks-1 (n=15, 28). (C) Average progeny per animal in wild type, rsks-1 and rsks-1 hermaphrodites mated to wild-type males. Average brood size for mated rsks-1(sv31) is 159.8±13.5 (n=11). The sperm number defect likely contributes to the low brood size phenotype (see Fig. 1A) since mating hermaphrodites to wild-type males partially restored brood size. (D) Average number of embryos laid versus larvae hatched in wild type and rsks-1 (n=10 each) over time. Total average number of embryos laid in wild type is 305.8±8.7 and larvae hatched is 305.5±8.6. Total average number of embryos laid in rsks-1 is 124.3±8.0 and larvae hatched is 104.4±7.5, a 16% hatching failure; most embryos that fail to hatch appear later in the reproductive period. (E) Representative (normal) oocyte morphology in wild type and rsks-1. Scale bar: 50 µm. (F) Summary of timing of germline development in wild type and rsks-1. Table indicates the earliest developmental stage and time (hours post-hatch) at which >40% of animals scored contain the indicated stage of post-mitotic germ cells (n≥35). (G) Detailed comparison of germline and somatic development in wild type (top) and rsks-1 (bottom). 'Stage' refers to both wild type and rsks-1. Time is hours from hatching and number of gonad arms scored is indicated below each time point (n). Percentage of gonad arms displaying each germ cell type is indicated for each time point. Solid black lines indicate 100%. Both germline and somatic development were delayed in rsks-1, but germline development was more delayed. Overall somatic development was delayed by ∼8 hours (measured by time to reach adult vulval morphology from the L1), whereas two delay points were found for germline development: (1) meiotic entry, occurring at the early L4 stage in rsks-1(sv31) compared with the mid-L3 stage in N2; and (2) spermatogenesis, delaying the timing of the spermatogenesis-to-oogenesis switch. These delays are additive, and fertilized embryos are found in the uterus in rsks-1(sv31) ∼20 hours after the adult molt, as compared with ∼8 hours after the adult molt in N2. ***P≤0.001, **P≤0.01, *P≤0.05, by two-tailed Student�s t-test. Error bars, s.e.m. Values are listed in Table S3.

• Supplemental Figure S2 -

  Fig. S2. Loss of rsks-1 does not affect adult GSC cell cycle but reduces proximal tumor size in gld-1(RNAi) adults. (A,B) Early adult stage (A) mitotic index and (B) S-phase index. Number of gonad arms and germ cells analyzed: (A) wild type, 28 and 5947, and rsks-1, 20 and 2141; (B) wild type, 22 and 4660, and rsks-1, 22 and 2491. n.s., not significant by Mann-Whitney U test. (C) (Left) Number of nuclei in gld-1(RNAi) proximal tumors scored 48 hours post-L4 in wild type and rsks-1(sv31). (Right) Representative DAPI-stained germ lines with proximal tumors outlined by dashed lines. Asterisk indicates distal end. Scale bar: 50 µm. ***P≤0.001 by two-tailed Student�s t-test. Error bars, s.e.m. Values are listed in Table S4.

• Supplemental Figure S3 -

  Fig. S3. Loss of rsks-1 enhances the phenotype of glp-1(rf). Percentage of gonad arms scored at the early adult stage containing ∼100, 50-100, 20-50, 1-20 or 0 proliferative zone nuclei in glp-1(e2141) and glp-1(e2141) rsks-1(sv31) at 20°C (reproduced from Fig. 3B), and in glp-1(e2141) treated with control or rsks-1 RNAi (n=216 and 121), and rrf-1(pk1417); glp-1(e2141) treated with control or rsks-1 RNAi (n=124 and 107). The 0 class of 'number of proliferative zone nuclei' includes both 'all sperm' (≤11%, background level) and otherwise 'distal-most pachytene' phenotypes.

• Supplemental Figure S4 -

  Fig. S4. rsks-1 acts largely independently of the daf-2 (insulin receptor) pathway. Number of proliferative zone nuclei in early adult (A) wild type, daf-2, rsks-1 and daf-2 rsks-1 at 20°C (see Fig. 4B for 25°), (B) rsks-1 and daf-2 fed control or daf-18 dsRNA. rsks-1(-) is rsks-1(sv31). ***P≤0.001, **P≤0.01; n.s., not significant by two-tailed Student�s t-test. Error bars, s.e.m. Values are listed in Table S6.

• Supplemental Figure S5 -

  Fig. S5. Germline phenotypes associated with rsks-1(T404A) and with reducing let-363 (TOR), daf-15 (RAPTOR) or eIF4E activity. (A) Alignment of the putative TOR phosphorylation site in C. elegans with the site in Drosophila and human; asterisk indicates the altered Thr residue. (B,C) Representative adult (B) rsks-1; naIs44pGC520, germline rsks-1(+) and (C) rsks-1; naIs48pGC609, germline rsks-1(T404A) gonad arms stained with DAPI and anti-GFP antibody (imaged in respective channels in first and second row), and overlaid DAPI, GFP and DIC (third row). Scale bar: 20 µm. Asterisk indicates distal end of the gonad, solid line indicates the proliferative zone/transition zone border, and dashed line outlines the proliferative zone. Bottom row shows RSKS-1::GFP and RSKS-1(T404A)::GFP expression in live animals where the GFP is difficult to detect in the distal end compared with oocytes. Scale bar: 20 µm. (D) Number of proliferative zone nuclei in early adult wild-type animals treated with control, let-363 or daf-15 RNAi starting from the maternal late-L4/adult molt at 25°C. (E,F) Mid-L4 stage mitotic index (E) and quantification of DNA content (F) in rrf-1 animals treated with control or let-363 RNAi from the maternal mid/late L4 stage at 25°C. Number of gonad arms and germ cells analyzed is (E) control, 32 and 3371, and let-363, 43 and 1303; (F) control, 10 and 1242, and let-363, 13 and 447. (G,H) Number of proliferative zone nuclei in (G) early adult wild type, ife-1(bn127), ife-2(ok306), ife-4(ok320), ife-5 (ok1934) at 20°C, and rrf-1 animals treated with control or ife-3 RNAi (pT72-ife-3; kind gift from Brett Keiper) from the maternal mid/late L4 stage at 25°C, and (H) wild type, rsks-1, ife-1, and rsks-1 ife-1 at 20°C. rsks-1(-) is rsks-1(sv31). ***P≤0.001, **P≤0.01, *P≤0.05; n.s., not significant by two-tailed Student�s t-test, except for three-sample comparison in D by one-way ANOVA and (E,F) Mann-Whitney U test. In G, comparisons are with wild type with the exception of ife-3, which is compared with control RNAi. Error bars, s.e.m. Values are listed in Table S7.

• Supplemental Figure S6 -

  Fig. S6. Effects of aak reduction on germline progenitors and brood size vis-à-vis rsks-1. (A,B) Number of proliferative zone nuclei in (A) early adult wild type, rsks-1(sv31) and rsks-1(sv31); aak-2(ok524) animals treated with control or aak-1 RNAi, and (B) wild type and aak-1(tm1944); aak-2(ok524) animals treated with control or rsks-1 RNAi. Note that in B the control RNAi results in a greater number of proliferative germ cells in the aak-1; aak-2 double mutant relative to wild type (P<0.05). The relative percent reduction by rsks-1(RNAi) compared with each control is similar, but slightly lower in the double mutant (26% versus 18%). Therefore, the possibility that removal of both aak genes weakly suppresses rsks-1 cannot be excluded. However, suppression of the rsks-1 mutant longevity phenotype has been reported with the aak-2 mutant alone and we see no suppression of the rsks-1 defect in the rsks-1; aak-2 double mutant (see Fig. 6C). This result supports our main conclusion that the longevity and germline phenotypes are likely to be acting through distinct pathways. (C) Average brood sizes for wild type, aak-2, rsks-1 and rsks-1;aak-2 are 305.5±8.6, 246.7±10.8, 94.0±12.0 and 103.5±3.9, respectively; n=10 broods for all except aak-2 where n=7. ***P≤0.001, **P≤0.01; n.s., not significant by Student�s t-test. Error bars, s.e.m. Values are listed in Table S8.

• Supplemental Figure S7 -

  Fig. S7. The eat-2 mutant causes a decrease in proliferative germ cell number that is partially mediated by rsks-1. Number of proliferative zone nuclei in early adult wild type, rsks-1(sv31), eat-2(ad1116), and eat-2(ad1116); rsks-1(sv31). ***P≤0.001 by Student�s t-test. Error bars, s.e.m. Values are listed in Table S9.