Fig. 3: HLH-30 regulates TGFβ signaling in response to nutrient cues.
a, hlh-30::mNeonGreen (hlh-30::mNG) expression pattern in ASI sensory neurons at 2 h of ARD and upon 1 h of refeeding 48-h ARD worms. Differential interference contrast (DIC) and fluorescence images of the head region. The arrow indicates an ASI neuron. Ph, terminal pharyngeal bulb. Scale bars, 10 μm. b, Representative images of daf-7p::GFP expression in the ASI neuron at 96 h of ARD and 48 h of refeeding after 96 h ARD. Fluorescence and DIC images of head regions. The arrow indicates an ASI neuron. Scale bars, 10 μm. c, Quantification of daf-7p::GFP expression in ASI neurons at 48, 72 and 96 h of ARD (80-ms exposure time) and after 48 h of refeeding (40 ms exposure time). Each dot represents daf-7p::GFP expression of one ASI neuron. BR = 3, one representative experiment shown. d, Quantification of whole-body daf-1p::GFP at 48 h of ARD and 48 h of refeeding measured with the COPAS Biosorter. Mean ± s.e.m., each dot represents one experiment. Two-way ANOVA followed by Fisher’s least significant difference post hoc test. e, Representative images of daf-1p::GFP expression in the DTC at 48 h of ARD and 48 h of refeeding. Fluorescence and DIC images of the distal gonad. The arrow indicates the DTC nucleus. Scale bars, 10 μm. f, Percentage of DTC positive for daf-1p::GFP expression of N2 and hlh-30(tm1978) at 48 h of ARD and after 48 h refeeding. BR = 4. One-tailed Mann–Whitney to compare the percentage of daf-1::GFP-positive DTC within ARD or refeeding and one-way ANOVA followed by Tukey’s post hoc test to compare genotypes across ARD and refeeding conditions. g, ChIP enrichment of HLH-30 binding to promotor regions of canonical TGFβ pathway genes. For each gene, one HLH-30 binding site (green) and the corresponding 3′ untranslated region (UTR) control (gray) was tested in this analysis. Data were normalized to 3′ UTR binding (dashed line)66. Mann–Whitney statistics (two sided) were performed on the non-normalized ∆∆ct values. One dot represents one biological repeat. BR = 4. h, Normalized mRNA expression from RNA-seq data of daf-14 and daf-5 in 48-h ARD comparing WT and hlh-30(tm1978) mutants. Data and statistics are presented in Supplementary Table 2. If not stated otherwise, data are the mean ± s.d. Two-tailed Mann–Whitney test.
