Intestine-specific removal of DAF-2 nearly doubles lifespan in Caenorhabditis elegans with little fitness cost

Abstract

Twenty-nine years following the breakthrough discovery that a single-gene mutation of daf-2 doubles Caenorhabditis elegans lifespan, it remains unclear where this insulin/IGF-1 receptor gene is expressed and where it acts to regulate ageing. Using knock-in fluorescent reporters, we determined that daf-2 and its downstream transcription factor daf-16 are expressed ubiquitously. Using tissue-specific targeted protein degradation, we determined that intracellular DAF-2-to-DAF-16 signaling in the intestine plays a major role in lifespan regulation, while that in the hypodermis, neurons, and germline plays a minor role. Notably, intestine-specific loss of DAF-2 activates DAF-16 in and outside the intestine, causes almost no adverse effects on development and reproduction, and extends lifespan by 94% in a way that partly requires non-intestinal DAF-16. Consistent with intestine supplying nutrients to the entire body, evidence from this and other studies suggests that altered metabolism, particularly down-regulation of protein and RNA synthesis, mediates longevity by reduction of insulin/IGF-1 signaling.

Fig. 1. Endogenous expression of the daf-2 gene detected in most or all C. elegans cells from embryos to adults.

a Schematic of two strategies to characterize the endogenous expression pattern of daf-2. The coding sequence of the mNeonGreen, mNeonGreen::degron (top panel), or NuGFP cassette (bottom panel) is knocked into the daf-2 genomic locus before the stop codon by CRISPR/Cas9 genome editing, which allows detection of daf-2 expression at the protein level or at the mRNA level, respectively. Blue boxes, coding regions; line, non-coding regions; gray boxes, 3’ untranslated regions. b Expression pattern of daf-2 at the protein level indicated by the DAF-2::mNeonGreen at day 1 of adulthood. A similar pattern of expression was observed in four independent experiments. c Expression pattern of daf-2 at the mRNA level indicated by the NuGFP reporter (NLS^SV40::GFP::mNeonGreen::NLS^EGL-13) at day 1 of adulthood. Left panel, overview of the expression of daf-2 mRNA throughout the whole body. H.N, head neuron; G, germ line, indicated by the circled red dotted line; In, intestine, indicated by short arrows; V, vulval cells, indicated by long arrow; E, embryo, indicated by the circled gray dashed line; H, hypodermal cells, indicated by triangles; T.N, tail neuron, indicated by the circled white dotted line; M, body wall muscle, indicated by asterisks. Right panel, local expression patterns of daf-2 mRNA. osm-6p::mCherry, ciliated sensory neuron marker; eak-4p::mCherry::NLS^EGL-13, XXX-cell-specific marker; myo-3p::H1::mCherry, body-wall-muscle-specific marker; lim-7p::mCherry::NLS^EGL-13, gonadal- sheath-specific marker. A similar pattern of expression was observed in two independent experiments. d Summary of the spatiotemporal expression pattern of NuGFP reporter. +, expression intensity of NuGFP; -, expression of NuGFP is not detectable.

Fig. 2. Ubiquitous presence of DAF-16 detected in the C. elegans soma and germline.

a Schematic of knocking in the coding sequence of GFP or GFP::degron into the daf-16 genomic locus before the stop codon using CRISPR/Cas9 genome editing. Blue boxes, coding regions; line, non-coding regions; gray boxes, 3’ untranslated regions. Expression patterns of DAF-16::GFP at day 1 of adulthood (b), embryonic stage (c), and L1 larval stage (d). A similar pattern of expression was observed in two independent experiments. e Summary of the spatiotemporal expression patterns of DAF-16::GFP. +, expression intensity of DAF-16::GFP; -, expression of DAF-16::GFP is not detectable.

Fig. 3. Intestine-specific degradation of DAF-2 extended C. elegans lifespan by 94.4%.

Degrading DAF-2 in the neurons (a), germline (b), and hypodermis (c) increased the lifespan by 18.6% (p < 0.0001), 6.4% (p = 0.014), and 13.7% (p = 0.001), respectively. d–f Degrading DAF-2 in the body wall muscle (d), gonadal sheath (e), or XXX cells (f) had no effect on WT lifespan (p > 0.05). g Degrading DAF-2 in the intestine increased WT lifespan by 94.4% (p < 0.0001). h Degrading DAF-2 in the whole body increased WT lifespan by 166.5% (green solid line versus green dashed line, p < 0.0001), which outlived the canonical hypomophic daf-2(e1370) mutant worms and the intestinal DAF-2 AID worms (gray solid line and red solid line, respectively). p values are calculated by log-rank tests. See survival statistics in Supplementary Data 1. Source data are provided as a Source Data file.

Fig. 4. Intestinal DAF-16 mediates daf-2(e1370) longevity.

a Degrading DAF-16 from the neurons shortened the daf-2(e1370) lifespan by 15.6% (p < 0.0001). b Degrading DAF-16 from the germline had no significant effect on the daf-2(e1370) lifespan (p = 0.347). c Degrading DAF-16 from the hypodermis shortened the daf-2(e1370) lifespan by 15.6% (p < 0.0001). Degrading DAF-16 in the body wall muscle (d), gonadal sheath (e), or XXX cells (f) had no effect on the lifespan of daf-2(e1370) (p > 0.05). g Degrading intestinal DAF-16 shortened the daf-2(e1370) lifespan by 40.1% (p < 0.0001). h Degrading DAF-16 in all tissues shortened the daf-2(e1370) lifespan by 57.8% (p < 0.0001). i Endogenously expressed DAF-16::GFP accumulated in the intestinal nuclei upon degrading DAF-2 from the intestine. A similar pattern of expression was observed in three independent experiments. j Degrading intestinal DAF-16 largely suppressed the longevity induced by degrading DAF-2 from the intestine (red dashed line versus red solid line, p < 0.0001). p values are calculated by log-rank tests. See survival statistics in Supplementary Data 1. Source data are provided as a Source Data file.

