1. Volume 39, Issue 2, Pages 209-223 (October 2016)
A C. elegans Thermosensory Circuit Regulates Longevity through crh-1/CREB- Dependent flp-6 Neuropeptide Signaling 
Yen-Chih Chen, Hung-Jhen Chen, Wei-Chin Tseng, Jiun-Min Hsu, Tzu-Ting Huang, Chun-Hao Chen, Chun-Liang Pan 
Developmental Cell 
Volume 39, Issue 2, Pages (October 2016)
DOI: /j.devcel
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2. Developmental Cell 2016 39, 209-223DOI: (10.1016/j.devcel.2016.08.021)
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3. Figure 1
crh-1 Acts in the AFD Thermosensory Neuron and the Intestine to Regulate Lifespan
(A) Lifespan of the crh-1(tz2) and crh-1(n3315) mutants. n = 50–70 animals.
(B) Lifespan of animals overexpressing crh-1 from its endogenous promoter. n = 60–150 animals.
(C) Epifluorescence-differential interference contrast (DIC) merged images of crh-1 expression revealed by the Pcrh-1::mCherry transgene.
(D) Confocal projection image showing that crh-1 was expressed in the AFD, ASE, and AWC neurons.
(E and F) smFISH for crh-1, with a GFP reporter (oyIs18) for AFD in (F). Dashed lines define the worm body (white) and the pharynx (yellow), respectively. Black and white arrowheads point to crh-1 mRNA granules.
(G) mCherry::CRH-1 was localized to the neuronal and intestinal nuclei.
(H and I) Lifespan of crh-1(tz2) animals expressing crh-1 from different promoters. n = 70 animals.
All scale bars represent 10 μm. For statistics and details of lifespan, see Table S1. See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
Thermosensory Transduction Is Necessary for Longevity Response to Temperature
(A–D) Lifespan of the ttx-1(oy26) (A), gcy-23(nj37) gcy-8(oy44) gcy-18(nj38) (B), cmk-1(oy21) (C), or crh-1(tz2); cmk-1(oy21) (D) mutants. n = 52–99 animals.
(E) Lifespan of the cmk-1 mutant with cmk-1 specifically expressed in the AFD. n = 70 animals.
(F) Lifespan of the crh-1(tz2); cmk-1(oy21) mutants with expression of wild-type or phosphomimetic CRH-1 in the AFD. n = 67 animals.
(G) Lifespan of the crh-1(tz2) mutant that expressed a phosphorylation-resistant CRH-1(S48A) in the AFD. n = 70 animals.
For statistics and details of lifespan, see Table S1. See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
flp-6 Acts Downstream of crh-1 to Regulate Longevity Response to Temperature
(A) Lifespan of the flp-6(ok3056) mutant. n = 41–80 animals.
(B) Lifespan of transgenic animals overexpressing flp-6 from its endogenous promoter. n = 55–70 animals.
(C) Lifespan of the crh-1(tz2); flp-6(ok3056) double mutant. n = 70 animals.
(D) Lifespan of the flp-6(ok3056) mutant that overexpressed crh-1. n = 60–120 animals.
(E) flp-6 was expressed in the AFD and the ASE neurons, as revealed by ynIs67(Pflp-6::GFP). These neurons also expressed crh-1.
(F) Lifespan of the flp-6(ok3056) mutant that overexpressed flp-6 from the endogenous flp-6 or the AFD-specific gcy-8 promoters. n = 70 animals.
(G) Lifespan of the animals in which flp-6 was specifically silenced in the AFD. n = 60–72 animals.
(H) Lifespan of cmk-1(oy21) mutants with or without flp-6 overexpression. n = 70 animals.
(I) Lifespan of the crh-1(tz2) mutant that overexpressed flp-6 from the endogenous flp-6 or the gcy-8 promoters. n = 70 animals.
(J) Confocal projection image showing that when expressed in the AFD (Pgcy-8::FLP-6::mCherry), FLP-6 was distributed in the soma as well as in the axon (arrowheads). Extracellular mCherry signal indicates that FLP-6 was secreted.
(K and L) Fluorescence and DIC merge images (K) and quantification of mCherry signals (L) in distal coelomocytes. ∗∗∗p < 0.001, Mann-Whitney U test.
(M) Fluorescence images and quantification of mCherry signals in the coelomocytes of animals expressing FLP-6::mCherry in the AFD. Numbers of coelomocytes being quantified for mCherry signals are indicated. Error bars denote SEM. ∗∗p < 0.01, Mann-Whitney U test. Arrowheads mark the coelomocytes in (K) and (M).
For statistics and details of lifespan, see Table S1. See also Figure S3.
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6. Figure 4 flp-6 Is a Transcriptional Target of crh-1
(A–C) Confocal fluorescence images (A) and quantification of smFISH signals (B, C) for flp-6 in the AFD (B) or the ASE (C). Dashed lines mark the AFD neurons. GFP that marked the AFD neuron is pseudocolored in blue, and smFISH signals derived from Quasar 670 are in gray. n = 18–24 neurons for each experiment. Error bars denote SEM. ∗∗∗p < , Mann-Whitney U test. n.s., not significant.
