Fig 1.
An SmFTZ-F1 homolog is essential for parasite vitality.
(A) A schematic diagram depicting RNAi screen strategy B and C, Images of control and Smftz-f1(RNAi) worms during in vitro culture. At day 20 of in vitro culture, control(RNAi) animals remain as male and female worm pairs, are firmly attached to the bottom of the plate, and move freely. By D9 of in vitro culture, Smftz-f1(RNAi) animals become unpaired and fail to firmly attach to the bottom of the place. Over time, movement of Smftz-f1(RNAi) worms diminishes and they stop moving altogether. At D9, the heads are curled (arrow). At D20 the worm’s full body is curled. n = 10 worm pairs, > 3 biological replicates (D) Whole mount in situ hybridization showing mRNA expression of Smftz-f1 in both male and female parasites. The expression is found throughout the bodies of both sexes and does not seem to be specific for a tissue. Scale Bars: B and C, 500 μm. D,100 μm.
Fig 2.
RNAseq reveals possible direct targets of SmFTZ-F1.
(A) Strategy for RNAseq, paired worms were subsequently separated and only male parasites were used. (B) Venn diagram of down-regulated RNAseq gene sets from Day 5 and 9. (C) Heat map of the 20 most differentially down-regulated genes following Smftz-f1(RNAi) treatment. n = 10 worms, 3 biological replicates. (D) Micrographs of EMSAs using biotin-labeled oligonucleotides corresponding to each of the candidate SmFTZ-F1 response elements. EMSAs were performed in presence or absence of in vitro translated SmFTZ-F1 protein ± the presence of 20-fold or 200-fold direct unlabeled competitor oligonucleotides. ns, non-specific oligo at 200-fold excess. n = 2 biological replicates. (E) Predicted DNA-binding motif sequence for nuclear receptor SmFTZ-F1. The height of the letter represents how frequently that nucleotide is observed in that position. The logo was generated using the MEME Suite.
Fig 3.
meg-8.3 is a putative target of SmFTZ-F1.
(A) Whole mount in situ hybridizations for putative SmFTZ-F1 target genes on D9 of RNAi. Smp_331380 is exclusively expressed in the worm esophageal gland. n = 10 worms, 3 biological replicates. (B) Images of control, Smftz-f1 and meg-8.3 RNAi-treated worms during in vitro culture. Control(RNAi) animals remain firmly attached to the bottom of the plate, and move freely. At D12 of meg-8.3(RNAi) animals fail to attach to the bottom of the culture plate. Their heads begin to curl inward on themselves (see inset). n > 10 worms, 3 biological replicates. (C) Plots depicting the number of animals that display normal or curled phenotypes following RNAi treatment at D15. (D) Whole mount in situ hybridization of D9 Smftz-f1 RNAi-treated worms using known esophageal gland markers. Control and RNAi-treated worms show no difference in esophageal gland staining. n > 10 worms, 3 biological replicates. (E) PNA labeling (green) of esophageal gland using RNAi treated worms at days 9, 12, 18. Maximum intensity projections are shown. n > 10 worms, 3 biological replicates (F) Cartoon of the ChIP-qPCR experiment (G) Schematic of the 2kb upstream region of the meg-8.3 promoter with the sequences and position of the response element and primer set used, length not proportional to the size. (H) ChIP-qPCR, shown as percentage of input DNA, for SmFTZ-F1 or preimmune serum antibodies at the putative promoter of meg-8.3. Data are mean ± SEM from 7 biological replicates. *p<0.05 by Student’s t-test. Scale bars, A,B,D, 100μm. E, 50 μm.
Fig 4.
meg-8.3 is essential for esophageal gland tissue maintenance.
(A) FISH of the gut marker ctsb (green), and fluorescent dextran (white) in the heads of control(RNAi), Smftz-f1(RNAi) and meg-8.3(RNAi) animals. Representative of >10 animals from 3 experiments (B) Three distinct phenotypic severities are observed following RNAi: Normal, Intermediate, and Severe. Normal animals possess an intact and well-organized gut as observed by ctsb expression and fluorescent dextran in lumen of the gut. Intermediate animals have normal gut structure and possess fluorescent dextran in the gut, but also contain dextran collecting in the protonephridial system in the head (arrows). Severe animals possess a relatively normal gut structure and very little, if any, dextran labeling in the gut. Instead, dextran labeling is found in the protonephridial system (see arrows). (C) Plots depicting the relative fraction of animals that display Normal, Intermediate, or Severe phenotypes at D5–D18 of RNAi. >12 animals from three separate experiments were observed for each time point. (D) Carmine-stained head regions of control(RNAi), Smftz-f1(RNAi), and meg-8.3(RNAi) treated worms at different time points (Arrows highlight “holes”). Representative of >10 animals from 4 experiments. Scale Bars: A, D 25 μm; B,100 μm.