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In a screen for suppressors of activated GOA-1 (Galpha(o)) under the control of the hsp-16.2 heat-shock promoter, we identified three genetic loci that affected heat-shock-induced GOA-1 expression. The cyl-1 mutants are essentially wild type in appearance, while hsf-1 and sup-45 mutants have egg-laying defects. The hsf-1 mutation also causes a temperature-sensitive developmental arrest, and hsf-1 mutants have decreased life span. Western analysis indicated that mutations in all three loci suppressed the activated GOA-1 transgene by decreasing its expression. Heat-shock-induced expression of hsp-16.2 mRNA was reduced in cyl-1 mutants and virtually eliminated in hsf-1 and sup-45 mutants, as compared to wild-type expression. The mutations could also suppress other transgenes under heat-shock control. cyl-1 and sup-45, but not hsf-1, mutations suppressed a defect caused by a transgene not under heat-shock control, suggesting a role in general transcription or a post-transcriptional aspect of gene expression. hsf-1 encodes the C. elegans homolog of the human heat-shock factor HSF1, and cyl-1 encodes a cyclin most similar to cyclin L. We believe HSF-1 acts in heat-shock-inducible transcription and CYL-1 acts more generally in gene expression.

Animals integrate sensory information from the environment and display various behaviors in response to external stimuli. In Caenorhabditis elegans hermaphrodites, 33 types of sensory neurons are responsible for chemosensation, olfaction, and mechanosensation. However, the functional roles of all sensory neurons have not been systematically studied due to the lack of facile genetic accessibility. A bipartite cGAL-UAS system has been previously developed to study tissue- or cell-specific functions in C. elegans. Here, we report a toolkit of new cGAL drivers that can facilitate the analysis of a vast majority of the 60 sensory neurons in C. elegans hermaphrodites. We generated 37 sensory neuronal cGAL drivers that drive cGAL expression by cell-specific regulatory sequences or intersection of two distinct regulatory regions with overlapping expression (split cGAL). Most cGAL-drivers exhibit expression in single types of cells. We also constructed 28 UAS effectors that allow expression of proteins to perturb or interrogate sensory neurons of choice. This cGAL-UAS sensory neuron toolkit provides a genetic platform to systematically study the functions of C. elegans sensory neurons. [ABSTRACT FROM AUTHOR]

Comparing genomic and biological characteristics across multiple species is essential to using model systems to investigate the molecular and cellular mechanisms underlying human biology and disease and to translate mechanistic insights from studies in model organisms for clinical applications. Building a scalable knowledge commons platform that supports cross-species comparison of rich, expertly curated knowledge regarding gene function, phenotype, and disease associations available for model organisms and humans is the primary mission of the Alliance of Genome Resources (the Alliance). The Alliance is a consortium of seven model organism knowledgebases (mouse, rat, yeast, nematode, zebrafish, frog, fruit fly) and the Gene Ontology resource. The Alliance uses a common set of gene ortholog assertions as the basis for comparing biological annotations across the organisms represented in the Alliance. The major types of knowledge associated with genes that are represented in the Alliance database currently include gene function, phenotypic alleles and variants, human disease associations, pathways, gene expression, and both protein–protein and genetic interactions. The Alliance has enhanced the ability of researchers to easily compare biological annotations for common data types across model organisms and human through the implementation of shared programmatic access mechanisms, data-specific web pages with a unified "look and feel", and interactive user interfaces specifically designed to support comparative biology. The modular infrastructure developed by the Alliance allows the resource to serve as an extensible "knowledge commons" capable of expanding to accommodate additional model organisms. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.005 Byline: Takao Inoue (a)(b), Paul W. Sternberg (b) Keywords: C. elegans; Organogenesis; Cell cycle regulation; Cell division; Cell proliferation; BED domain; Zinc finger; Transcription; Gene regulatory network (GRN) Abstract: The control of cell division is critical to organogenesis, but how this control is achieved is not fully understood. We found that mutations in bed-3, encoding a BED Zn-finger domain transcription factor, confer a phenotype where a specific set of cell divisions during vulval organogenesis is lost. Unlike general cell cycle regulators in Caenorhabditis elegans, the function of bed-3 is restricted to specific lineages. Transcriptional reporters suggest that bed-3 is expressed in a limited number of cell types including vulval cells whose divisions are affected in bed-3 mutants. A bed-3 mutation also affects the expression pattern of the cdh-3 cadherin gene in the vulva. The phenotype of bed-3 mutants is similar to the phenotype caused by mutations in cog-1 (Nkx6), a component of a gene regulatory network controlling cell type specific gene expression in the vulval lineage. These results suggest that bed-3 is a key component linking the gene regulatory network controlling cell-type specification to control of cell division during vulval organogenesis. Author Affiliation: (a) Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (b) HHMI and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 22 April 2009; Revised 9 November 2009; Accepted 4 December 2009

