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[Figure][1] H. Robert Horvitz THE 2001 GSA Medal is given to H. Robert Horvitz to honor his many contributions to genetics research and training. One of the pioneers in the Caenorhabditis elegans system, Bob has cut a genetic swath through many areas of developmental biology, cell biology

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.

Vulval differentiation in Caenorhabditis elegans is controlled by intercellular signalling mediated by a receptor tyrosine kinase and a ras gene product. The lin-45 gene encodes a homologue of the raf family of serine/threonine kinases and is necessary for vulval differentiation. The lin-45 raf gene product appears to act downstream of the ras protein in this pathway. A proto-oncogene-mediated signalling pathway may be a common feature of metazoan development.

Intercellular signaling and signal transduction underlie most aspects of development and behavior. To understand any specific case we must identify the ligands, receptors and transducers, as well as regulators that modulate the activity of the signaling pathway. To understand more general aspects of signaling, we have to address the questions: To what extent are there modular signaling pathways, i.e. pathways that act as coherent units? How many such pathways are there? What factors affect the action of a universal pathway in particular cases? How widely is a given component or pathway used? How are the effects of multiple signaling pathways integrated? Here I discuss the use of Caenorhabditis elegans molecular genetics to study problems of intercellular signaling and signal transduction.

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

Background The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention.

Reproducible cell-cell interactions contribute to the invariance of Caenorhabditis elegans development and allow high resolution study of molecular mechanisms of intercellular signaling. A number of new cell interactions have been discovered in the past year. The power of nematode molecular genetics has been increased through several technical advances and the genome project, and these new approaches are now being successfully applied both to familiar and new signaling mechanisms.

The lin-3 gene is necessary for induction of the Caenorhabditis elegans vulva by the anchor cell. It encodes a molecule similar to epidermal growth factor and to transforming growth factor-alpha and acts through the epidermal growth factor receptor homologue let-23. Expression of lin-3 in the anchor cell stimulates vulval induction; lin-3 may encode the vulval inducing signal.

Publisher Summary This chapter discusses pattern formation in Caenorhabditis elegans ( C. elegans ). C. elegans has two sexes, hermaphrodites and males. Hermaphrodites produce both oocytes and sperm and can reproduce by internal self-fertilization. Alternatively, males can fertilize hermaphrodites. The formation of any pattern specified by cell interactions involves the differentiation of two or more cells with equal developmental potentials. The formation of several developmental patterns can be events of sequential induction and cell interactions. For example, lateral signaling between two gonad precursor cells determines the anchor cell; the anchor cell then induces vulva1 formation; and the formation of the vulva then in turn induces several other developmental events including the branching of ventral cord motor neurons. The gonad of a C. elegans hermaphrodite or male starts to develop at the L1 larval stage from a gonadal primordium. The somatic gonad develops throughout the larval stages; the germ line continues to divide after the animals enter the adult stage. Cell interactions play important roles in the development of both the somatic gonad and germ line. Vulval pattern formation is proposed to be controlled by both inductive and lateral cell signals. An inductive signal from the anchor cell (AC) in the gonad to the ventral hypodermis controls the binary decision of vulval precursor cells (VPCs) between vulval cell types and the hypodermal cell type. Lateral signaling among vulval precursor cells controls the binary decision of the VPCs between two different vulval precursor cell types.

The let-60 gene of Caenorhabditis elegans controls the choice between vulval and hypodermal differentiation in response to an inductive signal from the gonad. let-60 encodes a ras protein that acts downstream of the let-23 receptor tyrosine kinase in a signal transduction pathway. Dominant-negative mutations of let-60 [let-60(dn)] cause a reduction of the gene activity in let-60(dn)/+ heterozygotes and a vulva-less mutant phenotype. We have found that nine let-60(dn) mutations cause replacements of conserved residues. Four are in two novel positions; others are in positions known previously to cause dominant-negative mutations in mammalian cells. The locations of these lesions suggest that they disrupt the ability of the ras protein to bind guanine nucleotides. Four let-60(dn) mutant genes were introduced into wild-type animals in the form of extrachromosomal arrays and were found to generate three dominant phenotypes--lethality, vulva-less, or multivulva--depending on gene dose and alleles. The dominant lethality caused by high-dose transgenic let-60(dn) genes suggests a toxic effect of these mutant genes in early development. The dominant-negative effects of these mutations in heterozygotes are likely to be caused by competition between let-60(dn) and let-60(+) protein for a positive regulator. All let-60(dn) mutations interfere with let-60(+) activity, but some alleles have partial constitutive activity, suggesting that the ability to interact with the activator is separable from the ability to exert a physiological effect (stimulation of vulval differentiation). These dn mutations might be useful for interfering with ras-mediated signal transduction pathways in other multicellular organisms.

