RNA-Seq Reveals Acute Manganese Exposure Increases Endoplasmic Reticulum Related and Lipocalin mRNAs in Caenorhabditis elegans.

Manganese (Mn) is an essential nutrient; nonetheless, excessive amounts can accumulate in brain tissues causing manganism, a severe neurological condition. Previous studies have suggested oxidative stress, mitochondria dysfunction, and impaired metabolism pathways as routes for Mn toxicity. Here, we used the nematode Caenorhabditis elegans to analyze gene expression changes after acute Mn exposure using RNA-Seq. L1 stage animals were exposed to 50 mM MnCl2 for 30 min and analyzed at L4. We identified 746 up- and 1828 downregulated genes (FDR corrected p < 0.05; two-fold change) that included endoplasmic reticulum related abu and fkb family genes, as well as six of seven lipocalin-related (lpr) family members. These were also verified by qRT-PCR. RNA interference of lpr-5 showed a dramatic increase in whole body vulnerability to Mn exposure. Our studies demonstrate that Mn exposure alters gene transcriptional levels in different cell stress pathways that may ultimately contribute to its toxic effects.

Chronic ethanol exposure increases cytochrome P-450 and decreases activated in blocked unfolded protein response gene family transcripts in caenorhabditis elegans.

Ethanol is a widely consumed and rapidly absorbed toxin. While the physiological effects of ethanol consumption are well known, the underlying biochemical and molecular changes at the gene expression level in whole animals remain obscure. We exposed the model organism Caenorhabditis elegans to 0.2 M ethanol from the embryo to L4 larva stage and assayed gene expression changes in whole animals using RNA-Seq and quantitative real-time PCR. We observed gene expression changes in 1122 genes (411 up, 711 down). Cytochrome P-450 (CYP) gene family members (12 of 78) were upregulated, whereas activated in blocked unfolded protein response (ABU) (7 of 15) were downregulated. Other detoxification gene family members were also regulated including four glutathione-S-transferases and three flavin monooxygenases. The results presented show specific gene expression changes following chronic ethanol exposure in C. elegans that indicate both persistent upregulation of detoxification response genes and downregulation of endoplasmic reticulum stress pathway genes.

PIWI-interacting RNA expression regulates pathogenesis in a Caenorhabditis elegans model of Lewy body disease.

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that regulate gene expression, yet their molecular functions in neurobiology are unclear. While investigating neurodegeneration mechanisms using human α-syn(A53T)Tg and AßTg;α-syn(A53T)Tg pan-neuronal overexpressing strains, we unexpectedly observed dysregulation of piRNAs. RNAi screening revealed that knock down of piRNA biogenesis genes improved thrashing behavior; further, a tofu-1 gene deletion ameliorated phenotypic deficits in α-syn(A53T)Tg and AßTg;α-syn(A53T)Tg transgenic strains. piRNA expression was extensively downregulated and H3K9me3 marks were decreased after tofu-1 deletion in α-syn(A53T)Tg and AßTg;α-syn(A53T)Tg strains. Dysregulated piRNAs targeted protein degradation genes suggesting that a decrease of piRNA expression leads to an increase of degradation ability in C. elegans. Finally, we interrogated piRNA expression in brain samples from PD patients. piRNAs were observed to be widely overexpressed at late motor stage. In this work, our results provide evidence that piRNAs are mediators in pathogenesis of Lewy body diseases and suggest a molecular mechanism for neurodegeneration in these and related disorders.

Overexpression of SUMO perturbs the growth and development of Caenorhabditis elegans.

Small ubiquitin-related modifiers (SUMOs) are important regulator proteins. Caenorhabditis elegans contains a single SUMO ortholog, SMO-1, necessary for the reproduction of C. elegans. In this study, we constructed transgenic C. elegans strains expressing human SUMO-1 under the control of pan-neuronal (aex-3) or pan-muscular (myo-4) promoter and SUMO-2 under the control of myo-4 promoter. Interestingly, muscular overexpression of SUMO-1 or -2 resulted in morphological changes of the posterior part of the nematode. Movement, reproduction and aging of C. elegans were perturbed by the overexpression of SUMO-1 or -2. Genome-wide expression analyses revealed that several genes encoding components of SUMOylation pathway and ubiquitin-proteasome system were upregulated in SUMO-overexpressing nematodes. Since muscular overexpression of SMO-1 also brought up reproductive and mobility perturbations, our results imply that the phenotypes were largely due to an excess of SUMO, suggesting that a tight control of SUMO levels is important for the normal development of multicellular organisms.

