Article Figures & Data
Figures
- Figure 1. Failure of LIN41 down-regulation explains multiple let-7–mutant phenotypes.
(A) Schematic illustration of let-7– and lin-41 3′ UTR–mutant alleles (not to scale) and of the let-7 silencing activities in the different mutant backgrounds. Red asterisks depict point mutations and the red dotted line indicates a deletion. For both let-7 target sites on the lin-41 3′ UTR in the lin-41(xe11); let-7(n2853) background, a wild-type G:C base pair is replaced by an A:U base pair. This rescues lin-41 down-regulation by let-7, although not to the full extent (Aeschimann et al, 2017). (B) Schematic of a section of the heterochronic pathway regulating the onset of events during the J/A transition. The experiments of Fig 1 test if these events are regulated by silencing of only one let-7 target (LIN41) or by silencing of any other combination of let-7 targets. (C) The percentage of burst adult worms of the indicated genotypes grown in synchronized populations at 25°C for 45 h. Data as mean ± SEM of N = 3 independent biological replicates with n ≥ 400 worms per genotype and replicate. (D) Example micrographs of young adult worms expressing transgenic scm::gfp to visualize seam cell nuclei. Branched lines indicate seam cells originating from extra cell divisions. Scale bar: 50 μm. (E) Quantification of seam cell nuclei at the late L4 larval (L4) or young adult (yA) stage, in worms of the indicated genetic backgrounds. Areas of bubbles represent the percentage of worms with the corresponding number of seam cells. n = 20 for L4, n > 50 for yA, worms grown at 25°C. (F) Example micrographs of tails in adult males of the indicated genetic background. Scale bar: 20 μm. (G) The percentage of young adult males of the indicated genotype with unretracted tails. n ≥ 100, worms grown at 25°C.
- Figure 2. A failure in LIN41 down-regulation results in a complete loss of male tail cell retractions.
(A, B) Example micrographs of male tails at different developmental time points in the indicated genetic backgrounds. The dashed arrow illustrates the anterior retraction of the epidermal cell at the tail tip. The full arrow and the arrowhead point to one of the rays and the fan, respectively. Scale bars: 20 μm. (C) Quantification of the male tail phenotypes of the indicated genotypes at the late L4 larval stage as illustrated with pictures (iii) in (A, B). Shown are the percentages of animals with over-retracted, partially retracted, or unretracted tails. n ≥ 100, except for lin-41(ma104) (n = 80). Worms were grown at 25°C. (B, C) lin-41(ma104) animals display a precocious male tail retraction phenotype and were included as a control.
- Figure S1. Novel mutant alleles and male tail phenotype experiments.
(A) Illustration of the mutant alleles (in blue) used in this study to disrupt the function of LIN41 target genes. The position of the endogenous GFP::3xFLAG inserted at the N terminus of LIN-29a (lin-29(xe63)) is indicated in green. The lin-29a(xe40) allele deletes coding exons two through four of lin-29 while causing a frameshift in the remaining lin-29a open reading frame, rendering it null for lin-29a without affecting expression of lin-29b. (B) Quantification of the male tail phenotypes at the young adult stage of the indicated genotypes. Shown are the percentages of animals with over-retracted, Lep, or unretracted tails. n ≥ 100, except for lin-41(ma104) (n = 55), which displays an over-retracted male tail phenotype and was included as a control. Worms were grown at 25°C.
- Figure 3. LIN41 controls male tail cell retraction through MAB-3 and DMD-3.
(A) MA plot for anti-FLAG RIP-seq experiments with FLAG::GFP::LIN41 and FLAG::GFP::SART-3 as a control. Semi-synchronous L3/L4 stage worm populations enriched in males (him-5(e1490) genetic background) were compared in three independent biological replicates. The plot compares the fold change (FC) in IP-to-input enrichments for RNA-sequencing reads in the FLAG::GFP::LIN41 versus the FLAG::GFP::SART-3 IP (y-axis) with the mean mRNA abundance (x-axis, CPM, counts per million). Genes passing the cutoff of FDR < 0.05 are highlighted in red and labeled. (B, C) Schematic depiction of the expression patterns of the LIN41 protein, the let-7 miRNA, and the LIN41 target proteins in the soma during development from larvae to adult worms, in the wild-type situation (B) and when let-7 fails to repress lin-41 (C). (D) Example micrographs of male tails at different developmental time points in the indicated genetic backgrounds. Scale bar: 20 μm. (E) Quantification of the male tail phenotypes at the late L4 larval stage of mab-10(0) lin-29(Δa) and mab-3(0); dmd-3(lf) animals. The data for wild-type and lin-41(∆LCS) males are re-plotted from Fig 1 for reference. Shown are the percentages of animals with over-retracted, partially retracted or unretracted tails. n ≥ 100, worms were grown at 25°C. (F, G) Confocal images of the male tail epidermis in young L3-stage male animals expressing nuclear-localized GFP(PEST)::H2B reporters, driven from the mab-3 (F) and dmd-3 (G) promoters and fused to their orthologous 3′ UTR sequences or the unregulated unc-54 3′ UTR as indicated. Animals were grown for 20 h at 25°C on lin-41 or mock RNAi bacteria. Scale bars: 10 μm.