Although transcriptomes have recently been used as phenotypes with which to perform epistasis analyses, they are not yet used to study intragenic function/structure relationships. We developed a theoretical framework to study allelic series using transcriptomic phenotypes. As a proof-of-concept, we apply our methods to an allelic series of dpy-22, a highly pleiotropic Caenorhabditis elegans gene orthologous to the human gene MED12, which encodes a subunit of the Mediator complex. Our methods identify functional units within dpy-22 that modulate Mediator activity upon various genetic programs, including the Wnt and Ras modules. [ABSTRACT FROM AUTHOR]

Bipartite expression systems, such as the GAL4-UAS system, allow fine manipulation of gene expression and are powerful tools for interrogating gene function. Recently, we established cGAL, a GAL4-based bipartite expression system for transgene control in Caenorhabditis elegans, where a single promoter dictates the expression pattern of a cGAL driver, which then binds target upstream activation sequences to drive expression of a downstream effector gene. Here, we report a split strategy for cGAL using the split intein gp41-1 for intersectional control of transgene expression. Split inteins are protein domains that associate, self-excise, and covalently ligate their flanking peptides together. We split the DNA binding domain and transcriptional activation domain of cGAL and fused them to the N terminal of gp41-1-N-intein and the C terminal of gp41-1-C-intein, respectively. In cells where both halves of cGAL are expressed, a functional cGAL driver is reconstituted via intein-mediated protein splicing. This reconstitution allows expression of the driver to be dictated by two promoters for refined spatial control or spatiotemporal control of transgene expression. We apply the split cGAL system to genetically access the single pair of MC neurons (previously inaccessible with a single promoter), and reveal an important role of protein kinase A in rhythmic pharyngeal pumping in C. elegans. Thus, the split cGAL system gives researchers a greater degree of spatiotemporal control over transgene expression, and will be a valuable genetic tool in C. elegans for dissecting gene function with finer cell-specific resolution. [ABSTRACT FROM AUTHOR]

The article presents the study on the transcriptional regulation of gene expression, which enables isogenic cells to phenotypic diversity.

Background: Over the last ten years, there has been explosive development in methods for measuring gene expression. These methods can identify thousands of genes altered between conditions, but understanding these datasets and forming hypotheses based on them remains challenging. One way to analyze these datasets is to associate ontologies (hierarchical, descriptive vocabularies with controlled relations between terms) with genes and to look for enrichment of specific terms. Although Gene Ontology (GO) is available for Caenorhabditis elegans, it does not include anatomical information. Results: We have developed a tool for identifying enrichment of C. elegans tissues among gene sets and generated a website GUI where users can access this tool. Since a common drawback to ontology enrichment analyses is its verbosity, we developed a very simple filtering algorithm to reduce the ontology size by an order of magnitude. We adjusted these filters and validated our tool using a set of 30 gold standards from Expression Cluster data in WormBase. We show our tool can even discriminate between embryonic and larval tissues and can even identify tissues down to the single-cell level. We used our tool to identify multiple neuronal tissues that are down-regulated due to pathogen infection in C. elegans. Conclusions: Our Tissue Enrichment Analysis (TEA) can be found within WormBase, and can be downloaded using Python's standard pip installer. It tests a slimmed-down C. elegans tissue ontology for enrichment of specific terms and provides users with a text and graphic representation of the results. [ABSTRACT FROM AUTHOR]