Nematode proteins related to the human epidermal growth factor receptor and Ras proteins act in a common pathway to control cell fates in response to an inductive signal. Analysis of these gene products during C. elegans vulval induction allows detailed study of their function in the context of a developing organism.

Developmental, genetic and molecular studies indicate that multiple intercellular signalling systems interact to specify the types and spatial patterns of cells generated during the formation of the vulva of the nematode Caenorhabditis elegans. Two classes of evolutionarily conserved transmembrane receptors and a Ras protein function in these signalling systems. The biology of vulval development provides a framework for understanding how cell interactions control the development of animals as diverse as nematodes, insects and mammals.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

Publisher Summary This chapter discusses the developmental and comparative studies that define the key issues in nematode development. It describes the evidence for intrinsic and extrinsic controls of cell fates, the studies of the mechanisms of cell fate specification, and the evolution of fate-specifying mechanisms. Most cell fates in nematodes are specified autonomously, and nematodes thus lie toward one end of a continuum of organisms whose development ranges from mostly conditional to mostly autonomous. Invariant cell interactions are experimentally useful: the experimenter knows what a cell will do before the cell becomes specified. As for understanding the autonomous specification of cell fate, the original attraction of nematodes still exists: asymmetric cell divisions, in which two daughter cells differ in their fates, presumably due to the asymmetric distribution of some factors at the cell division, can be studied in a variety of cells, from the first cleavage of the zygote to late in sexual maturation. Free-living soil nematodes develop during a relatively short period of embryogenesis, followed by a more extended period of postembryonic development. During postembryonic development, growth-associated increases in cell number and sexual differentiation occur. The invariant development of nematodes, such as Caenorhabditis elegans, is due not only to a deterministic cell lineage, but also to highly reproducible cell interactions. A rigidly determined cell lineage ensures that cells are born in identical positions in each animal, thereby allowing a precise fate specification by cell interactions.

Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism., Author Summary In the present study, we constructed a cDNA library enriched for molecules of the infective third-stage larva (L3) of Ascaris suum, the common roundworm of pigs. Using the method of suppressive-subtractive hybridization (SSH), we explored transcription of a subset of molecules by microarray analysis and conducted bioinformatic analyses to characterize these molecules, map them to biochemical pathways, and predict genetic interactions based on comparisons with Caenorhabditis elegans and/or other organisms. The results provide interesting insights into early molecular processes in A. suum. Approximately 60% of the L3-enriched molecules discovered had homologues in C. elegans. Probabilistic analyses suggested that a complex genetic network regulates or controls larval growth and development in A. suum L3s, some of which might be involved in or regulate the switch from the free-living to the parasitic stage. Functional studies of these molecules to elucidate developmental processes in Ascaris could assist in identifying new targets for intervention.

During induction of the Caenorhabditis elegans hermaphrodite vulva by the anchor cell of the gonad, six multipotent vulval precursor cells (VPCs) have two distinct fates: three VPCs generate the vulva and the other three VPCs generate nonspecialized hypodermis. Genes that control the fates of the VPCs in response to the anchor cell signal are defined by mutations that cause all six VPCs to generate vulval tissue (Multivulva or Muv) or that cause all six VPCs to generate hypodermis (Vulvaless or Vul). Seven dominant Vul mutations were isolated as dominant suppressors of a lin-15 Muv mutation. These mutations are dominant alleles of the gene let-60, previously identified only by recessive lethal mutations. Our genetic studies of these dominant Vul recessive lethal mutations, recessive lethal mutations, intragenic revertants of the dominant Vul mutations, and the closely mapping semi-dominant multivulva lin-34 mutations suggest that: (1) loss-of-function mutations of let-60 are recessive lethal at a larval stage, but they also cause a Vul phenotype if the lethality is rescued maternally by a lin-34 gain-of-function mutation. (2) The dominant Vul alleles of let-60 are dominant negative mutations whose gene products compete with wild-type activity. (3) lin-34 semidominant Muv alleles are either gain-of-function mutations of let-60 or gain-of-function mutations of an intimately related gene that elevates let-60 activity. We propose that let-60 activity controls VPC fates. In a wild-type animal, reception by a VPC of inductive signal activates let-60, and it generates into a vulval cell type; in absence of inductive signal, let-60 activity is low and the VPC generates hypodermal cells. Our genetic interaction studies suggest that let-60 acts downstream of let-23 and lin-15 and upstream of lin-1 and lin-12 in the genetic pathway specifying the switch between vulval and nonvulval cell types.

