Project description:Transcriptional profiling of P. pacificus worms from (1) dauer stage, or (2) dauer-exit at 12 hours stage, compared to mix-stage worms as a common reference. The goal was to determine genes regulated during dauer development and recovery or exit from dauer stage. This data was then compared to data generated for corresponding developmental stages in the C. elegans (see NCBI GEO series GSE30977) , to study evolution of developmental pathways regulating dauer development.
Project description:Transcriptional profiling of C. elegans worms from (1) dauer stage, or (2) dauer-exit at 12 hours stage, compared to mix-stage worms as a common reference. The goal was to determine genes regulated during dauer development and recovery or exit from dauer stage. This data was then compared to data generated for corresponding developmental stages in the nematode Pristionchus pacificus (see NCBI GEO series GSE31861) , to study evolution of developmental pathways regulating dauer development.
Project description:Transcriptional profiling of P. pacificus worms from (1) dauer stage, or (2) dauer-exit at 12 hours stage, compared to mix-stage worms as a common reference. The goal was to determine genes regulated during dauer development and recovery or exit from dauer stage. This data was then compared to data generated for corresponding developmental stages in the C. elegans (see NCBI GEO series GSE30977) , to study evolution of developmental pathways regulating dauer development. Two-condition experiments. Experiment 1 = Dauers vs. Mix-stage worms. 4 biological replicates for each condition, including 2 dye-swaps. Experiment 2 = Dauer-Exit at 12 hour time-point s vs. Mix-stage worms. 3 biological replicates for each condition, including 1 dye-swaps. Total samples from both experiments 1 and 2 = 7.
Project description:We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval stages (L1 to L4), dauer and L1/L4 post dauer. Histones were analyzed by our optimized middle-down protein sequencing platform using high mass accuracy tandem mass spectrometry. This allows quantification of intact histone tails and detailed characterization of distinct histone tails carrying co-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during development and it was mutually exclusive of the active marks H3K18ac, R26me1 and R40me1, suggesting a role for H3K23me3 in to silent chromatin. We observed distinct PTM profiles for normal L1 larvae and for L1-post dauer larvae, or L4 and L4 post-dauer, suggesting that histone PTMs mediate an epigenetic memory that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms.
Project description:Transcriptional profiling of C. elegans worms from (1) dauer stage, or (2) dauer-exit at 12 hours stage, compared to mix-stage worms as a common reference. The goal was to determine genes regulated during dauer development and recovery or exit from dauer stage. This data was then compared to data generated for corresponding developmental stages in the nematode Pristionchus pacificus (separate data-sets on a custom microarray platform designed by us and manufactured by Agilent) , to study evolution of developmental pathways regulating dauer development. Two-condition experiments. Experiment 1 = Dauers vs. Mix-stage worms. 4 biological replicates for each condition, including 2 dye-swaps. Experiment 2 = Dauer-Exit at 12 hour time-point s vs. Mix-stage worms. 4 biological replicates for each condition, including 2 dye-swaps. Total samples from both experiments 1 and 2 = 8.
Project description:Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions C. elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, P. pacificus, and S. ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer vs. non-dauer stages and infective larvae vs. free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved ‘dauer-infective’ expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest as well as long-term survival in free-living and parasitic nematodes.
Project description:Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions C. elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, P. pacificus, and S. ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer vs. non-dauer stages and infective larvae vs. free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved ‘dauer-infective’ expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest as well as long-term survival in free-living and parasitic nematodes. miRNA profiling in mixed and developmentally arrested stages (dauer/infective larvae) of nematodes by small RNA deep sequencing using Illumina GAII, Illumina HiSeq 2000, and ABI SOLiD.
Project description:Recent advances in (meta)genomic methods have provided new opportunities to examine host-microbe-environment interactions in the human gut. While opportunities exist to extract DNA from freshly sourced colonic tissue there are potentially valuable sources of DNA from historical studies that might also be examined. We examined how four different tissue DNA extraction methods employed in past clinical trials might impact the recovery of microbial DNA from a colonic tissue sample as assessed using a custom designed phylogenetic microarray for human gut bacteria and archaebacteria. While all methods of DNA extraction produced similar phylogenetic profiles some extraction specific biases were also observed. Real time PCR analysis targeting several bacterial groups substantiated this observation. These data suggest that while the efficacy of different DNA extraction methods differs somewhat all the methods tested produce an accurate representation of microbial diversity. This suggests that DNA samples archived in biobanks should be suitable for retrospective analyses. Three technical replicates per sample (extraction method) were analysed
Project description:Under adverse environmental conditions, nematodes arrest into dauer, an alternative developmental stage for diapause. Dauers endure unfavorable environment and interact with host animals to access favorable environments, thus playing a critical role in the survival of both free-living and parasitic nematodes. Here, we discovered that in Caenorhabditis elegans, daf-42 is essential for development into dauer stage, as the null mutant shows lethal phenotype during dauer entry. To examine the transcriptional changes accompanied by the absence of DAF-42 during dauer entry, we performed RNA-sequencing on daf-2 and daf-2; daf-42 worms at 52 hours after egg laying (HAE) and 60 HAE, the time at which 0% and about 40% of daf-2; daf-42 mutants form dead dauer at 25 degrees Celcius, respectively. daf-2 control worms develop into dauer stage after 60 HAE, and half of its population develop into dauer by 72 HAE, when raised at 25 degrees Celcius.