Project description:Post-embryonic development of the nematode C. elegans is governed by nutrient availability. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This “L1 arrest” (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. daf-16/FOXO had overlapping but distinct effects on gene expression in L1 arrest compared to daf-2/InsR adults. Notably, dbl-1/TGF-β, a ligand for the Sma/Mab pathway, and daf-36, which encodes an upstream component of the daf-12/NHR steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β and daf-36. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype, and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

Project description:Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Project description:Expression data from Caenorhabditis elegans let-418(RNAi), mep-1(RNAi) and gfp(RNAi) L1 larvae. The C. elegans genome encodes two homologs of the human protein Mi-2, namely LET-418 and CHD-3. LET-418 plays an essential role during development; its depletion leads to a pleiotropic and lethal phenotype that includes larval arrest, an everted vulva and sterility. Without maternal contribution, let-418 mutants stop their development at the L1 larval stage (von Zelewsky et al., 2000). We further characterized this arrest and showed that it is very similar to the L1 diapause induced by starvation; both germline and somatic cells remain in a quiescent state in let-418 L1 arrested larvae, indicating that LET-418 activity is required to bypass the L1 arrest in presence of food. The let-418 L1 larvae express ectopically the P granule component PGL-1 in somatic cells (Unhavaithaya et al., 2002). Interestingly, the phenotype of mep-1 mutants is remarkably similar to that of let-418: RNAi targeting mep-1 also induced an L1 arrest phenotype; furthermore, MEP-1 and LET-418 have been shown to physically interact (Unhavaithaya et al., 2002 and M. Passannante). The null allele mep-1(q660) is temperature sensitive and shows a more severe phenotype at higher temperatures. At 20Â°C, about 10% of mep-1 homozygotes derived from heterozygous mothers arrest as young larvae, whereas the remaining 90% develop into sterile adults (Belfiore et al., 2002). Later in development, the somatic gonad is affected in mep-1(q660) mutants. This results in an abnormal and disorganized gonad, a phenotype also observed in let-418(s1617) mutants. Both let-418 and mep-1 mutants produce a very limited number of oocytes and have pseudovulvae derived from P8.p (Belfiore et al., 2002; von Zelewsky et al., 2000 and C. Wicky, personal communication). Preliminary quantitative real-time PCR revealed that the expression of genes coding for P granule components was deregulated in both mep-1(RNAi) and let-418(RNAi) L1 larvae (data not shown). To further investigate this issue, we performed a complete gene expression analysis. Given the fact that mep-1(q660) mutants are sterile, we used RNA interference to generate mep-1 depleted worms. Bacteria expressing gfp dsRNA (pPE128.110 in HT115) were used as reference, since RNA interference may induce gene expression changes by itself. C. elegans L1 larvae treated with RNA interference were selected for RNA extraction and hybridization on Affymetrix microarrays. Synchronized wild type L4 animals were grown at 25Â° on bacteria expressing either gfp, let-418 or mep-1 dsRNA. Eggs were collected by bleaching gravid adults and allowed to hatch in the absence of food at 25Â°C. Newly hatched L1 larvae were fed on bacteria expressing the different dsRNA for three hours to recover from starvation. Three replicates per RNAi.

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Caenorhabditis elegans

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