Genetic analysis previously suggested that the let-60 gene controls the switch between vulval and hypodermal cell fates during C. elegans vulval induction. We have cloned the let-60 gene, and shown that it encodes a gene product identical in 84% of its first 164 amino acids to ras gene products from other vertebrate and invertebrate species. This conservation suggests that the let-60 product contains all the biochemical functions of ras proteins. Extrachromosomal arrays of let-60 ras DNA cause cell-type misspecification (extra vulval fates) phenotypically opposite to that caused by let-60 ras loss-of-function mutations (no vulval fates), and suppress the vulvaless phenotype of mutations in two other genes necessary for vulval induction. Thus, the level and pattern of let-60 ras expression may be under strict regulation; increase in let-60 ras activity bypasses or reduces the need for upstream genes in the vulval induction pathway.

Publisher Summary The inferred products of the four Caenorhabditis elegans developmental control genes whose sequences have been published include three with homeobox domains and one with similarity to growth factors or receptors. Moreover, some of the many unidentified gene products may involve novel biochemical activities or intriguing cell structures. It is clear that a majority of developmental decisions involve the basic components of the cellular regulatory machinery. The utility of developmental genetics as a tool for understanding cellular regulatory mechanisms is in embedding a particular gene product solidly into the context of a pathway. Genetics is at its most powerful in identifying interacting components, and study of such interacting components is necessary to complete the understanding of any mechanism. By elucidating detailed molecular mechanisms of cell-type determination and pattern formation either in C. elegans or in other organisms, and by understanding, in parallel, the genetic pathways controlling development in each of several organisms, it can be hoped that someday the genetic circuitry that underlies animal development can be unraveled.

Background The Caenorhabditis elegans male exhibits a stereotypic behavioral pattern when attempting to mate. This behavior has been divided into the following steps: response, backing, turning, vulva location, spicule insertion, and sperm transfer. We and others have begun in-depth analyses of all these steps in order to understand how complex behaviors are generated. Here we extend our understanding of the sperm-transfer step of male mating behavior. Results Based on observation of wild-type males and on genetic analysis, we have divided the sperm-transfer step of mating behavior into four sub-steps: initiation, release, continued transfer, and cessation. To begin to understand how these sub-steps of sperm transfer are regulated, we screened for ethylmethanesulfonate (EMS)-induced mutations that cause males to transfer sperm aberrantly. We isolated an allele of unc-18, a previously reported member of the Sec1/Munc-18 (SM) family of proteins that is necessary for regulated exocytosis in C. elegans motor neurons. Our allele, sy671, is defective in two distinct sub-steps of sperm transfer: initiation and continued transfer. By a series of transgenic site-of-action experiments, we found that motor neurons in the ventral nerve cord require UNC-18 for the initiation of sperm transfer, and that UNC-18 acts downstream or in parallel to the SPV sensory neurons in this process. In addition to this neuronal requirement, we found that non-neuronal expression of UNC-18, in the male gonad, is necessary for the continuation of sperm transfer. Conclusion Our division of sperm-transfer behavior into sub-steps has provided a framework for the further detailed analysis of sperm transfer and its integration with other aspects of mating behavior. By determining the site of action of UNC-18 in sperm-transfer behavior, and its relation to the SPV sensory neurons, we have further defined the cells and tissues involved in the generation of this behavior. We have shown both a neuronal and non-neuronal requirement for UNC-18 in distinct sub-steps of sperm-transfer behavior. The definition of circuit components is a crucial first step toward understanding how genes specify the neural circuit and hence the behavior.