- Figure 4. LIN41 controls self-renewal of seam cells through LIN-29a and its co-factor MAB-10.
(A) Quantification of seam cell nuclei of L4 stage and young adult (yA) animals of the indicated genetic backgrounds, as in Fig 1E. n = 20 for L4, n > 50 for yA, worms grown at 25°C. Results for wild-type and lin-41(∆LCS) animals are re-plotted from Fig 1 for reference. (B) Example micrographs of a young adult wild-type worm and a worm lacking LIN-29a and MAB-10 expressing transgenic scm::gfp. Branched lines indicate seam cell nuclei originating from extra cell divisions. Scale bar: 50 μm.
- Figure 5. LIN-29a and MAB-10 are involved in the vulva bursting phenotype.
(A, B) Quantification of burst worms of the indicated genotypes, grown in a synchronized population at 25°C for 45 h. Data as mean ± SEM of N = 3 independent biological replicates with n ≥ 400 worms counted for each genotype and replicate. In panel (A), results for wild-type and lin-41(∆LCS) animals are re-plotted from Fig 1 for reference.
- Figure 6. LIN-29a accumulation in the AC is independent of let-7 and increases the penetrance of vulval bursting.
(A, B) Example micrographs of worms expressing GFP-tagged LIN-29a, showing the epidermis of late L4-stage animals (A) and the anchor cell of L3-stage animals (B). Scale bar: 10 μm. Arrows point to seam cell nuclei, arrowheads to hyp7 nuclei. (C) Quantification of burst worms of the indicated genotypes, grown in a synchronized population at 25°C for 45 h. The xeSi417 transgene drives LIN-29a accumulation specifically in the anchor cell. Data as mean ± SEM of N = 3 independent biological replicates with n ≥ 400 worms counted for each genotype and replicate.
- Figure S2. The spatial activity of the ∆pes-10::ACEL promoter.
(A, B) Example micrographs to illustrate expression of the xeSi417 transgene in the vulval-uterine region and epidermis in the L3 (A) and L4 (B) larval stages. Transgene expression is monitored by nuclear-localized GFP, which is produced from an operon containing lin-29a followed by gfp::h2b. Operon expression is driven from the ∆pes-10 minimal promoter fused to the ACEL enhancer. Scale bars: 10 μm.
- Figure 7. LIN-29a accumulation in the AC is required for utse formation.
(A) Representative micrographs of the late L4-stage vulva in wild-type animals and mab-10(0) lin-29(Δa)– or lin-29(0)–mutant animals with and without AC production of LIN-29a from the xeSi417 transgene. Arrowheads point to the thin or thick tissue separating the vulva from the uterus. Scale bar: 10 μm. (B) Quantification of uterine-vulva connection phenotypes of late L4 larvae of the indicated genotypes. n ≥ 40, worms grown at 25°C. (C) Model for the output of the let-7–LIN41 pathway regulating two pairs of LIN41 targets involved in transcription. LIN-29a and MAB-10 stop seam cell divisions and prevent vulval rupturing. MAB-3 and DMD-3 drive cell retraction events to shape the male tail.
- Figure S3. The vulval phenotypes observed with different mutant alleles of lin-29 and mab-10.
(A, B) Quantification of Pvl (A) or Egl (B) phenotypes of animals of the indicated genotypes grown in a synchronized population at 25°C for 45 (A) or 60 (B) hours. Data as mean ± SEM of N = 3 independent biological replicates with n ≥ 400 worms counted for each genotype and replicate.
Supplementary Materials
Table S5 Worm strains used in this study.
Table S6 Plasmids used in this study.