Background: During the fourth larval (L4) stage, vulval cells of C. elegans undergo extensive morphogenesis accompanied by changes in gene expression. This phase of vulval development, occurring after the well-studied induction of vulval cells, is not well understood but is potentially a useful context in which to study how a complex temporal sequence of events is regulated during development. However, a system for precisely describing different phases of vulval development in the L4 stage has been lacking. Results: We defined ten sub-stages of L4 based on morphological criteria as observed using Nomarski microscopy (L4.0 ~ L4.9). Precise timing of each sub-stage at 20 °C was determined. We also re-examined the timing of expression for several gene expression markers, and correlated the sub-stages with the timing of other developmental events in the vulva and the uterus. Conclusions: This scheme allows the developmental timing of an L4 individual to be determined at approximately one-hour resolution without the need to resort to time course experiments. These well-defined developmental stages will enable more precise description of gene expression and other developmental events. [ABSTRACT FROM AUTHOR]

The Caenorhabditis elegans uterine seam cell (utse) is an H-shaped syncytium that connects the uterus to the body wall. Comprising nine nuclei that move outward in a bidirectional manner, this synctium undergoes remarkable shape change during development. Using cell ablation experiments, we show that three surrounding cell types affect utse development: the uterine toroids, the anchor cell and the sex myoblasts. The presence of the anchor cell (AC) nucleus within the utse is necessary for proper utse development and AC invasion genes fos-1, cdh-3, him-4, egl-43, zmp-1 and mig-10 promote utse cell outgrowth. Two types of uterine lumen epithelial cells, uterine toroid 1 (ut1) and uterine toroid 2 (ut2), mediate proper utse outgrowth and we show roles in utse development for two genes expressed in the uterine toroids: the RASEF ortholog rsef-1 and Trio/ unc-73 . The SM expressed gene unc- 53/NAV regulates utse cell shape; ablation of sex myoblasts (SMs), which generate uterine and vulval muscles, cause defects in utse morphology. Our results clarify the nature of the interactions that exist between utse and surrounding tissue, identify new roles for genes involved in cell outgrowth, and present the utse as a new model system for understanding cell shape change and, putatively, diseases associated with cell shape change. [ABSTRACT FROM AUTHOR]

The bacterium X enorhabdus nematophila is a mutualist of entomopathogenic S teinernema carpocapsae nematodes and facilitates infection of insect hosts. X . nematophila colonizes the intestine of S . carpocapsae which carries it between insects. In the X . nematophila colonization-defective mutant nilD6:: Tn 5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X . nematophila strains from S . anatoliense and S . websteri nematodes. Only nilD from the S . carpocapsae strain of X . nematophila rescued the colonization defect of the nilD6:: Tn 5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I- E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression. [ABSTRACT FROM AUTHOR]

Background: The identity of each neuron is determined by the expression of a distinct group of genes comprising its terminal gene battery. The regulatory sequences that control the expression of such terminal gene batteries in individual neurons is largely unknown. The existence of a complete genome sequence for C. elegans and draft genomes of other nematodes let us use comparative genomics to identify regulatory sequences directing expression in the DVA interneuron. Methodology/Principal Findings: Using phylogenetic comparisons of multiple Caenorhabditis species, we identified conserved non-coding sequences in 3 of 10 genes (fax-1, nmr-1, and twk-16) that direct expression of reporter transgenes in DVA and other neurons. The conserved region and flanking sequences in an 85-bp intronic region of the twk-16 gene directs highly restricted expression in DVA. Mutagenesis of this 85 bp region shows that it has at least four regions. The central 53 bp region contains a 29 bp region that represses expression and a 24 bp region that drives broad neuronal expression. Two short flanking regions restrict expression of the twk-16 gene to DVA. A shared GA-rich motif was identified in three of these genes but had opposite effects on expression when mutated in the nmr-1 and twk-16 DVA regulatory elements. Conclusions/Significance: We identified by multi-species conservation regulatory regions within three genes that direct expression in the DVA neuron. We identified four contiguous regions of sequence of the twk-16 gene enhancer with positive and negative effects on expression, which combined to restrict expression to the DVA neuron. For this neuron a single binding site may thus not achieve sufficient specificity for cell specific expression. One of the positive elements, an 8-bp sequence required for expression was identified in silico by sequence comparisons of seven nematode species, demonstrating the potential resolution of expanded multi-species phylogenetic comparisons. [ABSTRACT FROM AUTHOR]

Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it can adopt one of two developmental fates (adulthood or diapause) depending on environmental conditions. The steroid hormone dafachronic acid (DA) directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We used a combination of physiological and molecular biology techniques to demonstrate that commitment to reproductive adult development occurs when DA levels, produced in the neuroendocrine XXX cells, exceed a threshold. Furthermore, imaging and cell ablation experiments demonstrate that the XXX cells act as a source of DA, which, upon commitment to adult development, is amplified and propagated in the epidermis in a DAF-12 dependent manner. This positive feedback loop increases DA levels and drives adult programs in the gonad and epidermis, thus conferring the irreversibility of the decision. We show that the positive feedback loop canalizes development by ensuring that sufficient amounts of DA are dispersed throughout the body and serves as a robust fate-locking mechanism to enforce an organism-wide binary decision, despite noisy and complex environmental cues. These mechanisms are not only relevant to C. elegans but may be extended to other hormonal-based decision-making mechanisms in insects and mammals. [ABSTRACT FROM AUTHOR]

Background: The success of a biological control agent depends on key traits, particularly reproductive potential, environmental tolerance, and ability to be cultured. These traits can deteriorate rapidly when the biological control agent is reared in culture. Trait deterioration under laboratory conditions has been widely documented in the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora (Hb) but the specific mechanisms behind these genetic processes remain unclear. This research investigates the molecular mechanisms of trait deterioration of two experimental lines of Hb, an inbred line (L5M) and its original parental line (OHB). We generated transcriptional profiles of two experimental lines of Hb, identified the differentially expressed genes (DEGs) and validated their differential expression in the deteriorated line. Results: An expression profiling study was performed between experimental lines L5M and OHB of Hb with probes for 15,220 ESTs from the Hb transcriptome. Microarray analysis showed 1,185 DEGs comprising of 469 down- and 716 up-regulated genes in trait deteriorated nematodes. Analysis of the DEGs showed that trait deterioration involves massive changes of the transcripts encoding enzymes involved in metabolism, signal transduction, virulence and longevity. We observed a pattern of reduced expression of enzymes related to primary metabolic processes and induced secondary metabolism. Expression of sixteen DEGs in trait deteriorated nematodes was validated by quantitative reverse transcription-PCR (qRT-PCR) which revealed similar expression kinetics for all the genes tested as shown by microarray. Conclusion: As the most closely related major entomopathogen to C. elegans, Hb provides an attractive near-term application for using a model organism to better understand interspecies interactions and to enhance our understanding of the mechanisms underlying trait deterioration in biological control agents. This information could also be used to improve the beneficial traits of biological control agents and better understand fundamental aspects of nematode parasitism and mutualism. [ABSTRACT FROM AUTHOR]

Background: The entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests. This nematode-bacterium-insect association represents an emerging tripartite model for research on mutualistic and parasitic symbioses. Elucidation of mechanisms underlying these biological processes may serve as a foundation for improving the biological control potential of the nematode-bacterium complex. This large-scale expressed sequence tag (EST) analysis effort enables gene discovery and development of microsatellite markers. These ESTs will also aid in the annotation of the upcoming complete genome sequence of H. bacteriophora. Results: A total of 31,485 high quality ESTs were generated from cDNA libraries of the adult H. bacteriophora TTO1 strain. Cluster analysis revealed the presence of 3,051 contigs and 7,835 singletons, representing 10,886 distinct EST sequences. About 72% of the distinct EST sequences had significant matches (E value < 1e-5) to proteins in GenBank's non-redundant (nr) and Wormpep190 databases. We have identified 12 ESTs corresponding to 8 genes potentially involved in RNA interference, 22 ESTs corresponding to 14 genes potentially involved in dauer-related processes, and 51 ESTs corresponding to 27 genes potentially involved in defense and stress responses. Comparison to ESTs and proteins of free-living nematodes led to the identification of 554 parasitic nematode-specific ESTs in H. bacteriophora, among which are those encoding F-box-like/WDrepeat protein theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein. Gene Ontology terms were assigned to 6,685 of the 10,886 ESTs. A total of 168 microsatellite loci were identified with primers designable for 141 loci. Conclusion: A total of 10,886 distinct EST sequences were identified from adult H. bacteriophora cDNA libraries. BLAST searches revealed ESTs potentially involved in parasitism, RNA interference, defense responses, stress responses, and dauerrelated processes. The putative microsatellite markers identified in H. bacteriophora ESTs will enable genetic mapping and population genetic studies. These genomic resources provide the material base necessary for genome annotation, microarray development, and in-depth gene functional analysis. [ABSTRACT FROM AUTHOR]