Nematode sinusoidal movement has been used as a phenotype in many studies of C. elegans development, behavior and physiology. A thorough understanding of the ways in which genes control these aspects of biology depends, in part, on the accuracy of phenotypic analysis. While worms that move poorly are relatively easy to describe, description of hyperactive movement and movement modulation presents more of a challenge. An enhanced capability to analyze all the complexities of nematode movement will thus help our understanding of how genes control behavior. We have developed a user-friendly system to analyze nematode movement in an automated and quantitative manner. In this system nematodes are automatically recognized and a computer-controlled microscope stage ensures that the nematode is kept within the camera field of view while video images from the camera are stored on videotape. In a second step, the images from the videotapes are processed to recognize the worm and to extract its changing position and posture over time. From this information, a variety of movement parameters are calculated. These parameters include the velocity of the worm's centroid, the velocity of the worm along its track, the extent and frequency of body bending, the amplitude and wavelength of the sinusoidal movement, and the propagation of the contraction wave along the body. The length of the worm is also determined and used to normalize the amplitude and wavelength measurements. To demonstrate the utility of this system, we report here a comparison of movement parameters for a small set of mutants affecting the Go/Gq mediated signaling network that controls acetylcholine release at the neuromuscular junction. The system allows comparison of distinct genotypes that affect movement similarly (activation of Gq-alpha versus loss of Go-alpha function), as well as of different mutant alleles at a single locus (null and dominant negative alleles of the goa-1 gene, which encodes Go-alpha). We also demonstrate the use of this system for analyzing the effects of toxic agents. Concentration-response curves for the toxicants arsenite and aldicarb, both of which affect motility, were determined for wild-type and several mutant strains, identifying P-glycoprotein mutants as not significantly more sensitive to either compound, while cat-4 mutants are more sensitive to arsenite but not aldicarb. Automated analysis of nematode movement facilitates a broad spectrum of experiments. Detailed genetic analysis of multiple alleles and of distinct genes in a regulatory network is now possible. These studies will facilitate quantitative modeling of C. elegans movement, as well as a comparison of gene function. Concentration-response curves will allow rigorous analysis of toxic agents as well as of pharmacological agents. This type of system thus represents a powerful analytical tool that can be readily coupled with the molecular genetics of nematodes.

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., Author Summary During development, in which the single-celled egg generates a whole organism, cells become different from each other and form patterns of types of cells. It is these spatially defined fate patterns that underlie the formation of complex organs. Regulatory molecules called transcription factors influence the fate patterns that cells adopt. Understanding the role of these transcription factors and their interactions with other genes could tell us how cells establish a certain pattern of cell fates. This study focuses on studying how the seven cell types of the Caenorhabditis elegans vulva arise. This organ is one of the most intensively studied, and while the signaling network that initiates vulval development and sets the gross pattern of cell differentiation is well understood, the network of transcription factors that specifies the final cell fates is not understood. Here, we identify nhr-67, a new transcription factor that regulates patterning of cell fates in this organ. Transcription factors do not necessarily act alone, and we explore how NHR-67 works with three other regulatory factors (each with human homologs) to specify the different properties of the vulval cells. We also demonstrate that the interconnections of these transcription factors differ between these seven diverse cell types, which may partially account for how these cells acquire a certain pattern of cell fates.

The publication of the draft genome sequence of Caenorhabditis briggsae improves the annotation of the genome of its close relative Caenorhabditis elegans and will facilitate comparative genomics and the study of the evolutionary changes during development.

Sir-Several animal taxa display a consistent left-right asymmetry of the body plan. In nematodes, dextrality predominates. However, we have now found a nematode species that has sinistral populations.

Previous studies have shown that the development of the vulva of the C. elegans hermaphrodite involves six multipotential hypodermal cells as well as the gonadal anchor cell, which induces vulval formation. Our further examination of the interactions among these seven cells has led to the following model. Each hypodermal precursor cell becomes determined to adopt one of its three potential fates; each of these fates is to generate a particular cell lineage. In the absence of cellular interactions each precursor cell will generate the nonvulval cell lineage; an inductive signal from the anchor cell is required for a precursor cell to generate either of the two types of vulval cell lineages. The inductive signal is spatially graded, and the potency of the signal specifies which lineage is expressed by each of the tripotential precursor cells.