Trans fat diet causes decreased brood size and shortened lifespan in Caenorhabditis elegans delta-6-desaturase mutant fat-3.

Trans-fatty acids (TFAs) enter the diet through industrial processes and can cause adverse human health effects. The present study was aimed to examine the effects of dietary cis- and trans-fatty acids on the model organism Caenorhabditis elegans. Cis- or trans-18:1n9 triglycerides (25 µM) caused no apparent changes in the numbers of viable progeny of wild-type N2 animals. However, in fat-3 mutants lacking delta-6-desaturase, the trans-isomer caused modest decreases in lifespan and progeny after three generations. Long-chain polyunsaturated fatty acids (PUFA) profiles were significantly altered in fat-3 mutants compared to wild type but were not altered after exposure to dietary cis- or trans-18:1n9. Genome-wide expression analysis of fat-3 mutants revealed hundreds of changes. Several genes involved in fat metabolism (acs-2, fat-7, mdt-15) were significantly increased by cis- or trans-18:1n9 without discrimination between isomers. These results provide support for the hypothesis that dietary trans fats are detrimental to development and aging.

TDP-1/TDP-43 potentiates human α-Synuclein (HASN) neurodegeneration in Caenorhabditis elegans.

TAR DNA binding protein (TDP-43) is a DNA/RNA binding protein whose pathological role in amyotrophic lateral sclerosis (ALS) and frontal temporal lobe dementia (FTLD) via formation of protein aggregates is well established. In contrast, knowledge concerning its interactions with other neuropathological aggregating proteins is poorly understood. Human α-synuclein (HASN) elicits dopaminergic neuron degeneration via protein aggregation in Parkinson's disease. HASN protein aggregates are also found in TDP-43 lesions and colocalize in Lewy Body Dementia (LBD). To better understand the interactions of TDP-43 and HASN, we investigated the effects of genetic deletion of tdp-1, the Caenorhabditis elegans ortholog of human TDP-43, as well as overexpression of TDP-43, in transgenic models overexpressing HASNWT and HASNA53T. Tdp-1 deletion improved the posture, movement, and developmental delay observed in transgenic animals pan-neuronally overexpressing HASNA53T, and attenuated the loss and impairment of dopaminergic neurons caused by HASNA53T or HASNWT overexpression. Tdp-1 deletion also led to a decrease in protein level, mRNA level and aggregate formation of HASN in living animals. RNA-seq studies suggested that tdp-1 supports expression of lysosomal genes and decreases expression of genes involved in heat shock. RNAi demonstrated that heat shock proteins can mediate HASN neuropathology. Co-overexpression of both human TDP-43 and HASNWT resulted in locomotion deficits, shorter lifespan, and more severe dopaminergic neuron impairments compared to single transgenes. Our results suggest TDP-1/TDP-43 potentiates HASN mediated neurodegeneration in C. elegans. This study indicates a multifunctional role for TDP-1/TDP-43 in neurodegeneration involving HASN.

Whole genome microarray analysis of C. elegans rrf-3 and eri-1 mutants.

We performed genome wide gene expression analysis on L4 stage Caenorhabditis elegans rrf-3(pk1426) and eri-1(mg366) mutant strains to study the effects caused by loss of their encoded proteins, which are required for the accumulation of endogenous secondary siRNAs. Mutant rrf-3 and eri-1 strains exhibited 72 transcripts that were co-over-expressed and 4 transcripts co-under-expressed (>2-fold, P<0.05) compared to N2 wild type strain. Ontology analysis indicated these transcripts were over represented for protein phosphorylation and sperm function genes. These results provide additional support for the hypothesis that RRF-3 and ERI-1 act together in the endo-siRNA pathway, and furthermore suggests their involvement in additional biological processes.

Chronic MeHg exposure modifies the histone H3K4me3 epigenetic landscape in Caenorhabditis elegans.