Fig. 5. Intestine-specific degradation of DAF-2 had no adverse effect on development and reproduction.

a Intestinal degradation of DAF-2 did not cause dauer arrest at 25 °C. b The intestinal DAF-2 AID worms developed faster than the daf-2(e1370) worms. Data are represented as mean ± SEM of three biological replicates. c The brood size of intestinal DAF-2 AID worms is comparable to that of the control animals. Data are represented as mean ± SD of two biological replicates. p values are calculated by unpaired two-sample t-test. d Degradation of intestinal DAF-2 elevates the triacylglycerol (TAG) content by 2.4-fold relative to the control animals. Data are represented as mean ± SEM of four biological replicates. p values are calculated by unpaired two-sample t-test. Source data are provided as a Source Data file.

Fig. 6. Intestine-specific degradation of DAF-2 evokes a comparatively simple pro-longevity transcriptional program.

a Transcriptome analysis of daf-2(e1370) versus WT (N2) worms. Left panel, overlap of the canonical DAF-16 targets and DEGs (q value < 0.05) of daf-2(e1370). Middle panel, enriched KEGG pathways among Class I and Class II DAF-16 targets by Over Representation Analysis (ORA). Only pathways with adjusted p value < 0.001 are shown. For multiple comparisons, adjustments were made with Benjamini–Hochberg (BH) method. Right panel, GSEA of daf-2(e1370) worms. Pathways with p-value < 0.01 are shown, and those related to protein metabolism (blue color) and RNA metabolism (orange color) are highlighted. See statistics in Source data and Supplementary Data 2. b GSEA analysis of worms subjected to tissue-specific degradation of DAF-2. Pathways with q-value < 0.01 (labeled by *) in at least one sample are shown, and those related to protein metabolism (blue color) and RNA metabolism (orange color) are highlighted. For multiple comparisons, adjustments were made with Benjamini–Hochberg (BH) method. c qRT-PCR analysis of ribosomal RNAs (left panel) as well as their precursors (right panel) in daf-2(e1370) and N2 worms. Data are represented as mean ± SEM of three biological replicates. p values are calculated by unpaired two-sample t-test. d Knocking down of genes related to RNA metabolism (fib-1 and M28.5) by RNAi further extended the lifespan of WT worms. See survival statistics and biological replicates in Supplementary Data 1. Source data are provided as a Source Data file.

Fig. 7. Loss of intestinal DAF-2 triggered gene expression changes in other tissues through cross-tissue DAF-2 to DAF-16 signaling.

a Isolation of neurons, hypodermis, body wall muscle (BWM), and intestine cells from intestinal DAF-2 AID worms for RNA-seq. Left panel, tissue-specific transgenic reporters in the intestinal DAF-2 AID strain used to isolate tissue-specific cells. Neurons, labeled by rgef-1p::NuGFP; hypodermis, labeled by dpy-7p::NLS^SV40::GFP; BWM, labeled by myo-3p::NuGFP; intestine, labeled by ges-1p:: NuGFP. Right panel, representative images of tissue-specific cells isolated by FACS for RNA-seq. A similar pattern of expression was observed in two independent experiments. b GSEA analysis of up-regulated and down-regulated GO terms enriched in each isolated tissue. GO terms with q-value < 0.01 in at least two tissues are shown. For multiple comparisons, adjustments were made with Benjamini–Hochberg (BH) method. c Degrading DAF-2 from the intestine induces DAF-16 nuclear accumulation in the hypodermis at the L2 larval stage. A similar pattern of expression was observed in three independent experiments. d Five DEGs selected to verify the cross-tissue effect of intestinal IIS on non-intestinal tissues by mCherry transgenic reporters in the intestinal DAF-2 AID strain. Representative images of each reporter are shown in the bottom panel. A similar pattern of expression was observed in three independent experiments. e Schematic of the combination of tissue-specific GFP nanobody-mediated ZIF-1 system and tissue-specific AID system to simultaneously degrade DAF-2 and DAF-16 in two different tissues, the intestine and non-intestinal tissues (neurons or hypodermis). f Lifespan phenotypes following simultaneous degradation of intestinal DAF-2 and non-intestinal DAF-16. Degrading intestinal DAF-2 by GFP nanobody-mediated ZIF-1 system extended lifespan by 51.8% (top panel, p < 0.0001). Degrading DAF-16 in the hypodermis (bottom panel, p = 0.003), but not in the neurons (middle panel, p = 0.337), moderately but significantly decreased the lifespan of the worms in which the intestinal DAF-2 level was reduced. p-values are calculated by log-rank tests. See survival statistics in Supplementary Data 1. g Motif enrichment analysis of transcriptional binding sites among 1-kb promoter sequence of the intestinal, hypodermal, and neuronal DEGs. See statistics in Supplementary Data 5. Source data are provided as a Source Data file.