Each of the six C. elegans vulval precursor cells (VPCs) has three potential fates (1 degree, 2 degrees, or 3 degrees). The fate of each VPC depends on two types of signals: a graded inductive signal that acts at a distance and a short-range lateral signal among the VPCs. We describe interactions among mutations that cause different misspecifications of VPC fates. Particular combinations of mutations cause all six VPCs to have a single fate independent of their positions. Our results suggest that specification of the three VPC fates is accomplished by two binary decisions, each effected by one of the two signaling pathways. The gene lin-12 acts in the lateral signaling pathway and specifies 2 degrees. The "vulvaless" and "multivulva" genes act in the inductive signaling pathway and specify 1 degree independently of lin-12 and 2 degrees via lin-12. We describe a model for the regulatory circuitry underlying VPC determination that includes a role for lin-12 in both autocrine and paracrine VPC signaling.

Transcription of the yeast HO gene requires five genes, SWI1, 2, 3, 4, 5 . We present evidence that some SWI products activate HO by antagonizing negative regulatory activities encoded by the SIN genes. sin − mutants (defining six genes) were identified because they express HO in the absence of particular SWI products. We argue that SWI5 activates HO by antagonizing SIN3 and that SWI4 activates HO by antagonizing SIN6 . HO is expressed in sin3 − daughter cells, hence we infer that the SIN3 product represses HO in wild-type daughter cells and that SWI5 and SIN3 are responsible for the cell-lineage-dependent expression of HO . HO is transcribed only when all types of repression are absent: in mother cells, where SWI5 antagonizes SIN3 ; in late G1, when SWI4 antagonizes SIN6 ; and in a or α cells, where a1-α2 repression is absent.

The identification of a gene necessary for the asymmetry of cell division would be an important first step toward understanding how sister cells come to differ in their developmental fates. The lin-17 gene of the nematode Caenorhabditis elegans is an excellent candidate for being such a gene. lin-17 mutations cause several blast cells that normally generate sister cells of two distinct types to generate instead sister cells of the same type. Moreover, lin-17 mutations cause sister cells to be equal in size as well as equivalent in developmental fate, suggesting that lin-17 acts at or prior to the asymmetric cell division. The lin-17 gene product is involved in asymmetric cell divisions in a variety of tissues, indicating that lin-17 functions in a general mechanism for the establishment of cellular asymmetry in parent cells.

We have described 19 genes that affect neural cell lineages and cell fates during the development of C. elegans. These genes differ markedly in the nature, breadth, and specificity of their effects. Their only obvious common characteristic is that they all lack specificity for the nervous system, affecting both neural and nonneural development. For some of these genes (lin-5, lin-6, unc-59, unc-85), this nonspecificity probably reflects a general utilization of their products in cellular replication. In contrast, most of these genes appear to be highly specific in their effects, but their specificity is not on the basis of cell type but rather on the basis of some particular aspect of development. Specifically, unc-83 and unc-84 mutations affect certain precursor cells that generate both neural and nonneural descendants; lin-22 and lin-26 mutants lead to the generation of supernumerary neural cells with a concomitant loss of nonneural cells; lin-4, lin-14, lin-28, and lin-29 mutants perturb global aspects of developmental timing, altering the time of appearance (or preventing the appearance) of both neural and nonneural cells; lin-1, lin-8, lin-9, and lin-15 mutations affect the cell lineages of certain nonneuron -producing ectoblasts in hermaphrodites and of homologous neuron-producing ectoblasts in males; lin-12 mutations affect many sets of nonidentical homologs (cells of similar lineage history that express different fates), only some of which are neural; ced-3 mutations prevent all programmed cell deaths, again only some of which are neural. Of these 19 genes, only unc-86 is specific for neural as opposed to nonneural cell lineages. However, some unc-86 mutants are abnormal in chromosome segregation at meiosis, indicating that this gene also may affect nonneural aspects of development. One implication of these observations is that genes (and molecules) involved in neural development are likely to function in nonneural development as well. The genes lin-22, lin-12, unc-86, and ced-3 may play decision-making roles during C. elegans neurogenesis, as mutations in each of these genes cause specific transformations in the fates of particular cells. These genes and others like them may act within a hierarchy to effect decisions at different levels within cell lineages. For example, lin-22 animals display transformations affecting entire postembryonic cell lineages, unc-86 animals are altered at an intermediate level of certain cell lineages, and ced-3 animals are affected only in the ultimate fates of cells produced by terminal cell divisions.(ABSTRACT TRUNCATED AT 400 WORDS)