Regulation of spatio-temporal gene expression in diverse cell and tissue types is a critical aspect of development. Progression through Caenorhabditis elegans vulval development leads to the generation of seven distinct vulval cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), each with its own unique gene expression profile. The mechanisms that establish the precise spatial patterning of these mature cell types are largely unknown. Dissection of the gene regulatory networks involved in vulval patterning and differentiation would help us understand how cells generate a spatially defined pattern of cell fates during organogenesis. We disrupted the activity of 508 transcription factors via RNAi and assayed the expression of ceh-2, a marker for vulB fate during the L4 stage. From this screen, we identified the tailless ortholog nhr-67 as a novel regulator of gene expression in multiple vulval cell types. We find that one way in which nhr-67 maintains cell identity is by restricting inappropriate cell fusion events in specific vulval cells, namely vulE and vulF. nhr-67 exhibits a dynamic expression pattern in the vulval cells and interacts with three other transcriptional regulators cog-1 (Nkx6.1/6.2), lin-11 (LIM), and egl-38 (Pax2/5/8) to generate the composite expression patterns of their downstream targets. We provide evidence that egl-38 regulates gene expression in vulB1, vulC, vulD, vulE, as well as vulF cells. We demonstrate that the pairwise interactions between these regulatory genes are complex and vary among the seven cell types. We also discovered a striking regulatory circuit that affects a subset of the vulval lineages: cog-1 and nhr-67 inhibit both one another and themselves. We postulate that the differential levels and combinatorial patterns of lin-11, cog-1, and nhr-67 expression are a part of a regulatory code for the mature vulval cell types. [ABSTRACT FROM AUTHOR]

Explains the genetic control of cell lineage during the developmental stages of nematodes. Patterns of cell divisions and cell fates; Invariance and cell autonomy; Discussion of cell lineage mutants.

The nematode Caenorhabditis elegans utilizes chemosensation to navigate an ever-changing environment for its survival. A class of secreted small-molecule pheromones, termed ascarosides, play an important role in olfactory perception by affecting biological functions ranging from development to behavior. The ascaroside #8 (ascr#8) mediates sex-specific behaviors, driving avoidance in hermaphrodites and attraction in males. Males sense ascr#8 via the ciliated male-specific cephalic sensory (CEM) neurons, which exhibit radial symmetry along dorsal–ventral and left–right axes. Calcium imaging studies suggest a complex neural coding mechanism that translates stochastic physiological responses in these neurons to reliable behavioral outputs. To test the hypothesis that neurophysiological complexity arises from differential expression of genes, we performed cell-specific transcriptomic profiling; this revealed between 18 and 62 genes with at least twofold higher expression in a specific CEM neuron subtype vs both other CEM neurons and adult males. These included two G protein–coupled receptor (GPCR) genes, srw-97 and dmsr-12, that were specifically expressed in nonoverlapping subsets of CEM neurons and whose expression was confirmed by GFP reporter analysis. Single CRISPR-Cas9 knockouts of either srw-97 or dmsr12 resulted in partial defects, while a double knockout of both srw-97 and dmsr-12 completely abolished the attractive response to ascr#8. Together, our results suggest that the evolutionarily distinct GPCRs SRW-97 and DMSR-12 act nonredundantly in discrete olfactory neurons to facilitate male-specific sensation of ascr#8. [ABSTRACT FROM AUTHOR]

