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The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO—a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations—evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)—mechanistic models of molecular “pathways” (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

The human gene FAM136A (family with sequence similarity 136 member A) has been associated with familial Meniere’s disease (MD) (Requena et al., 2015), a chronic disorder of the inner ear (Nakashima et al., 2016). There is also a suggestion that the expression of the gene could be associated with lung cancer prognosis (Zhao et al., 2020). FAM136A is evolutionary conserved, with orthologs identified in various metazoan species (Alliance of Genome Resources, 2022). However, with the exception of some expression data and omics level analysis (Alliance of Genome Resources, 2022; Stuckenholz et al., 2009), very little is known about FAM136A.

Steinernema hermaphroditum entomopathogenic nematodes (EPN) and their Xenorhabdus griffiniae symbiotic bacteria have recently been shown to be a genetically tractable system for the study of both parasitic and mutualistic symbiosis. In their infective juvenile (IJ) stage, EPNs search for insect hosts to invade and quickly kill them with the help of the symbiotic bacteria they contain. The mechanisms behind these behaviors have not been well characterized, including how the nematodes sense their insect hosts. In the well-studied free‑living soil nematode Caenorhabditis elegans, ciliated amphid neurons enable the worms to sense their environment, including chemosensation. Some of these neurons have also been shown to control the decision to develop as a stress-resistant dauer larva, analogous to the infective juveniles of EPNs, or to exit from dauer and resume larval development. In C. elegans and other nematodes, dye-filling with DiI is an easy and efficient method to label these neurons. We developed a protocol for DiI staining of S. hermaphroditum sensory neurons. Using this method, we could identify neurons positionally analogous to the C. elegans amphid neurons ASI, ADL, ASK, ASJ, as well as inner labial neurons IL1 and IL2. Similar to findings in other EPNs, we also found that the IJs of S. hermaphroditum are dye-filling resistant.

The Alliance of Genome Resources (the Alliance) is a combined effort of 7 knowledgebase projects: Saccharomyces Genome Database, WormBase, FlyBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database, and the Gene Ontology Resource. The Alliance seeks to provide several benefits: better service to the various communities served by these projects; a harmonized view of data for all biomedical researchers, bioinformaticians, clinicians, and students; and a more sustainable infrastructure. The Alliance has harmonized cross-organism data to provide useful comparative views of gene function, gene expression, and human disease relevance. The basis of the comparative views is shared calls of orthology relationships and the use of common ontologies. The key types of data are alleles and variants, gene function based on gene ontology annotations, phenotypes, association to human disease, gene expression, protein–protein and genetic interactions, and participation in pathways. The information is presented on uniform gene pages that allow facile summarization of information about each gene in each of the 7 organisms covered (budding yeast, roundworm Caenorhabditis elegans, fruit fly, house mouse, zebrafish, brown rat, and human). The harmonized knowledge is freely available on the alliancegenome.org portal, as downloadable files, and by APIs. We expect other existing and emerging knowledge bases to join in the effort to provide the union of useful data and features that each knowledge base currently provides.

The efficient CRISPR/Cas9 genome editing toolkit in the nematode Caenorhabditis elegans has made the creation of null mutants easier than ever. The putative lipid hydrolase lips-6 is known to function in the DAF-12/DIN-1 pathway to promote fat mobilization during periods of starvation in adult worms (Tao et al. 2016). Similarly, it was found to be strongly downregulated in response to dauer ascarosides given during the L1 to L2d decision in larval worms (Cohen et al. 2021). Here, we have created a mutant of the putative lipase lips-6 and studied its lifespan as well as its ascaroside profile.

C. elegans larvae choose between dauer diapause and immediate reproductive development in two phases, the first being a choice between L2 and L2d, and the second between dauer and L3 (Golden and Riddle 1984). We used RNA-seq to study the transcriptomic response of well-fed C. elegans when exposed to a cocktail of diapause-inducing ascarosides (ascr#2, ascr#3, and ascr#8) during the L1 larval stage when they first receive cues from their environment and then adapt via developmental programming (Kaplan et al. 2011). This research was performed in N2 wild-type worms and daf-22(ok693) mutants that lack an enzyme necessary for the biosynthesis of ascaroside pheromones (reviewed by Ludewig and Schroeder 2013). We found that early exposure to ascarosides triggers a pervasive transcriptional response that may drive diapause induction within as little as three hours. In N2, 362 genes were significantly differentially expressed at p = 0.001, while in daf-22, 1,007 genes were significantly differentially expressed at p = 0.001. The daf-22 mutant may have more significantly expressed genes when exposed to ascarosides indicating a stronger response as it has never encountered ascaroside signaling previously, even at low levels.