Fertilization, a critical step in animal reproduction, is triggered by a series of specialized sperm-egg interactions. However, the molecular mechanisms underlying fertilization are not well understood. Here, we identify a sperm-enriched C. elegans TRPC homolog, TRP-3. Mutations in trp-3 lead to sterility in both hermaphrodites and males due to a defect in their sperm. trp-3 mutant sperm are motile, but fail to fertilize oocytes after gamete contact. TRP-3 is initially localized in intracellular vesicles, and then translocates to the plasma membrane during sperm activation. This translocation coincides with a marked increase in store-operated calcium entry, providing an in vivo mechanism for the regulation of TRP-3 activity. As C. elegans oocytes lack egg coats, our data suggest that some TRPC family channels might function to mediate calcium influx during sperm-egg plasma membrane interactions leading to fertilization.

We find that C. elegans egl-30 encodes a heterotrimeric G protein a subunit more than 80% identical to mammalian Gqalpha family proteins, and which can function as a Gqalpha subunit in COS-7 cells. We have identified new egl-30 alleles in a selection for genes involved in the C. elegans acetylcholine response. Two egl-30 alleles specify premature termination of Gqalpha and are essentially lethal in homozygotes. Animals homozygous for six other egl-30 alleles are viable and fertile, but exhibit delayed egg laying and leave flattened tracks. Overexpression of the wild-type egl-30 gene produces the opposite behavior. Analysis of these mutants suggest that their phenotypes reflect defects in the muscle or neuromuscular junction.

C. elegans male mating behavior comprises a series of steps: response to contact with the hermaphrodite, backing along her body, turning around her head or tail, location of the vulva, insertion of the two copulatory spicules into the vulva, and sperm transfer. By ablation of male-specific copulatory structures and their associated neurons, we have identified sensory structures and neurons that participate in each of these steps: the sensory rays mediate response to contact and turning; the hook, the postcloacal sensilla, and the spicules mediate vulva location; the spicules also mediate spicule insertion and regulate sperm transfer. Generally, successful completion of each step places the male in a position to receive a cue for the next step in the pathway. However, the high degree of sensory regulation allows the male to execute some steps independently.

Background: Reciprocal signaling between distinct tissues is a general feature of organogenesis. Despite the identification of developmental processes in which coordination requires reciprocal signaling, little is known regarding the underlying molecular details. Here, we use the development of the uterine–vulval connection in the nematode Caenorhabditis elegans as a model system to study reciprocal signaling. Results: In C. elegans , development of the uterine–vulval connection requires the specification of uterine uv1 cells and morphogenesis of 1°-derived vulval cells. LIN-3, an epidermal growth factor (EGF) family protein, is first produced by the gonadal anchor cell to induce vulval precursor cells to generate vulval tissue. We have shown that lin -3 is also expressed in the 1° vulval lineage after vulval induction and that the 1° vulva is necessary to induce the uv1 uterine cell fate. Using genetic and cell biological analyses, we found that the specification of uterine uv1 cells is dependent on EGF signaling from cells of the 1° vulval lineages to a subset of ventral uterine cells of the gonad. RAS and RAF are necessary for this signaling. We also found that EGL-38, a member of the PAX family of proteins, is necessary for transcription of lin -3 in the vulva but not in the anchor cell. A let -23 mutation that confers ligand-independent activity bypasses the requirement for EGL-38 in specification of the uv1 cell fate. Conclusions: We have shown how relatively simple EGF signals can be used reciprocally to specify the uterine–vulval connection during C. elegans development.

SummarySleep is characterized by behavioral quiescence, homeostasis, increased arousal threshold, and rapid reversibility. Understanding how these properties are encoded by a neuronal circuit has been difficult, and no single molecular or neuronal pathway has been shown to be responsible for the regulation of sleep. Taking advantage of the well-mapped neuronal connections of Caenorhabditis elegans and the sleep-like states in this animal, we demonstrate the changed properties of both sensory neurons and downstream interneurons that mediate sleep and arousal. The ASH sensory neuron displays reduced sensitivity to stimuli in the sleep-like state, and the activity of the corresponding interneurons in ASH’s motor circuit becomes asynchronous. Restoration of interneuron synchrony is sufficient for arousal. The multilevel circuit depression revealed provides an elegant strategy to promote a robust decrease in arousal while allowing for rapid reversibility of the sleep state.

