Project description:Whole L1 larvae were collected from GC1459 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III ?; daf-18(ok480) IV] and GC1171 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III] strains up to two hours after hatching without food.
Project description:This experiment was designed to identify early targets of daf-16/FoxO during L1 starvation in C. elegans. Previous work (Baugh et al 2009) examined temporal dynamics of gene expression in fed and starved L1 larvae using the same platform and methods. Here, a single time point was analyzed (~3 hr after hatching) in fed and starved larvae also comparing wild-type and a daf-16 null mutant. Statistical analysis includes pairwise comparisons between the four conditions as well as a two-factor analysis to explicitly identify genes affected by nutrient availability in daf-16-dependent fashion.
Project description:Embryos of wild type N2, daf-16(mu86), daf-18(ok480) and daf-16(mu86); daf-18(ok480) were collected by hypochlorite-treat gravid adult worms and were put in virgin S-basal with 0.1% EtOH at 20 °C. After hatching, those embryos entered L1 arrest. Arrested L1s were collected 16 hr post hypochlorite treatment (about 4 hr of starvation).
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:Whole starved L1 larvae from four genotypes (N2, daf-2(e1370), irld-39(duk1);irld-52(duk17), and daf-2(e1370); irld-39(duk1); irld-52(duk17)) were collected after 12 hours of starvation
Project description:hrde-1(tm1200) animals were bleached and embryos were plated on empty vector, daf-2, or pitr-1 RNAi (HT115). Animals were allowed to develop for 72 hr, after which embryos were released via bleaching and allowed to hatch in the absence of food. Arrested L1 larvae were snap frozen in liquid nitrogen ~24 hr after bleaching.
Project description:Insulin/IGF-1 signaling (IIS) mediates metabolic and developmental acclimation to stressful conditions including starvation. The transcription factor DAF-16/FoxO actuates many of the physiological effects of reduced IIS, yet the specific contributions of DAF-16 target genes to stress resistance remain poorly understood. We explore the function of C. elegans H1 linker histone variant hil-1/H1.0, a DAF-16 target that is upregulated during starvation. The HIL-1 sequence is divergent from the other eight annotated C. elegans H1 variants, and the others are not so highly responsive to nutrient availability and DAF-16 activity, suggesting distinct function. Using knock-in reporters, we find that HIL-1 is expressed ubiquitously in nuclei of L1 and dauer larvae during starvation, but that expression is largely undetectable in fed larvae. Disrupting hil-1 activity through mutation or auxin-inducible degradation led to reduced growth after extended L1 starvation, revealing reduced starvation resistance. RNA-seq of hil-1 mutants showed that hil-1 affects expression of relatively few genes. However, hil-1 activates genes involved in the innate immune response, Pseudomonas aeruginosa infection, and components of the nipi-3/TRIB1 immunity pathway. hil-1 mutants display compromised survival upon exposure to P. aeruginosa under reduced IIS, and genes activated by hil-1 promote resistance to P. aeruginosa. Together these results suggest that DAF-16/FoxO activates transcription of hil-1 during starvation to promote resistance to starvation and pathogens. We demonstrate conditional regulation of an H1 histone, and we reveal a novel mechanism for how IIS promotes stress resistance by identifying a histone variant that connects nutrient sensing to immunity.
Project description:Reduced insulin/IGF signaling (IIS) in C. elegans increases starvation resistance in daf-16/FoxO-dependent fashion, but it is unclear whether the effects of reduced IIS are entirely dependent on daf-16/FoxO or if another effector(s) of IIS may be involved. We used RNA sequencing (RNA-seq) and phenotypic analysis of L1 starvation resistance to assess epistasis between daf-2/InsR and daf-16/FoxO. Essentially all differential gene expression caused by disruption of daf-2/InsR during starvation is daf-16-dependent. The effects of daf-2/InsR on starvation survival also appear entirely dependent on daf-16/FoxO, and the effect of daf-2/InsR on growth following starvation is largely daf-16-dependent. However, we also show daf-16-independent effects on growth and reproduction following recovery from starvation. These results support the conclusion that the effects of reduced IIS during L1 starvation are essentially daf-16/FoxO-dependent, but that reduced IIS engages one or more additional effectors during development following starvation.
