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. 2018 Mar 20;7:e33221. doi: 10.7554/eLife.33221

Figure 3. An RNAi screen identifies zfp-1 and zfp-1–1 as genes required for the production of tsp-2+ cells.

(A) Cartoon depicting the RNAi screening strategy used to identify regulators of tegument progenitor specification. Candidate genes were knocked-down using RNAi, worms were pulsed with EdU for 4 hrs and then fixed. Neoblasts and tegument progenitor cells were observed using EdU detection and tsp-2 RNA FISH, respectively. (B) Results of control RNAi experiments. Negative control RNAi preserves tsp-2 cells and neoblasts whereas h2b RNAi results in a loss of neoblasts and tsp-2 cells. brca1, bard, fgfr1/4, and p53 RNAi results in a partial depletion of neoblasts and a proportional decrease in tsp-2+cells. Representative maximum intensity confocal projections are shown. Numbers represent the fraction of parasites displaying the observed phenotype. (C) Maximum intensity projection showing tsp-2 expression and EdU incorporation in zfp-1(RNAi) or zfp-1-1(RNAi) parasites. (D) Quantification of the number of tsp-2+ cells per mm of worm. Control(RNAi) n = 17, zfp-1(RNAi) n = 19, zfp-1-1(RNAi) n = 15. (E) Maximum intensity projection showing nanos2 expression and EdU incorporation in zfp-1(RNAi) or zfp-1-1(RNAi) parasites. (F) Quantification of the number of EdU+ cells per mm worm. Control(RNAi) n = 17, zfp-1(RNAi) n = 19, zfp-1-1(RNAi) n = 15. (G) Maximum intensity projection showing tsp-2 and sm13 expression in zfp-1(RNAi) or zfp-1-1(RNAi) parasites. (H) Quantification of the number of sm13+ cells per mm worm. Control(RNAi) n = 17, zfp-1(RNAi) n = 19, zfp-1-1(RNAi) n = 15. (I) WISH showing zfp-1 expression in adult male worm. (J) Double FISH showing expression of zfp-1 relative to nanos2 (a neoblast marker), zfp-1–1, and tsp-2. (K) WISH showing zfp-1–1 expression in adult male worm. (L) Double FISH showing expression of zfp-1–1 relative to tsp-2, a mixture tegument-specific markers (tegument), and nanos2 (a neoblast marker). (M) 3D rendering showing expression of zfp-1–1 in a subset of tsp-2+ cells. The dorsal and ventral surfaces of the animal are oriented towards the top and the bottom of the image, respectively, as indicated by the arrows in the first panel. Scale bars: B, C, G, I, K 50 µm; E, J, L, M 10 µm. Error bars represent 95% confidence intervals, **p<0.01 (Student’s t-test).

Figure 3.

Figure 3—figure supplement 1. RNAi screen of candidate tegument biogenesis regulators.

Figure 3—figure supplement 1.

Results of knocking down candidate transcripts that are dispensable for normal neoblast function and tsp-2+ cell production. Representative maximum intensity confocal projections are shown. Numbers represent the fraction of parasites displaying the observed phenotype. Scale bars: 50 µm.
Figure 3—figure supplement 2. Quantification of gene expression in zfp-1(RNAi) and zfp-1-1(RNAi) parasites.

Figure 3—figure supplement 2.

Quantitative real time PCR analysis of the effects of zfp-1 and zfp-1–1 RNAi on the expression of (A) zfp-1, (B) zfp-1–1, (C) sm13, and (D) tsp-2. Each bar represents the expression of the indicated gene from an individual biological replicate relative to the expression from a control RNAi treatment group. Expression levels of indicated genes were normalized to both Cytochrome C Oxidase (Smp_900000) and Proteasome Subunit Beta Type-4 (Smp_056500). *p<0.05, **p<0.01 (Student’s t-test).
Figure 3—figure supplement 3. Gene expression patterns of sm13 and zfp-1–1.

Figure 3—figure supplement 3.

Representative double FISH demonstrating the localization of sm13 expression relative to zfp-1–1 expression. 1 of 248 sm13+ cells was zfp-1–1 positive. Image represents a z-projection. Scale bar: 10 µm.