Many invertebrates are capable of jumping, such as locusts, fleas, mantids, and cockroaches. Some invertebrates can jump without the use of their legs, such as click beetles and springtails (Mo et al., 2020). Nematodes, which are non-segmented roundworms, are capable of a variety of complex behaviors, though jumping is arguably the most visually striking. Jumping is a rare behavior among nematodes, with only a few species in the genus Steinernema being reported as capable of jumping (Campbell and Kaya, 1999). The kinematic performance of S. carpocapsae jumping was first described in 1999, with the peak velocity and peak acceleration reported as 1.13 m/s and 164 g, respectively (Campbell and Kaya, 1999). These data were not calculated based on direct observation and high-speed videography, but were estimated based on the distance, height, and timing of the nematode jumps. This nematode is reported to be capable of jumping a distance of 4.8 mm, and a height of 3.9 mm (Campbell and Kaya, 1999).

Accumulating evidence demonstrates that mutations in ALDH1A3 (the aldehyde dehydrogenase 1 family, member A3) are associated with developmental defects. The ALDH1A3 enzyme catalyzes retinoic acid biosynthesis and is essential to patterning and neuronal differentiation in the development of embryonic nervous system. Several missense mutations in ALDH1A3 have been identified in family studies of autosomal recessive microphthalmia, autism spectrum disorder, and other neurological disorders. However, there has been no evidence from animal models that verify the functional consequence of missense mutations in ALDH1A3. Here, we introduced the equivalent of the ALDH1A3 C174Y variant into the Caenorhabditis elegans ortholog, alh-1, at the corresponding locus. Mutant animals with this missense mutation exhibited decreased fecundity by 50% compared to wild-type animals, indicating disrupted protein function. To our knowledge, this is the first ALDH1A3 C174Y missense model, which might be used to elucidate the effects of ALDH1A3 C174Y missense mutation in the retinoic acid signaling pathway during development.

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. egl-20/Wnt is expressed in the tail (Whangbo and Kenyon, 2000) and forms a posterior-to-anterior concentration gradient (Coudreuse et al., 2006). It has previously been shown that EGL-20 acts instructively during vulva development by imparting directional information, as opposed to being permissive, where it would only be required for polarization (Green et al., 2008; Minor et al., 2013). By moving the source of egl-20 expression from the posterior of the worm to the anchor cell, the axis of symmetry of the developing vulva, we can reorient the daughter cells of P5.p and P7.p toward the center in a wild-type configuration. Expression of egl-20 from the center of the axis of symmetry partially suppresses the lin-17(n671) phenotype (Green et al., 2008; Table 1). To test whether LRP-2 acts downstream of EGL-20, we ectopically expressed egl-20 from the anchor cell in a lin-17(n671); lrp-2(gk272) double mutant background and compared it to a lin-17(n671); lrp-2(+) strain. If LRP-2 acts downstream of EGL-20, then anteriorly-expressed EGL-20 will not be able to suppress the lin-17 phenotype, with is the result observed (Table 1). Thus, like CAM-1 and VANG-1, LRP-2 likely acts downstream of EGL-20.

Recently, the GAL4-UAS system (cGAL) has been adapted for use in C. elegans for control of gene expression across 15°C - 25°C (Wang et al., 2017). In order to create a desired gene expression pattern, one crosses a transgenic strain containing a driver construct with another strain containing an effector gene. Here we mapped several cGAL driver and effector integrations. We first crossed each of the cGAL driver and effector strains with N2 males, picked the heterozygous male progeny, crossed them with hermaphrodites of the mapping strain (DA438), picked L4 hermaphrodites with the corresponding transgenic marker of the driver or effector strain and scored the progeny in the next generation. The DA438 strain contains six recessive mutations, each of which locates on one of the six chromosomes and produces visible phenotypes (Bli on chromosome I, Rol on II, Vab on III, Unc on IV, Dpy on V, and Lon on X (Avery, 1993). F2 progeny with each of the six phenotypes were selected and examined for the presence or absence of the dominant marker associated with the transgene. In the cases where the dominant transgene marker is unlinked to the recessive phenotypic marker, about three quarters of the F2 progeny will have the dominant marker. If the two markers are linked, very few or no animals are expected to have the dominant transgenic marker. The following tables summarize the mapping results for each cGAL strain, stating the ratios of the F2 mutant progeny with and without the dominant transgenic marker.