Methylmercury (MeHg) is a persistent environmental pollutant that occurs in the food chain, at occupational sites, and via medical procedures. Exposure in humans and animal models results in renal, neuro, and reproductive toxicities. In this study, we demonstrate that chronic exposure to MeHg (10µM) causes epigenetic landscape modifications of histone H3K4 trimethylation (H3K4me3) marks in Caenorhabditis elegans using chromatin immuno-precipitation sequencing (ChIP-seq). The modifications correspond to the locations of 1467 genes with enhanced and 508 genes with reduced signals. Among enhanced genes are those encoding glutathione-S-transferases, lipocalin-related protein and a cuticular collagen. ChIP-seq enhancement of these genes was confirmed with increased mRNA expression levels revealed by qRT-PCR. Furthermore, we observed enhancement of H3K4me3 marks in these genes in animals exposed to MeHg in utero and assayed at L4 stage. In utero exposure enhanced marks without alterations in mRNA expression except for the lpr-5 gene. Finally, knockdown of lipocalin-related protein gene lpr-5, which is involved in intercellular signaling, and cuticular collagen gene dpy-7, structural component of the cuticle, by RNA interference (RNAi) resulted in increased lethality of animals after MeHg exposure. Our results provide new data on the epigenetic landscape changes elicited by MeHg exposure, as well as describe a unique model for studying in utero effects of heavy metals. Together, these findings may help to understand the toxicological effects of MeHg at the molecular level.

Increased lifespan in transgenic Caenorhabditis elegans overexpressing human alpha-synuclein.

alpha-Synuclein is a short 14-kDa protein found in pathological lesions of age-related neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and multiple system atrophy. Its overexpression in transgenic mice, rats, Drosophila melanogaster, and Caenorhabditis elegans recapitulates many of the pathologic features observed in human Parkinson's disease including loss of dopaminergic neurons and motor deficits. Integrated transgenic C. elegans lines were generated that overexpress either human wildtype (WT) or mutant (A53T) forms. These transgenic lines demonstrated approximately 25% increase in lifespan (p<0.0001) compared to controls. When the transgenes were crossed into long-lived daf-2 (m577) or daf-2 (e1370) genetic backgrounds, the lifespan increase was also approximately 25% in comparison to the corresponding daf-2 strains (p<0.05). Pharyngeal pumping and egg laying were significantly decreased in the overexpressing transgenic lines, and lifespan increases were attenuated when lines were grown on thick bacterial lawns, suggesting that caloric restriction may explain some of the effects on lifespan. These studies provide initial evidence for a beneficial role of human alpha-synuclein in influencing lifespan.

Inhibition of Excessive Oxidative Protein Folding Is Protective in MPP(+) Toxicity-Induced Parkinson's Disease Models.

AIMS: Protein misfolding occurs in neurodegenerative diseases, including Parkinson's disease (PD). In endoplasmic reticulum (ER), an overload of misfolded proteins, particularly alpha-synuclein (αSyn) in PD, may cause stress and activate the unfolded protein response (UPR). This UPR includes activation of chaperones, such as protein disulphide isomerase (PDI), which assists refolding and contributes to removal of unfolded proteins. Although up-regulation of PDI is considered a protective response, its activation is coupled with increased activity of ER oxidoreductin 1 (Ero1), producing harmful hydroperoxide. The objective of this study was to assess whether inhibition of excessive oxidative folding protects against neuronal death in well-established 1-methyl-4-phenylpyridinium (MPP(+)) models of PD. RESULTS: We found that the MPP(+) neurotoxicity and accumulation of αSyn in the ER are prevented by inhibition of PDI or Ero1α. The MPP(+) neurotoxicity was associated with a reductive shift in the ER, an increase in the reduced form of PDI, an increase in intracellular Ca(2+), and an increase in Ca(2+)-sensitive calpain activity. All these MPP(+)-induced changes were abolished by inhibiting PDI. Importantly, inhibition of PDI resulted in increased autophagy, and it prevented MPP(+)-induced death of dopaminergic neurons in Caenorhabditis elegans. INNOVATION AND CONCLUSION: Our data indicate that although inhibition of PDI suppresses excessive protein folding and ER stress, it induces clearance of aggregated αSyn by autophagy as an alternative degradation pathway. These findings suggest a novel model explaining the contribution of ER dysfunction to MPP(+)-induced neurodegeneration and highlight PDI inhibitors as potential treatment in diseases involving protein misfolding. Antioxid. Redox Signal. 25, 485-497.

Selective sensitivity of Caenorhabditis elegans neurons to RNA interference.