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion. [ABSTRACT FROM AUTHOR]

Summary: Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly upregulated during recovery from growth-arrested states. This expression pattern is anticorrelated to nonoperon genes, consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. We provide evidence that operons become advantageous because, by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes. [Copyright &y& Elsevier]

Abstract: In C aenorhabditis elegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9–11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1°, 2°, or 3°), while eight anterior cells, P(1–8).p, fuse with the hyp7 epidermal syncytium. Here we show that activation of the canonical BAR-1 β-catenin pathway of Wnt signaling alters the competence of P(3–8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1–8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-12 or EGF signaling. While increased Wnt signaling induces predominantly 1° HCG fates, increased LIN-12 or EGF signaling in combination with MAB-5 overexpression promotes 2° HCG fates in anterior Pn.p cells, suggesting distinctive functions of Wnt, LIN-12, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9–11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development. [ABSTRACT FROM AUTHOR]

Neuropeptides are evolutionarily conserved modulators of many aspects of animal behavior and physiology, and expand the repertoire of processes that can be controlled by a limited number of neurons. Deciphering the neuropeptidergic codes that govern distinct processes requires systematic functional analyses of neuropeptides and their cognate receptors. Even in well-studied model organisms like Caenorhabditis elegans, however, such efforts have been precluded by a lack of mutant reagents. Here, we generated and screened 21 C. elegans neuropeptide G-protein coupled receptor mutants with no pre-existing reagents for the touch-evoked escape response, and implicated six receptors expressed in diverse neuron classes representing multiple circuit levels in this behavior. We further characterized the mutant with the most severe phenotype, frpr-14, which was defective in multiple behavioral paradigms. We leveraged this range of phenotypes to reveal that FRPR-14 modulation of different precommand interneuron classes, AVH and AIB, can drive distinct behavioral subsets, demonstrating cellular context-dependent roles for FRPR-14 signaling. We then show that Caenorhabditis briggsae CBR-FRPR-14 modulates an AVH-like interneuron pair to regulate the same behaviors as C. elegans but to a smaller extent. Our results also suggest that differences in touch-evoked escape circuit architecture between closely related species results from changes in neuropeptide receptor expression pattern, as opposed to ligand-receptor pairing. This study provides insights into the principles utilized by a compact, multiplexed nervous system to generate intraspecific behavioral complexity and interspecific variation. [ABSTRACT FROM AUTHOR]

When Caenorhabditis elegans larvae hatch from the egg case in the absence of food, their development is arrested (L1 arrest), and they show increased stress resistance until food becomes available. To study nutritional control of larval development, we analyzed growth and gene expression profiles during L1 arrest and recovery. Larvae that were fed responded relatively slowly to starvation compared with the rapid response of arrested larvae to feeding. Chromatin immunoprecipitation of RNA polymerase II (Pol II) followed by deep sequencing showed that during L1 arrest, Pol II continued transcribing starvation-response genes, but the enzyme accumulated on the promoters of growth and development genes. In response to feeding, promoter accumulation decreased, and elongation and messenger RNA levels increased. Therefore, accumulation of Pol II at promoters anticipates nutritionally controlled gene expression during C. elegans development. [ABSTRACT FROM AUTHOR]

To mitigate the deleterious effects of temperature increases on cellular organization and proteotoxicity, organisms have developed mechanisms to respond to heat stress. In eukaryotes, HSF1 is the master regulator of the heat shock transcriptional response, but the heat shock response pathway is not yet fully understood. From a forward genetic screen for suppressors of heatshock- induced gene expression in Caenorhabditis elegans, we found a new allele of hsf-1 that alters its DNA-binding domain, and we found three additional alleles of sup-45, a previously molecularly uncharacterized genetic locus. We identified sup-45 as one of the two hitherto unknown C. elegans orthologs of the human AF4/FMR2 family proteins, which are involved in regulation of transcriptional elongation rate. We thus renamed sup-45 as affl-2 (AF4/FMR2-Like). Through RNA-seq, we demonstrated that affl-2 mutants are deficient in heat-shock-induced transcription. Additionally, affl-2 mutants have herniated intestines, while worms lacking its sole paralog (affl-1) appear wild type. AFFL-2 is a broadly expressed nuclear protein, and nuclear localization of AFFL-2 is necessary for its role in heat shock response. affl-2 and its paralog are not essential for proper HSF-1 expression and localization after heat shock, which suggests that affl-2 may function downstream of, or parallel to, hsf-1. Our characterization of affl-2 provides insights into the regulation of heat-shock-induced gene expression to protect against heat stress. [ABSTRACT FROM AUTHOR]

