Insulin signaling primarily affects cell cycle. (A) The distance in cell diameters from the distal tip to the transition zone in L4 N2 worms, daf-2, hcf-1 and glp-1 mutants. Error bars indicate s.e.m.; P<0.001, two-tailed Student's t-test versus N2 control. (B) Mitotic index in the same individuals as in A. The number of gonad arms and number of nuclei for both A and B is: N2, 53 and 8144; daf-2(e1370), 54 and 6373; daf-2(m577), 25 and 3235; daf-2(e1368), 25 and 3146; hcf-1(ok559), 36 and 3789; glp-1(e2141), 40 and 2641; glp-1(bn18), 38 and 2743; daf-2(e1370) glp-1(e2141), 35 and 2832. ^**P<0.01; ^***P<0.001; ^****P<0.0001; otherwise P>0.1; two-tailed Mann-Whitney U-test versus N2 control. (C) Representative DAPI-stained mid- to mid/late-L4 worms grown as in A, B. Insets show vulvae from the same individual worms in each panel. (D) EdU labeling of S-phase nuclei. The number of gonad arms and number of nuclei is: N2, 26 and 3996; and daf-2(e1370), 25 and 3419. P<0.0001, two-tailed Mann-Whitney U-test. See Fig. S1 in the supplementary material for box plots of data in A, C and D. Labels and scale in C and D are as in Fig. 1B. (E) Quantification of DNA content in N2 and daf-2(e1370) L4 larvae. The number of gonad arms and number of nuclei is: N2, 15 and 1448; and daf-2(e1370), 15 and 1270. ^*P<0.05; ^**P<0.01; ^***P<0.001; ^****P<0.0001; otherwise P>0.05; two-tailed Mann-Whitney U-test versus N2 control.

DAF-2 signaling pathway components act in the germ line for robust larval germline proliferation. (A) Number of nuclei in proliferative zone of early adult daf-2(e1370) and rrf-1(pk1417);daf-2(e1370) worms treated with RNAi targeting daf-18 or daf-16. Error bars indicate s.e.m.; P<0.0001, two-tailed Student's t-test versus appropriate L4440 negative control. (B) Number of nuclei in proliferative zone of early adult N2, rrf-1(pk1417) and daf-16(mu86) worms treated with RNAi targeting daf-2 or hcf-1. Error bars indicate s.e.m. Statistics: ^**P<0.01; ^***P<0.001; otherwise P>0.1; two-tailed Student's t-test versus appropriate L4440 control. (C) Number of nuclei in proliferative zone of early adult worms in indicated genotypes. `Ex' genotypes express the DAF-16::GFP fusion driven by the indicated promoter and non-array-bearing siblings are shown as comparisons (sibling pairs are indicated by vertical lines). Tissue-specific activity of the promoters is indicated; the lag-2 promoter is not exclusive to the DTC. For the rpl-11.1 promoter (`Is' genotype) the daf-16(mu86);daf-2(e1370) double mutant background strain is shown for comparison. Note, expression of DAF-16::GFP in GC1144 naIs43[Prpl-11.1::DAF-16::GFP] has since been silenced in this line (see Table S1 in the supplementary material for details). Error bars indicate s.e.m. ^*P<0.05; ^****P<0.0001; otherwise, P>0.1. Top pair, two-tailed Student's t-test versus daf-16, daf-2; all others, one-tailed Student's t-test versus siblings. See Fig. S2 in the supplementary material for corresponding box plots.

ins-3 and ins-33 are required in the soma and depend on daf-18 and daf-16 to promote robust larval germline proliferation. (A) Number of proliferative zone nuclei in germ lines of adult N2 worms raised from hatching on bacteria carrying L4440 (black line and squares) or RNAi targeting ins-3 (dashed line, diamonds) or ins-33 (dotted line, triangles). Error bars indicate s.e.m. (B) Number of proliferative zone nuclei in germ lines of early adult N2, rrf-1(pk1417), daf-18(ok480) and daf-16(mu86) worms grown from hatching on L4440 or RNAi targeting ins-1 (negative control), ins-3 or ins-33. Error bars indicate s.e.m. Statistics: ^*P<0.05; ^****P<0.0001; unmarked, P>0.05; two-tailed Student's t-test versus appropriate L4440 control. (C) Number of proliferative zone nuclei in germ lines of early adult ins-3 or ins-33 mutants on indicated RNAi. Error bars indicate s.e.m. ^***P<0.001; ^****P<0.0001; unmarked, P>0.05 except ins-3(RNAi) in ins-3(tm2488) (P=0.02); two-tailed Student's t-test versus appropriate L4440 control. (D) The L4 mitotic index in the same strains as in C, and in these strains carrying extrachromosomal arrays containing ins-3(+) or ins-33(+). Siblings that had lost the array (non-Rol) are indicated as ins-3(0) and ins-33(0), respectively. `Ex[ins-33(+)]' and `Ex[ins-3(+)]' refer to naEx197 and naEx187, respectively. Similar results were obtained from naEx186 and naEx195 (see Table S3 in the supplementary material). The number of gonad arms and number of nuclei is: N2, 53 and 8144; ins-33(tm2988), 38 and 5138; ins-3(ok2488), 40 and 4840; ins-33(tm2988) with naEx187, 25 and 3728, and siblings, 25 and 3364; ins-3(ok2488) with naEx197, 25 and 3704, and siblings, 25 and 3339. ^***P<0.001; ^****P<0.0001; Mann-Whitney U-test (two-tailed for mutants versus N2; one-tailed for sibling pairs). P>0.1 for Ex strains versus N2. See Fig. S5 in the supplementary material for box plots corresponding to B, C and D.

daf-16 is not required for the regulation of germline proliferation by the somatic gonadal sheath. Representative unablated control (left) and 2SS-cell ablated (right) gonad arms of GC678 tnIs6[Plim-7::GFP];qIs19[Plag-2::GFP] worms raised on bacteria carrying L4440 (top) or daf-16(RNAi)-inducing (bottom) plasmids. For L4440 and daf-16 RNAi, respectively, seven out of seven and nine out of nine successfully ablated gonads were underdeveloped as adults. Unoperated gonad arms from operated animals developed normally, as did an additional 14 L4440 and 24 daf-16 RNAi unoperated control worms. Scale bar: 20 μm.