RNA interference is a new approach to knockdown gene expression, but effectiveness varies depending on the organism, cell type or target sequence. Studies with Caenorhabditis elegans have shown that subsets of cells including neurons are often resistant to RNA interference. We measured RNA interference using green fluorescent protein reporter strains and feeding, soaking and injection delivery methods in a number of Caenorhabditis elegans neuron subtypes (dopaminergic, GABAergic, cholinergic, glutamatergic, touch). The sensitivity to RNA interference varied: GABAergic and dopaminergic neurons showed greater resistance while cholinergic, glutamatergic and touch neurons were more sensitive. Dysfunctional RRF-3, a putative RNA-directed RNA polymerase, had a significant effect on increasing neuron sensitivity in most subtypes. These results demonstrate that Caenorhabditis elegans neurons vary in their sensitivity to RNA interference.

Identification of genes regulated by memantine and MK-801 in adult rat brain by cDNA microarray analysis.

In this study, we monitored gene expression profiles using cDNA microarrays after an acute systemic administration of the high affinity N-methyl-D-aspartate (NMDA) uncompetitive antagonist MK-801 (1 mg/kg; 4 h), and the clinically used moderate affinity antagonist memantine (25 mg/kg; 4 h) in adult rat brains. From a microarray containing 1090 known genes, 13 genes were regulated by both treatments of which 12 were upregulated and one was downregulated. In addition, 28 and 34 genes were regulated (> or = 1.5- or < or = 0.67-fold change) by either memantine or MK-801, respectively. Genes commonly regulated by both treatments and not previously reported were confirmed by in situ hybridization (ISH) and include regenerating liver inhibitory factor-1 (RL/IF-1), GDP-dissociation inhibitor 1 (GDI-1), neural visinin Ca2+-binding protein 2 (NVP-2), neuromedin B receptor, and Na+/K+ transporting ATPase 2beta. ISH with memantine (5-50 mg/kg) revealed regulation of these genes in other cortical and hippocampal regions. RL/IF-1 induction occurred at 1 h and returned to basal levels by 8 h, consistent with the profile of an immediate early gene. Western blot analysis showed increases (approximately 30-65%) in GDI-1 protein present in both cytosolic and membrane fractions that were significant in the 84-kDa Rab bound form, suggesting that memantine influences Ras-like GTPase function. Genes regulated by a 5 mg/kg dose of memantine might be important in its therapeutic effects. These findings increase the number of known, differentially altered genes after treatment of uncompetitive NMDA receptor antagonists and suggest broader actions of these agents than previously realized.

Chronic antipsychotic drug treatment induces long-lasting expression of fos and jun family genes and activator protein 1 complex in the rat prefrontal cortex.

We have characterized the effects of chronic clozapine and haloperidol treatments on the expression of fos (c-fos, fosB, fra-2) and jun (c-jun, junB, junD) family genes in the rat forebrain. The effects of chronic (17d) clozapine and haloperidol on mRNA expression were determined two hours, 24 hours, and six days after the last drug injection, and the DNA-binding activity of the activator protein-1 (AP-1) complex was studied after washout periods of 24 hours and six days. Chronic clozapine treatment with a 6 d washout period induced the expression of several fos and jun family genes in cortical regions, including the prefrontal cortex (PFC), and in the caudate putamen and nucleus accumbens. Moreover, the DNA-binding activity of the AP-1 complex was greatly increased in the anterior cingulate cortex-PFC in mobility shift assays already after 24 h, and remained increased after a 6d washout period. Chronic administration of haloperidol upregulated fos and jun family mRNA expression that was detectable 24 h and 6 d after cessation of the treatment mainly in the cortex. However, the DNA-binding activity of the AP-1 complex was not altered in the anterior cingulate cortex-PFC by chronic haloperidol administration at any of the time points studied. Thus, chronic treatments with clozapine and haloperidol induce a long-lasting enhancement of fos and jun family transcription factors that continues for several days after the cessation of the treatments in the cortex. These lasting effects might represent events that are potentially involved in the mechanisms of antipsychotic drug action.

Subunit selective decrease of AMPA and metabotropic glutamate receptor mRNA expression in rat brain by systemic administration of the NMDA receptor blocker MK-801.