(D and E) Fluorescence images of animals expressing Pflp-6::GFP and Pgcy-8::mCherry (D) and quantification of GFP in the AFD neurons (E). Arrows label the AFD neurons. n = 29–30 pairs of AFD/ASE neurons for each experiment at designated temperatures. Error bars denote SEM. ∗∗∗p < , Mann-Whitney U test. n.s., not significant.
(F and G) Fluorescence images of animals expressing Pgcy-8::mCherry and either Pflp-6::GFP or Pflp-6::GFP that lacked the CRE (F) and GFP quantification in the AFD (G). Arrows label the AFD neurons. n = 29–30 pairs of AFD/ASE neurons for each experiment at designated temperatures. Error bars denote SEM. ∗∗∗p < , Mann-Whitney U test. n.s., not significant.
(H) Lifespan of flp-6(ok3056) animals overexpressing flp-6 from the full-length or mutant flp-6 promoters that lacked the CRE. n = 70 animals.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
The AIY Neuron Mediates the Longevity Effects of CRH-1 and FLP-6
(A) Lifespan of the ttx-3(ot22) mutant. n = 39–73 animals.
(B and C) Lifespan of the ttx-3(ot22) mutant in combination with the crh-1(tz2) (B) or the flp-6(ok3056) (C) mutations. n = 60–120 animals.
(D and E) Lifespan of the ttx-3(ot22) mutant in the presence of crh-1 (D) or flp-6 (E) overexpression. n = 70 animals.
For statistics and details of lifespan, see Table S1. See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6 crh-1 and flp-6 Genetically Interact with the daf-9 Pathways
(A and B) Lifespan of the daf-9(rh50) mutant with or without crh-1 (A) or flp-6 (B). n = 60–120 animals.
(C and D) Lifespan of the daf-9(rh50) mutants overexpressing crh-1 (C) or flp-6 (D). n = 23–155 animals.
(E) Lifespan of the crh-1(tz2) mutant overexpressing daf-9 in the XXX cells. n = 70 animals.
(F and G) Quantification of DAF-9::GFP in XXX cells. ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney U test. n.s., not significant. Numbers in bars indicate numbers of XXX cells scored. Error bars denote SEM.
For statistics and details of lifespan, see Table S1.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
flp-6 Regulates Multiple Genes Related to DAF-16 Signaling that Are Known to Influence Longevity
(A) Log-scale scatterplot of gene-expression profiles in the N2 and the flp-6(ok3056) mutant at 25°C. Upregulated and downregulated genes are shaded in blue and red, respectively. Examples of highly upregulated genes are indicated. For a complete list of differentially expressed genes, see Table S2.
(B) Gene ontology (GO) analysis of the upregulated or downregulated genes in the flp-6(ok3056) mutant. Numbers for each GO term are indicated.
(C) Real-time qPCR of indicated genes in the wild-type, crh-1(tz2), and flp-6(ok3056) mutants, and of ins-7 in the ttx-3(ot22) mutant. One of the three or six biological replicas is shown. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.001, t test.
(D–F) Lifespan of the wild-type or the flp-6 mutant subjected to control or dod-19 (D), dod-20 (E), or dod-23 (F) feeding RNAi at 25°C. n = 31–60 animals.
For statistics and details of lifespan, see Table S1.
Developmental Cell  , DOI: ( /j.devcel )
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10. Figure 8 flp-6 Represses Intestinal ins-7 to Promote Longevity
(A) Lifespan of the wild-type or the flp-6(ok3056) mutant with or without an additional ins-7(tm2001) mutation. n = 60 or 61 animals. For statistics and details of lifespan, see Table S1.
(B) ins-7 expression pattern, by yxIs13(Pins-7::GFP). Scale bar, 50 μm.
(C) Intestinal ins-7 expression was upregulated by temperature. Scale bar, 40 μm.
(D and E) Intestinal ins-7 expression of the wild-type, crh-1(tz2), flp-6(ok3056), or AIY-ablated animals (D), as well as in those with excess crh-1 or flp-6 (E), at 25°C. The two foregut cells shown here were used for quantification of epifluorescent Pins-7::GFP signals and data are normalized to that of the wild-type. Exposure times of 40 ms and 150 ms were used for images in (D) and (E), respectively, to avoid oversaturation of the images. Error bars denote SEM. Numbers in bars indicate numbers of pairs of foregut cells quantified. ∗∗p < 0.001, ∗∗∗p < , t test. Scale bar, 40 μm.
(F) Confocal projection images and quantification of ins-7 smFISH. Arrowheads mark ins-7 mRNA granules pseudocolored in gray, with intestinal nuclei pseudocolored in blue. n = 34–38 intestinal cells per genotype. ∗∗p < 0.001, ∗∗∗p < , t test.
(G) Schematic model of the neuronal CRH-1 and FLP-6 regulating temperature-dependent longevity through repressing intestinal INS-7.
See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
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