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. 2012;8(8):e1002836.
doi: 10.1371/journal.pgen.1002836. Epub 2012 Aug 16.

Cell-nonautonomous Signaling of FOXO/DAF-16 to the Stem Cells of Caenorhabditis Elegans

Free PMC article

Cell-nonautonomous Signaling of FOXO/DAF-16 to the Stem Cells of Caenorhabditis Elegans

Wenjing Qi et al. PLoS Genet. .
Free PMC article


In Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Lifespan of daf-16 transgenic animals.
(A) shc-1(ok198);Is[daf-16::gfp] animals show reduced lifespan. (B) Treatment with FUDR extends the lifespan of both shc-1(ok198);Is[daf-16::gfp] and Is[daf-16::gfp] animals. (C) Knock-down of glp-1 extends lifespan of shc-1(ok198);Is[daf-16::gfp] animals (See also Figure S1, S2, S3 and Table S1). (D) The lifespan extension by FUDR does not require the adapter protein KRI-1. (E) In the absence of FUDR extrachromosomal daf-16::gfp transgene does not extend lifespan of wild type animals. (F) In the presence of FUDR extrachromosomal daf-16::gfp transgene extends lifespan of wild type animals. In the lifespan assay with FUDR treatment, animals were transferred onto the agar plates containing 0.1 mg/ml FUDR and E.coli 24 hours after the L4 larval stage. These were raised on the FUDR containing plates for four days and then transferred onto new plates without FUDR. The mean lifespan and statistical analyses in this figure are summarized in Table 1.
Figure 2
Figure 2. shc-1(ok198);Is[daf-16::gfp] animals display pleiotropic defects in the reproductive system.
(A–C) DIC images of the gonad of one day adult animals. The white arrowhead denotes accumulated germ cells in the pseudocoelom (B) and a DTC migration defect (C). mito: mitotic region of the germline; meiot: meiotic region of the germline; o: oocyte; sp: sperm; em: embryo. (D and E) DIC images of the whole anterior gonad arm of L3 larvae. The white arrowhead denotes the germ cells leaking out from the gonad (E). (F and G) MitoTracker Red staining of the gonadal basement membrane. The white arrowhead points to the intact basement membrane in wild type (F) and disrupted basement membrane in shc-1(ok198);Is[daf-16::gfp] (G) animals. (H and I) DIC image (H) and anti-PGL-1 antibody staining (I) of shc-1(ok198);Is[daf-16::gfp] L3 larva. The white arrowhead indicates the anterior gonad with leakage (I). (J) Knock-down of shc-1 gene enhances the defect in the basement membrane of animals carrying daf-16 transgene. In this and the following figures data of defect in the basement membrane are presented as mean±SD; the mean values and statistic analysis are summarized in Table 2 and Table 3; the symbol * means P<0.01, ** means P<0.001 and *** means P<0.0001. (K) The gonad arms with disrupted basement membrane of shc-1(ok198);Is[daf-16::gfp] animals contain more germ cells than those with intact basement membrane at mid-L3 larval stage. For quantitative assessments PGL-1 antibody staining was performed to label the germ cells. Numbers of germ cells per gonad ±SD: wild type N2: 33±4 (n = 18); Is[daf-16::gfp]: 34±3 (n = 8); intact gonad of shc-1;Is[daf-16::gfp] animals: 32±5 (n = 24); disrupted gonad of shc-1;Is[daf-16::gfp] animals: 49±13 (n = 29) (P<0.0001 compared to N2, Is[daf-16::gfp] or intact gonad of shc-1;Is[daf-16::gfp] animals). (L and M) DAPI staining of wild type (L) and shc-1(ok198);Is[daf-16::gfp] (M) one day adult animals. The white arrowheads point to the chromosomes in the diakinetic oocytes of wild type animals and DNA in the endomitotic germ cells of shc-1(ok198);Is[daf-16::gfp] animals, respectively. Scale bar 10 µm.
Figure 3
Figure 3. Active DAF-16 derogates integrity of the gonadal basement membrane.
(A) Knock-down of daf-16 by RNAi suppressed the early lethality of shc-1(ok198);Is[daf-16::gfp] animals. Animals died within the first five days of adulthood: shc-1(ok198);Is[daf-16::gfp](L4440) as control: 45.7%, shc-1(ok198);Is[daf-16::gfp]+daf-16 RNAi: 1.4%. (B) daf-16 mutation suppresses the defects in gonad integrity of shc-1 mutant animals. (C) Transgenic expression of a constitutively nuclearly located daf-16(4A)::gfp results in disruption of the gonad in shc-1(−), but not in wild type animals (See also Figure S7 and Table 3). (D) SHC-1 does not affect the subcellular localization of DAF-16(4A)::GFP. In both wild type and shc-1(ok198) animals DAF-16(4A)::GFP is constitutively located in the nucleus. The mean lifespan and statistical analyses in this figure are summarized in Table 1. byEx represents extrachromosomal transgenic alleles which are summarized in Table 3. Mean values and statistic analysis of defect in gonadal integrity are summarized in Table 2 and Table 3. (E) SHC-1 does not affect expression level of transgenic daf-16(4A)::gfp. Columns represent pooled normalised values of three independent experiments plus standard deviation (SD). Mann Whitney test. Normalized GFP intensity: byEx797: 1.00±2.5 (n = 31); shc-1;byEx797: 1.00±2.1 (n = 33), P = 0.9670; byEx798: 1.00±0.19 (n = 37); shc-1;byEx798:1.06±0.22 (n = 48), P = 0.1591.
Figure 4
Figure 4. Active DAF-2, AKT-2 and SGK-1 antagonize AKT-1 mediated inhibition of DAF-16 to derogate gonad integrity.
(A) DAF-16 activity affecting gonad integrity is inhibited by AKT-1. (B) Inactivation of IIR DAF-2 suppresses the defect in the gonadal basement membrane (See also Figure S8 and Table S2). (C) Inactivation of daf-2 extends lifespan of shc-1(ok198);Is[daf-16::gfp] animals. (D) daf-2 mutation suppresses gonad disruption in akt-1;shc-1 animals (See also Figure S9). (E) AKT-2 and SGK-1 antagonize AKT-1. (F) shc-1(ok198);sgk-1(ok538);Is[daf-16::gfp] L3 larvea have less germ cells than shc-1(ok198);Is[daf-16::gfp] L3 larvea. shc-1;Is[daf-16::gfp] animals: 49±13 (n = 29); shc-1;sgk-1;Is[daf-16::gfp] animals: 33±6 (n = 23) (P<0.0001). (See also Figure S10). The mean lifespan and statistical analyses in this figure are summarized in Table 1. Mean values and statistic analysis of gonad disruption are summarized in Table 2.
Figure 5
Figure 5. JNK signaling antagonizes hypodermal DAF-16 to ensure gonadal integrity.
(A) daf-18 mutation enhances disruption of the gonad in shc-1(ok198);akt-1(ok525) but not in akt-1(ok525) one day adult animals. (B) Knock-down of mek-1 and kgb-1 enhances the defect in Is[daf-16::gfp] animals. The mean values and statistic analysis are summarized in Table 2. (C) Expression of hypodermal daf-16(4A)::gfp leads to disruption of the gonadal basement membrane. byEx represents extrachromosomal transgene alleles which are summarized in Table 3. Mean values and statistic analysis of defect in gonadal integrity are summarized in Table 3.

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This work was funded through German Research Foundation (DFG) Collaborative Research Centres (CRC) 746 and 850, European Union Network of Excellence LIFESPAN, and the German Research Foundation (DFG) Excellence Initiative (FRIAS LIFENET and BIOSS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.