Glutamate mediates its effects in mammals through both ionotropic and metabotropic receptors. Antagonists of ionotropic N-methyl-D-aspartate (NMDA) glutamate receptors elicit neuroprotective and neurotropic effects that have been attributed to Ca2+ block through the membrane ion channel. Nonetheless, molecular and biochemical effects of NMDA receptor antagonism on other glutamate receptor subunits remain poorly understood. We investigated the effects of acute administration of the noncompetitive NMDA receptor antagonist MK-801 on the mRNA expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate receptor (mGluR) subunits to determine the contribution of different glutamate receptors in response to blockade of NMDA receptor channels. In situ hybridization to rat brain sections revealed that AMPAreceptor subunits GluR3 and GluR4, and mGluR3 were modestly but significantly decreased approximately 10-20%, 8 h following 5 mg/kg MK-801 administration. Atime course and dose response study revealed that the effect on mGluR3 was reversed by 24 h and occurred significantly at a dose range from 1 to 5 mg/kg. These results indicate that selected AMPA and mGluR subunit mRNAs respond at the RNA level to the blockade of NMDA receptors.

Overexpression of the full-length neurotrophin receptor trkB regulates the expression of plasticity-related genes in mouse brain.

Significant body of evidence indicates an important role for brain-derived neurotrophic factor (BDNF) in the hippocampal synaptic plasticity; however, the exact mechanisms how the BDNF signal is converted to plastic changes during memory processes are under an intense investigation. To specifically address the role of the trkB receptor, we have previously generated transgenic mice overexpressing the full-length trkB receptor and observed a continuous activation of the trkB.TK+ receptor, improved learning and memory but an attenuated LTP in these mice. In this study, we describe the trkB.TK+ mRNA and protein distribution in the transgenic mice, showing the most prominent increase in the full-length trkB expression in the cortical layer V pyramidal neurons and dentate gyrus of the hippocampus. In addition, we have analyzed the mRNA expression patterns of a group of genes associated with both plastic changes in the nervous system and BDNF signaling. Regulated expression of immediate early genes c-fos, fra-2 and junB was observed in the transgenic mice. Furthermore, the mRNA expression of alpha-Ca2+/calmodulin-dependent kinase II (alpha-CaMKII) was reduced in both the hippocampus and parietal cortex, whereas growth-associated protein 43 (GAP-43) mRNA expressions were induced in the corresponding regions. Conversely, the mRNA expression of the transcription factor cAMP response element binding protein (CREB) was not altered in the trkB.TK+mice. Finally, the density of neuropeptide Y (NPY)-expressing cells was increased in the trkB.TK+ mice dentate hilus. Altogether, these results demonstrate in vivo that the increased trkB.TK+ signaling regulates several important plasticity-related genes.

Transcriptional profiling reveals differential expression of a neuropeptide-like protein and pseudogenes in aryl hydrocarbon receptor-1 mutant Caenorhabditis elegans.

The aryl hydrocarbon receptor (AHR) functions in higher organisms in development, metabolism and toxic responses. Its Caenorhabditis elegans (C. elegans) ortholog, AHR-1, facilitates neuronal development, growth and movement. We investigated the effect of AHR mutation on the transcriptional profile of L4 stage C. elegans using RNA-seq and quantitative real time PCR in order to understand better AHR-1 function at the genomic level. Illumina HiSeq 2000 sequencing yielded 51.1, 61.2 and 54.0 million reads from wild-type controls, ahr-1(ia03) and ahr-1(ju145) mutants, respectively, providing detection of over 18,000 transcripts in each sample. Fourteen transcripts were over-expressed and 125 under-expressed in both ahr-1 mutants when compared to wild-type. Under-expressed genes included soluble guanylate cyclase (gcy) family genes, some of which were previously demonstrated to be regulated by AHR-1. A neuropeptide-like protein gene, nlp-20, and a F-box domain protein gene fbxa-192 and its pseudogenes fbxa-191 and fbxa-193 were also under-expressed. Conserved xenobiotic response elements were identified in the 5' flanking regions of some but not all of the gcy, nlp-20, and fbxa genes. These results extend previous studies demonstrating control of gcy family gene expression by AHR-1, and furthermore suggest a role of AHR-1 in regulation of a neuropeptide gene as well as pseudogenes.

Methylmercury exposure increases lipocalin related (lpr) and decreases activated in blocked unfolded protein response (abu) genes and specific miRNAs in Caenorhabditis elegans.