SummaryMore than a quarter of the world's population is infected with nematode parasites, and more than a hundred species of nematodes are parasites of humans [1–3]. Despite extensive morbidity and mortality caused by nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood, largely because of the lack of genetically tractable model systems. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of Heterorhabditis, which contain Photorhabdus in their gut, can infect and kill Drosophila larvae. We show that infection activates an immune response in Drosophila that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by Photorhabdus. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion and that is necessary for successful parasitism. We find that the chemosensory neurons and signaling pathways that control dauer recovery in Caenorhabditis elegans also control IJ recovery in Heterorhabditis, suggesting conservation of these developmental processes across free-living and parasitic nematodes.

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1 degree, 2 degrees, 3 degrees), and 2 degrees HCG fate specification is mediated by LIN-12/Notch. We show that 2 degrees HCG specification depends on the presence of a cell with the 1 degree fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1 degree and 2 degrees HCG fate. A requirement for EGF signaling during 1 degree fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1 degree lineage, and LIN-12/NOTCH induces a 2 degrees lineage. Wnt signaling is also required for execution of the 1 degree and 2 degrees HCG lineages. lin-17 and bar-1/beta-catenin are preferentially expressed in the presumptive 1 degree cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.

With support from the Institutes and Centers forming the NIH Blueprint for Neuroscience Research, we have designed and implemented a new initiative for integrating access to and use of Web-based neuroscience resources: the Neuroscience Information Framework. The Framework arises from the expressed need of the neuroscience community for neuroinformatic tools and resources to aid scientific inquiry, builds upon prior development of neuroinformatics by the Human Brain Project and others, and directly derives from the Society for Neuroscience’s Neuroscience Database Gateway. Partnered with the Society, its Neuroinformatics Committee, and volunteer consultant- collaborators, our multi-site consortium has developed: (1) a comprehensive, dynamic, inventory of Web-accessible neuroscience resources, (2) an extended and integrated terminology describing resources and contents, and (3) a framework accepting and aiding concept-based queries. Evolving instantiations of the Framework may be viewed at http://nif.nih.gov, http://neurogateway.org, and other sites as they come on line.

Small groups of multipotent cells share the same set of potential developmental fates, with the fate of each cell specified by cell-cell interactions. Genetic studies in the nematode have identified two pathways that distinguish between two alternative cell fates in such ‘equivalence groups'. In a group whose members choose between two fates, only a single pathway operates. In groups whose members choose among three fates, both pathways operate, suggesting that complex decisions might result from the combination of binary decisions.

During Caenorhabditis elegans vulval induction the anchor cell of the gonad specifies a spatial pattern of three cell types among a set of six multipotent epidermal cells, the vulval precursor cells (VPCs). Previous studies suggested that the anchor cell produces a graded inductive signal which can directly stimulate VPCs away from a ground state (type 3) to become type 1 or type 2 depending on their distance from the anchor cell. Here, we investigate the interactions among VPCs in a mutant, lin-15, in which VPC fates are rendered partially independent of the inductive signal, and show that type 1 cells actively inhibit adjacent cells from also becoming type 1 cells. The fate of each VPC therefore depends on the combined action of two intercellular signals: a graded inductive signal from the anchor cell, and a lateral inhibitory signal from at least some of its neighbours. Pattern formation among the VPCs lin-15 mutant is analogous to the establishment of the pattern of neuroblasts and dermatoblasts during early insect neurogenesis, suggesting that the similarities in inferred molecular structure of the lin-12 and Notch gene products, which are involved in these two instances of pattern formation, might extend to similarities in function.

The postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus are described and compared with those of Caenorhabditis elegans . The newly hatched larvae of P. redivivus females and males and C. elegans hermaphrodites and males are very similar. An almost identical set of blast cells divides postembryonically in P. redivivus and C. elegans to produce similar changes in the neuronal, muscular, hypodermal, and digestive systems. Most of these cell lineages are invariant; however, there is substantial variability in the number of cell divisions in the relatively extensive lineages of the lateral hypodermis of P. redivivus . Typically, in P. redivivus females, 55 blast cells generate 635 surviving progeny and 29 cell deaths; in P. redivivus males, 59 blast cells generate 758 surviving progeny and 35 cell deaths. The lineages generating the cells of the male tails of P. redivivus and C. elegans are almost identical; thus, the grossly different characteristics of these structures must reflect differences in the morphogenesis of cells equivalent in lineage history. Laser ablation experiments demonstrate that the gonad induces vulva development and that cell-cell interactions are important in specifying the fates of hypodermal precursor cells. The lateral hypodermal lineages provide striking examples of the apparent construction of complex lineages from modular sublineages; one simple pattern of cell divisions and cell fates occurs 70 times in the P. redivivus female. The differences in cell lineage between P. redivivus and C. elegans are relatively minor, and many appear to have involved two types of evolutionary change: the replacement of sublineages, and the modification of sublineages by the four classes of lineage transformations previously proposed based on a comparison of P. redivivus and C. elegans gonadal cell lineages (Sternberg and Horvitz, 1981). These types of differences suggest that the genetic programming of cell lineage includes instructions specifying where and when a particular sublineage is utilized, and other instructions specifying the nature of that sublineage.

Background Protein kinases regulate a plethora of essential signalling and other biological pathways in all eukaryotic organisms, but very little is known about them in most parasitic nematodes. Methods Here, we defined, for the first time, the entire complement of protein kinases (kinome) encoded in the barber’s pole worm (Haemonchus contortus) through an integrated analysis of transcriptomic and genomic datasets using an advanced bioinformatic workflow. Results We identified, curated and classified 432 kinases representing ten groups, 103 distinct families and 98 subfamilies. A comparison of the kinomes of H. contortus and Caenorhabditis elegans (a related, free-living nematode) revealed considerable variation in the numbers of casein kinases, tyrosine kinases and Ca2+/calmodulin-dependent protein kinases, which likely relate to differences in biology, habitat and life cycle between these worms. Moreover, a suite of kinase genes was selectively transcribed in particular developmental stages of H. contortus, indicating central roles in developmental and reproductive processes. In addition, using a ranking system, drug targets (n = 13) and associated small-molecule effectors (n = 1517) were inferred. Conclusions The H. contortus kinome will provide a useful resource for fundamental investigations of kinases and signalling pathways in this nematode, and should assist future anthelmintic discovery efforts; this is particularly important, given current drug resistance problems in parasitic nematodes. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1231-5) contains supplementary material, which is available to authorized users.

Background In the present study, we reconstructed the insulin/insulin-like growth factor 1 signalling (IIS) pathway for Haemonchus contortus, which is one of the most important eukaryotic pathogens of livestock worldwide and is related to the free-living nematode Caenorhabditis elegans. Methods We curated full-length open-reading frames from assembled transcripts, defined the complement of genes that encode proteins involved in this pathway and then investigated the transcription profiles of these genes for all key developmental stages of H. contortus. Results The core components of the IIS pathway are similar to their respective homologs in C. elegans. However, there is considerable variation in the numbers of isoforms between H. contortus and C. elegans and an absence of AKT-2 and DDL-2 homologs from H. contortus. Interestingly, DAF-16 has a single isoform in H. contortus compared with 12 in C. elegans, suggesting novel functional roles in the parasitic nematode. Some IIS proteins, such as DAF-18 and SGK-1, vary in their functional domains, indicating distinct roles from their homologs in C. elegans. Conclusions This study paves the way for the further characterization of key signalling pathways in other socioeconomically important parasites and should help understand the complex mechanisms involved in developmental processes. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1341-8) contains supplementary material, which is available to authorized users.

The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.

Background Proteins of the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 (CAP) superfamily are recognized or proposed to play roles in parasite development and reproduction, and in modulating host immune attack and infection processes. However, little is known about these proteins for most parasites. Results In the present study, we explored CAP proteins of Toxocara canis, a socioeconomically important zoonotic roundworm. To do this, we mined and curated transcriptomic and genomic data, predicted and curated full-length protein sequences (n = 28), conducted analyses of these data and studied the transcription of respective genes in different developmental stages of T. canis. In addition, based on information available for Caenorhabditis elegans, we inferred that selected genes (including lon-1, vap-1, vap-2, scl-1, scl-8 and scl-11 orthologs) of T. canis and their interaction partners likely play central roles in this parasite’s development and/or reproduction via TGF-beta and/or insulin-like signaling pathways, or via host interactions. Conclusion In conclusion, this study could provide a foundation to guide future studies of CAP proteins of T. canis and related parasites, and might assist in finding new interventions against diseases caused by these parasites. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1642-y) contains supplementary material, which is available to authorized users.