ABSTRACT: Analysis of gene expression between monomethyl branched-chain fatty acid deficient (elo-5) and wild type N2 C. elegans L1 larvae hatched on food-free NGM plates
Project description:Wild-type (N2) C. elegans embryos were plated with or without E. coli OP50 as food and with 100mM or 500mM NaCl at 25C (4 conditions). They were allowed to develop for 24hr and collected as L1 larvae for RNA-seq.
Project description:Gene expresssion is not globally shut down in elo-5 as compared to N2 Many changes in metabolic and regulatory gene expression were detected between elo-5 and N2 L1 larvae. RNA samples for hybridization with the C. elegans Affymetrix Gene Chips were prepared as follows. elo-5(gk208) and N2 control animals were grown from eggs for one generation on NGM plates spotted with 300µl OP50 overnight liquid culture supplemented with 1mM C17ISO in DMSO at 20°C. Gravid adults were washed off the plates with M9 and bleached. Eggs were plated on OP50/NGM plates supplemented with 1mM of either C13ISO or C17ISO and maintained at 20°C to obtain gravid adults. Adults were washed off the plates and bleached, and eggs were then plated on food-free NGM or OP50/NGM plates at 20°C. Twenty-four hours later, L1s from food-free plates were collected for RNA isolation with Trizol and Qiagenâ??s RNAse Easy Kit, according to the manufacturersâ?? protocols. On the following days, animals on OP50/NGM were checked for phenotypes to confirm C17ISO-deficiency in elo-5(gk208) grown with C13ISO supplement. Two biological replicates were prepared for each condition; elo-5(gk208) with C13ISO and C17ISO supplements and a control N2 with C13ISO supplement. Data analysis was performed with dChip analytical software (Li and Wong, 2001).
Project description:To systematically evaluate the effect of embryo lysates feeding, we conducted RNA sequencing (RNA-seq) on day 7 N2 worms treated with embryo lysates. Our findings revealed that embryo lysates affected the expression of genes related to fatty acid metabolism, degradation of branched-chain amino acids, and lysosome functions. Notably, worms treated with embryo lysates showed increased fatty acid degradation gene expression and decreased fatty acid elongation gene expression.
Project description:When C. elegans larvae hatch in the absence of food they persist in a stress resistant, developmentally arrested state (L1 arrest). We characterized mRNA expression genome-wide in a pair of bifurcating time series starting in the late embryo and proceeding through the hatch in the presence and absence of food (E. coli). We used Affymetrix C. elegans expression arrays to measure gene expression in 18 total timepoint/conditions in three biological replicates. Experiment Overall Design: Nematode populations were staged such that they hatched withing a 2.5 hr window, and time point zero (0 hr) corresponds to when 50% of the population had hatched. RNA was isolated with TRIzol and 100 ng was used for T7 RNA polymerase-based mRNA amplification and biotin labeling using MessageAmp II-Biotin Enhanced kit (Ambion). Labeled cRNA was fragmented, hybridized to the Affymetriz array, and scanned according to the manufacturerâs protocol.
Project description:Gene expresssion is not globally shut down in elo-5 as compared to N2 Many changes in metabolic and regulatory gene expression were detected between elo-5 and N2 L1 larvae.
Project description:Our understanding of cellular mechanisms by which animals regulate their response to starvation is limited despite the close relevance of the problem to major human health issues. L1 diapause of Caenorhabditis elegans, where newly hatched first stage larval arrested in response to food-less environment, is an excellent system to study the problem. We found through genetic manipulation and lipid analysis that ceramide biosynthesis, particularly those with longer fatty acid side chains, critically impacts animal survival during L1 diapause. Genetic and expression analyses indicate that ceramide likely regulate this response by affecting gene expression and activity in multiple regulatory pathways known to regulate starvation-induced stress, including the insulin-IGF-1 signaling (IIS) pathway, Rb and other pathways that mediate pathogen/toxin/oxidative stress responses. These findings provide an important insight into the roles of sphingolipid metabolism in not only starvation response but also aging and food-response related human health problems.
Project description:Wild-type (N2) and tps1;tps2 double mutant embryos were resuspended at 1 embryo/uL in S-basal with or without 50mM trehalose. Samples were collected after 24 hours after bleach as L1 larvae
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: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 20°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 25° 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 25°C. Newly hatched L1 larvae were fed on bacteria expressing the different dsRNA for three hours to recover from starvation. Three replicates per RNAi.
