Functional genomic analysis of C. elegans molting.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development

Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability

Evolutionarily conserved mechanisms that control aging are predicted to have prereproductive functions in order to be subject to natural selection. Genes that are essential for growth and development are highly conserved in evolution, but their role in longevity has not previously been assessed. We screened 2,700 genes essential for Caenorhabditis elegans development and identified 64 genes that extend lifespan when inactivated postdevelopmentally. These candidate lifespan regulators are highly conserved from yeast to humans. Classification of the candidate lifespan regulators into functional groups identified the expected insulin and metabolic pathways but also revealed enrichment for translation, RNA, and chromatin factors. Many of these essential gene inactivations extend lifespan as much as the strongest known regulators of aging. Early gene inactivations of these essential genes caused growth arrest at larval stages, and some of these arrested animals live much longer than wild-type adults. daf-16 is required for the enhanced survival of arrested larvae, suggesting that the increased longevity is a physiological response to the essential gene inactivation. These results suggest that insulin-signaling pathways play a role in regulation of aging at any stage in life

Dual Regulation of the lin-14 Target mRNA by the lin-4 miRNA

microRNAs (miRNAs) are ∼22 nt regulatory RNAs that in animals typically bind with partial complementarity to sequences in the 3′ untranslated (UTR) regions of target mRNAs, to induce a decrease in the production of the encoded protein. The relative contributions of translational inhibition of intact mRNAs and degradation of mRNAs caused by binding of the miRNA vary; for many genetically validated miRNA targets, translational repression has been implicated, whereas some analyses of other miRNA targets have revealed only modest translational repression and more significant mRNA destabilization. In Caenorhabditis elegans, the lin-4 miRNA accumulates during early larval development, binds to target elements in the lin-14 mRNA, and causes a sharp decrease in the abundance of LIN-14 protein. Here, we monitor the dynamics of lin-14 mRNA and protein as well as lin-4 miRNA levels in finely staged animals during early larval development. We find complex regulation of lin-14, with the abundance of lin-14 mRNA initially modestly declining followed by fluctuation but little further decline of lin-14 mRNA levels accompanied by continuing and more dramatic decline in LIN-14 protein abundance. We show that the translational inhibition of lin-14 is dependent on binding of the lin-4 miRNA to multiple lin-4 complementary sites in the lin-14 3′UTR. Our results point to the importance of translational inhibition in silencing of lin-14 by the lin-4 miRNA

Regulation of the C. elegans molt by pqn-47

AbstractC. elegans molts at the end of each of its four larval stages but this cycle ceases at the reproductive adult stage. We have identified a regulator of molting, pqn-47. Null mutations in pqn-47 cause a developmental arrest at the first larval molt, showing that this gene activity is required to transit the molt. Mutants with weak alleles of pqn-47 complete the larval molts but fail to exit the molting cycle at the adult stage. These phenotypes suggest that pqn-47 executes key aspects of the molting program including the cessation of molting cycles. The pqn-47 gene encodes a protein that is highly conserved in animal phylogeny but probably misannotated in genome sequences due to much less significant homology to a yeast transcription factor. A PQN-47::GFP fusion gene is expressed in many neurons, vulval precursor cells, the distal tip cell (DTC), intestine, and the lateral hypodermal seam cells but not in the main body hypodermal syncytium (hyp7) that underlies, synthesizes, and releases most of the collagenous cuticle. A functional PQN-47::GFP fusion protein localizes to the cytoplasm rather than the nucleus at all developmental stages, including the periods preceding and during ecdysis when genetic analysis suggests that pqn-47 functions. The cytoplasmic localization of PQN-47::GFP partially overlaps with the endoplasmic reticulum, suggesting that PQN-47 is involved in the extensive secretion of cuticle components or hormones that occurs during molts. The mammalian and insect homologues of pqn-47 may serve similar roles in regulated secretion

TheCaenorhabditis elegansOrphan Nuclear Hormone Receptor Genenhr-2Functions in Early Embryonic Development

AbstractWe have identified aCaenorhabditis elegansgene,nhr-2,that is a member of the nuclear hormone receptor superfamily of transcription factors and defines a new subclass of the superfamily.nhr-2messenger RNA is expressed in the maternal germline and during the first half of embryogenesis. Zygotic expression ofnhr-2begins by the 16-cell stage, making it one of the earliest genes known to be transcribed in the embryo. Immunolocalization detects NHR-2 protein in embryonic nuclei as early as the 2-cell stage. The protein is present in every nucleus until the 16- to 20-cell stage. Subsequently, expression continues in many, but not all, cell lineages, becoming progressively restricted to the anterior and dorsal regions of the embryo and disappearing during the initial stages of morphogenesis. Disruption ofnhr-2function with antisense RNA results in embryonic and early larval arrest, indicating that the gene has an essential function in embryonic development.nhr-2does not correspond to known mutations mapped to the same genetic interval, and will provide an entry point for further study of a heretofore uncharacterized zygotic gene regulatory pathway

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues

Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability

Evolutionarily conserved mechanisms that control aging are predicted to have prereproductive functions in order to be subject to natural selection. Genes that are essential for growth and development are highly conserved in evolution, but their role in longevity has not previously been assessed. We screened 2,700 genes essential for Caenorhabditis elegans development and identified 64 genes that extend lifespan when inactivated postdevelopmentally. These candidate lifespan regulators are highly conserved from yeast to humans. Classification of the candidate lifespan regulators into functional groups identified the expected insulin and metabolic pathways but also revealed enrichment for translation, RNA, and chromatin factors. Many of these essential gene inactivations extend lifespan as much as the strongest known regulators of aging. Early gene inactivations of these essential genes caused growth arrest at larval stages, and some of these arrested animals live much longer than wild-type adults. daf-16 is required for the enhanced survival of arrested larvae, suggesting that the increased longevity is a physiological response to the essential gene inactivation. These results suggest that insulin-signaling pathways play a role in regulation of aging at any stage in life

Functional Genomic Analysis of C. elegans Molting

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development

Acceleration Profiles and Processing Methods for Parabolic Flight

Parabolic flights provide cost-effective, time-limited access to "weightless" or reduced gravity conditions experienced in space or on planetary surfaces, e.g. the Moon or Mars. These flights facilitate fundamental research - from materials science to space biology - and testing/validation activities that support and complement infrequent and costly access to space. While parabolic flights have been conducted for decades, reference acceleration profiles and processing methods are not widely available - yet are critical for assessing the results of these activities. Here we present a method for collecting, analyzing, and classifying the altered gravity environments experienced during a parabolic flight. We validated this method using a commercially available accelerometer during a Boeing 727-200F flight with $20$ parabolas. All data and analysis code are freely available. Our solution can be easily integrated with a variety of experimental designs, does not depend upon accelerometer orientation, and allows for unsupervised and repeatable classification of all phases of flight, providing a consistent and open-source approach to quantifying gravito-intertial accelerations (GIA), or $g$ levels. As academic, governmental, and commercial use of space increases, data availability and validated processing methods will enable better planning, execution, and analysis of parabolic flight experiments, and thus, facilitate future space activities.Comment: Correspondence to C.E. Carr ([email protected]). 15 pages, 4 figures, 3 supplemental figures. Code: https://github.com/CarrCE/zerog, Dataset: https://osf.io/nk2w4

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues