Extended Data Fig. 5: HERD-1 is required for germline RNAi but limits the transgenerational inheritance of RNAi.
a, WT or herd-1(-) animals were subjected to dpy-6 RNAi treatment. Young adult animals, with or without dpy-6 RNAi, were shown. n = 3 biological replicates. b, Fluorescence imaging of -3 to -1 oocytes in WT, hrde-1(-), and herd-1(-) animals expressing GFP::H2B (pkIS32) treated with gfp RNAi are shown. Scale bar, 10 μm. c, Quantification of GFP fluorescence intensity in (b). n = 10 animals. Mean values ± s.d. Student’s t-test, two-tailed. P = 0.0002 (herd-1(ths351) gfp RNAi vs WT gfp RNAi), 0.0002 (herd-1(ths351) gfp RNAi vs hrde-1(ths682) gfp RNAi), 0.00013 (herd-1(ths351) gfp RNAi vs control RNAi). d-e, Progeny of animals from (b) were grown on non-RNAi plates for the indicated generations. Fluorescence imaging (d) and quantification of animals with silenced GFP (e) are shown. Scale bar, 10 μm. n = 3 biological replicates (50 animals per replicate). Error bar, ± s.e.m. Log-rank test. P = 0.0253 (hrde-1(ths685)), and <0.0001(herd-1(ths351)). f, Fluorescence imaging of oocytes at indicated times after gfp RNAi treatment from (b) shows % of animals with silenced GFP. Scale bar, 10 μm. g, WT and herd-1(-) animals expressing single-copy mCherry::H2B in the germline were subjected to control or mCherry RNAi, and fluorescence was imaged. Scale bar, 10 μm. h, Fluorescence intensity from -1 to -3 oocytes was quantified from (g). n = 10 animals. mean values ± s.d. Student’s t-test, two-tailed. P = 8.19 Ă— 10−8 (WT gfp RNAi vs control RNAi), 0.004 (herd-1(ths351) gfp RNAi vs control RNAi, and 0.0005 (herd-1(ths351) gfp RNAi vs WT gfp RNAi). i, Progenies from (g) were grown on non-RNAi plates. Representative imaging of oocytes from animals at indicated generations shows % of animals with silenced mCherry. Scale bar, 10 μm. j, The percentage of animals that failed to express mCherry::H2B was scored under a 63X oil objective every generation from (i). n = 3 biological replicates. Error bar, ± s.e.m. Log-rank test. P = 0.0455 (hrde-1(tm1200)), and <0.0001 (herd-1(ths351)). (k–l) The pUG PCR assay detected pUG RNA in the P0 and F1 (k), as well as the F15 and F30 (l) generations, after WT, znfx-1(-), hrde-1(-), or herd-1(-) animals expressing pie-1::gfp::h2b (mjIS31) were exposed to gfp RNAi. n = 3 biological replicates. (m–n) The pUG PCR assay detected pUG RNA in the P0 and F1 (m), as well as the F15 and F30 (n) generations, after WT, hrde-1(-), or herd-1(-) animals expressing pie-1::gfp::h2b (pkIS32) were exposed to gfp RNAi. (o–p) The pUG PCR assay detected pUG RNA in the P0 and F1 (o), as well as the F15 and F30 (p) generations, after WT, znfx-1(-), hrde-1(-), or herd-1(-) animals expressing pie-1::mcherry::h2b were exposed to mcherry RNAi. q, smFISH detected pUG RNA in animals with indicated genotype. Scale bar, 5 μm. n = 3 biological replicates. r, Fluorescence imaging of -3 to -1 oocytes in young adult animals with indicated genotype expressing a piRNA sensor. Scale bar, 10 μm. s, Classification of genes differentially targeted by endo siRNAs in herd-1(ths351) relative to WT animals. * P < 0.05, ** P < 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Source numerical data and unprocessed gels are available in source data.