Summary The genetic basis of sleep regulation remains poorly understood. In C. elegans , cellular stress induces sleep through epidermal growth factor (EGF)-dependent activation of the EGF receptor in the ALA neuron. The downstream mechanism by which this neuron promotes sleep is unknown. Single-cell RNA sequencing of ALA reveals that the most highly expressed, ALA-enriched genes encode neuropeptides. Here we have systematically investigated the four most highly enriched neuropeptides: flp - 7 , nlp - 8 , flp - 24 , and flp - 13 . When individually removed by null mutation, these peptides had little or no effect on stress-induced sleep. However, stress-induced sleep was abolished in nlp - 8 ; flp - 24 ; flp - 13 triple-mutant animals, indicating that these neuropeptides work collectively in controlling stress-induced sleep. We tested the effect of overexpression of these neuropeptide genes on five behaviors modulated during sleep—pharyngeal pumping, defecation, locomotion, head movement, and avoidance response to an aversive stimulus—and we found that, if individually overexpressed, each of three neuropeptides ( nlp - 8 , flp - 24 , or flp - 13 ) induced a different suite of sleep-associated behaviors. These overexpression results raise the possibility that individual components of sleep might be specified by individual neuropeptides or combinations of neuropeptides. [ABSTRACT FROM AUTHOR]

Abstract: The vaccinia-related kinases (VRKs) are highly conserved throughout the animal kingdom and phosphorylate several chromatin proteins and transcription factors. In early Caenorhabditis elegans embryos, VRK-1 is required for proper nuclear envelope formation. In this work, we present the first investigation of the developmental role of VRKs by means of a novel C. elegans vrk-1 mutant allele. We found that VRK-1 is essential in hermaphrodites for formation of the vulva, uterus, and utse and for development and maintenance of the somatic gonad and thus the germ line. VRK-1 regulates anchor cell polarity and the timing of anchor cell invasion through the basement membranes separating vulval and somatic gonadal cells during the L3 larval stage. VRK-1 is also required for proper specification and proliferation of uterine cells and sex myoblasts. Expression of the fibroblast growth factor-like protein EGL-17 and its receptor EGL-15 is reduced in vrk-1 mutants, suggesting that VRK-1 might act at least partially through activation of FGF signaling. Expression of a translational VRK-1∷GFP fusion protein in the ventral nerve cord and vulva precursor cells restores vulva and uterus formation, suggesting both cell autonomous and non-autonomous roles of VRK-1. [Copyright &y& Elsevier]

Abstract: Hox genes are highly conserved segmental identity genes well known for their complex expression patterns and divergent targets. Here we present an analysis of cis-regulatory elements in the Caenorhabditis elegans Hox gene egl-5, which is expressed in multiple tissues in the posterior region of the nematode. We have utilized phylogenetic footprinting to efficiently identify cis-regulatory elements and have characterized these with gfp reporters and tissue-specific rescue experiments. We have found that the complex expression pattern of egl-5 is the cumulative result of the activities of multiple tissue or local region-specific activator sequences that are conserved both in sequence and near-perfect order in the related nematode Caenorhabditis briggsae. Two conserved regulatory blocks analyzed in detail contain multiple sites for both positively and negatively acting factors. One of these regions may promote activation of egl-5 in certain cells via the Wnt pathway. Positively acting regions are repressed in inappropriate tissues by additional negative pathways acting at other sites within the promoter. Our analysis has allowed us to implicate several new regulatory factors significant to the control of egl-5 expression. [Copyright &y& Elsevier]