Fig. 4: Regulation of growth signaling protects against a senescence-like state.
a, Hermaphrodite distal gonad schematic. Germ cells in gray. The progenitor zone contains a distal pool of stem cells (area of sygl-1 expression, yellow) and a proximal pool of differentiating cells. Forward growth signaling induces LAG-2 secretion from the DTC to GSCs, initiating mitotic cell division and sygl-1 expression via GLP-1. SYGL-1 abundance declines with increasing distance to the DTC. b, Quantification of lag-2p::mKate2::PH expression intensity in the DTC cap at 48 h of ARD. Each dot represents lag-2p::mKate2::PH expression in one DTC per worm. c, lag-2p::mKate2::PH expression in DTC at 48 h of ARD and after 48 h refeeding. Fluorescence images. Contrast and intensity were adjusted to visualize DTC processes. Scale bars, 10 μm. d, Quantification of SYGL-1 abundance in the distal gonad of 48-h-ARD worms and of 24-h refed worms, based on intensity of α-FLAG staining. Average intensity values (y axis) were plotted against distance (μm) from the DTC (x axis). Lines indicate mean intensity; shaded areas are the s.e.m.; BR = 3, one representative experiment. e, Quantification of the area under the curve (AUC) of SYGL-1 expression. AUCs were normalized to the N2 AUC for each biological replicate. One-tailed Mann–Whitney test. BR = 3. f, Quantification of germ cell nucleolar area at 96 h of ARD. Each dot represents the nucleolar area in μm2 of one germ cell per worm. BR = 3, one representative experiment. g, Number of M-phase cells in the distal gonad arms (progenitor zone) at 48 h of ARD and 24 h of refeeding. BR = 3, one representative experiment. One dot indicates the number of M-phase-positive cells per gonad arm. h, Quantification of GSCs positive for RAD-51 foci in the sygl-1-positve area per gonad. One dot represents the number of positive GSCs per gonad arm per worm. Pooled from three BRs. i, Biological age prediction (BiT) from transcriptomes at 48 h of ARD. Each point represents one replicate. j, Correlation plot of hlh-30(tm1978)/N2 and hlh-30 daf-1(m40)/hlh-30 DEGs at 48 h of ARD. Significantly regulated genes (adjusted P < 0.05) highlighted in red (genes down in hlh-30 and reversed by daf-1, 2,347 DEGs, quadrant 2) or blue (genes up in hlh-30 and reversed by daf-1, 2,083 DEGs, quadrant 4). Simple linear regression line in gray. Equation Y = −0.48X + 0.13; R2 = 0.27. k, Working model. In WT ARD HLH-30/TFEB is active and downregulates TGFβ signaling at multiple levels. Consequently, Notch signaling is inhibited via LAG-2/DSL downregulation in the DTCs, resulting in GSC quiescence. Upon refeeding, HLH-30 becomes inactive, resulting in reactivation of TGFβ and downstream signaling. Subsequently, stem cell niche remodeling (DTC outgrowth) is initiated, GSCs proliferate and worms reproduce. cGMP and insulin–IGF signaling (IIS) work upstream or in parallel to TGFβ signaling. Data and statistics are presented in Supplementary Table 2. If not stated otherwise, data are the mean ± s.d. One-way ANOVA followed by Tukey’s post hoc test. Genotypes N2, hlh-30(tm1978), hlh-30 daf-1(m40).
