Figure 1.

A Novel Inheritance Paradigm Demonstrates that Transgenerational Inheritance Is Associated with Continued Small RNA Production

(A) A diagram of the Hrde sensor inheritance paradigm. Green animals illustrate the germline-expressed GFP sensor, whereas black worms represent silenced animals.

(B) Representative images showing the germline-expressed transgene. The left panel shows the germline of an animal fed control vector, whereas the right panel shows the germline of an animal whose parent was treated with dsRNA targeting the GFP transgene. Arrows show the developing oocytes.

(C) Graph showing the percentage of GFP-silenced animals following exposure to GFP RNAi. Wild-type worms do not contain the hrde sensor; P0-F4 animals carry the sensor and differ only in their exposure to dsRNA. GFP fluorescence of the transgene and the percentage of silenced animals per plate were determined using a large particle biosorter and FlowJo. Ten silenced worms were selected from each plate to produce the next generation. At least 1,000 worms were analyzed per plate with the following number of replicates: P0 (GFP vector) n = 3, F1 n = 18, F2 n = 11, F3 n = 8, F4 n = 5. Error bars represent the SEM. Silencing was normalized to wild-type to account for autofluorescence of the intestine.

(D) qRT-PCR showing levels of nascent, unspliced pre-mRNA and mRNA for the GFP transgene in silenced, GFP RNAi treated F2 and control worms. Fold change is shown relative to control and normalized to ama-1 expression. n = 4, 3, 4, 4 for pre-mRNA control, gfp, mRNA control and gfp, respectively.

(E) Small RNA reads with unique perfect match in the transgene construct and no perfect match in the reference genome are shown for P0 and F4 L4 stage animals. F4 animals are the offspring of silenced animals in previous generations. Antisense and sense reads are shown in red and blue, respectively. Profiles indicate number of reads per million. Schematic indicates the transgene structure. Blue bars are pie-1 genomic DNA, 5′ and 3′ UTR, and exon (thin, medium, thick), respectively. Thick green/yellow bars represent GFP/his-58, respectively.

(F) Size distribution of small RNA reads in (E). For each size, the relative contribution of small RNAs with a particular 5′ nucleotide is represented in colors as indicated.

Error bars represent SEM. See also Figures S1 and S2.