Methylmercury (MeHg) is a persistent environmental and dietary contaminant that causes serious adverse developmental and physiologic effects at multiple cellular levels. In order to understand more fully the consequences of MeHg exposure at the molecular level, we profiled gene and miRNA transcripts from the model organism Caenorhabditis elegans. Animals were exposed to MeHg (10 µM) from embryo to larval 4 (L4) stage and RNAs were isolated. RNA-seq analysis on the Illumina platform revealed 541 genes up- and 261 genes down-regulated at a cutoff of 2-fold change and false discovery rate-corrected significance q < 0.05. Among the up-regulated genes were those previously shown to increase under oxidative stress conditions including hsp-16.11 (2.5-fold), gst-35 (10.1-fold), and fmo-2 (58.5-fold). In addition, we observed up-regulation of 6 out of 7 lipocalin related (lpr) family genes and down regulation of 7 out of 15 activated in blocked unfolded protein response (abu) genes. Gene Ontology enrichment analysis highlighted the effect of genes related to development and organism growth. miRNA-seq analysis revealed 6-8 fold down regulation of mir-37-3p, mir-41-5p, mir-70-3p, and mir-75-3p. Our results demonstrate the effects of MeHg on specific transcripts encoding proteins in oxidative stress responses and in ER stress pathways. Pending confirmation of these transcript changes at protein levels, their association and dissociation characteristics with interaction partners, and integration of these signals, these findings indicate broad and dynamic mechanisms by which MeHg exerts its harmful effects.

The draft genome and transcriptome of Panagrellus redivivus are shaped by the harsh demands of a free-living lifestyle.

Nematodes compose an abundant and diverse invertebrate phylum with members inhabiting nearly every ecological niche. Panagrellus redivivus (the "microworm") is a free-living nematode frequently used to understand the evolution of developmental and behavioral processes given its phylogenetic distance to Caenorhabditis elegans. Here we report the de novo sequencing of the genome, transcriptome, and small RNAs of P. redivivus. Using a combination of automated gene finders and RNA-seq data, we predict 24,249 genes and 32,676 transcripts. Small RNA analysis revealed 248 microRNA (miRNA) hairpins, of which 63 had orthologs in other species. Fourteen miRNA clusters containing 42 miRNA precursors were found. The RNA interference, dauer development, and programmed cell death pathways are largely conserved. Analysis of protein family domain abundance revealed that P. redivivus has experienced a striking expansion of BTB domain-containing proteins and an unprecedented expansion of the cullin scaffold family of proteins involved in multi-subunit ubiquitin ligases, suggesting proteolytic plasticity and/or tighter regulation of protein turnover. The eukaryotic release factor protein family has also been dramatically expanded and suggests an ongoing evolutionary arms race with viruses and transposons. The P. redivivus genome provides a resource to advance our understanding of nematode evolution and biology and to further elucidate the genomic architecture leading to free-living lineages, taking advantage of the many fascinating features of this worm revealed by comparative studies.

Caenorhabditis Elegans Mutants Predict Regulation of Fatty Acids and Endocannabinoids by the CYP-35A Gene Family.

BACKGROUND: Cytochrome P450s (CYPs) are mono-oxygenases that metabolize endogenous compounds, such as fatty acids and lipid signaling molecules, and furthermore have a role in metabolism of xenobiotics. In order to investigate the role of CYP genes in fat metabolism at the molecular level, four Caenorhabditis elegans mutants lacking functional CYP-35A1, CYP-35A2, CYP-35A4, and CYP-35A5 were characterized. Relative amounts of fatty acids, as well as endocannabinoids, which regulate weight gain and accumulation of fats in mammals, were measured while fat contents in worms were visualized using Oil-Red-O staining. RESULTS: The cyp-35A1 and cyp-35A5 mutants had a significantly lower intestinal fat content than wild-type animals, whereas cyp-35A2 and cyp-35A4 mutants appeared normal. The overall fatty acid compositions of CYP mutants did not alter dramatically, although modest but significant changes were observed. cyp-35A1 and cyp-35A5 mutants had significantly higher levels of C18:1n7 and lower C18:2n6c. All four mutants had higher relative amounts of C18:1n7 than the wild-type. In the cyp-35A5 mutant, the levels of the endocannabinoid anandamide were found to be 4.6-fold higher than in wild-type. Several fatty acid synthesis genes were over-expressed in cyp-35A1 including fat-2. Feeding oleic or elaidic triglycerides to wild-type animals demonstrated that cyp-35A1 transcriptional levels are insensitive to environmental exposure of these fats, while cyp-35A2, cyp-35A4, and cyp-35A5 were significantly down regulated. CONCLUSION: These results demonstrate a dynamic role for CYP-35A subfamily members in maintaining the diversity of fatty acid profiles in C. elegans, and more generally highlight the importance of CYPs in generating both structural and signaling fatty acid functions in other organisms.