cGAL, a recently developed temperature-robust bipartite GAL4-UAS system in C. elegans, consists of two components: a cGAL “driver” that expresses the cGAL protein in specific cells using a promoter (i.e. neuron-specific or tissue-specific), and an “effector” that carries a gene of interest downstream of UAS (Wang et al., 2017). Crossing or combining a driver with an effector leads to the expression of the gene of interest in a cell-specific or tissue-specific manner. Here we report a new cGAL driver for the DVC interneuron. The ceh-63 promoter was chosen due to its restricted expression in the DVC neuron (Feng et al. 2012). The DVC interneuron driver construct containing the ceh-63 promoter (646 bp upstream of ATG translation start site) was injected into N2 and an integrated DVC driver line was generated. When crossed with the UAS-GFP effector strain (PS6843), the ceh-63 cGAL driver dictated GFP expression in the single DVC neuron (Figure 1), in addition to GFP in the coelomocyte from Punc-122::gfp co-injection marker. We did not observe GFP expression in uterus as reported by Feng et al., 2012.

WormBase curates four different types of gene-to-gene interaction data: genetic, regulatory, physical, and predicted. These data are found in the Interactions widget in each gene page. Aside from the predicted interactions, the other three types are curated with direct experimental evidence from the literature. In WormBase, genetic interaction data is defined as a phenotypic deviation of double mutants (or any other genetic perturbations) from single mutant phenotypes and the control phenotype. Regulatory interactions are defined by how perturbation of one gene or gene product affects the expression of another gene or the localization of its gene product. Physical interactions represent molecular associations between genes and gene products from C. elegans (Grove et al. 2018). Each type of interaction data is essential to understanding certain aspects of the biological process mediated by the two interacting genes. However, integrating information from these three types of interaction data is critical to reading the biological context. Understanding the logical relations between different types of interaction data provides a vital clue on how to tackle a gene-to-gene interaction within this context. To achieve this goal, we introduce a new tool for analyzing these logical relations among the interaction data using a Venn diagram, named Vennter (Venn diagram for interaction). Venn diagrams are very useful for displaying similarities and distinctions between different data sets of interest, especially when they are area-proportional to the amount of information presented. Vennter consists of three circles, each representing one of the three different interaction data types. The size of any area within the interactive Venn diagram corresponds to the number of unique interactor genes pertaining to that region. Figure 1A shows an example of a Vennter diagram for selected genes (daf-2, daf-15, daf-16, and let-363) which play key roles in the Insulin/IGF-1 and TOR-dependent signaling pathway. These results exhibit distinct patterns of overlap among their interaction data. In some cases, the differences might reflect differences in prior scientific approaches to studying these genes as well as the availability of annotated interaction data in WormBase. Vennter is fully interactive for analyzing interactor genes from different sets of interaction data. By clicking on any single or multiple area in combination, one can easily obtain all gene names corresponding to the selected area (represented by hashed lines) in Vennter. The number of selected genes is shown next to the ‘View selected genes’ button (see arrows). From there, one can open a popup window called ‘Browse gene set’ which contains a comprehensive list of interactor genes shown in alphabetic order. This list can be copied or downloaded in diverse formats, and for the user’s convenience, each gene name is linked to its unique WormBase gene page. Under the same ‘Browse gene set’ window, Vennter also offers other functions for further analysis of selected genes, such as direct links to the batch analysis tools ‘SimpleMine’ and ‘Gene-set Enrichment Analysis’ (Figure 1B) which can help to query the gene list more conveniently and efficiently. To demonstrate yet another aspect of Vennter, we can look at the high-throughput physical protein-protein interaction data, which comprise long lists of genes. To date, WormBase has curated 86.9% of physical protein-protein interaction data from these high-throughput studies (Cho et al. 2018). However, identifying the functional relevance of such large amounts of interacting gene candidates can often be quite challenging to researchers. To improve this, Vennter provides more organized and relevant information to researchers by evaluating overlapping information between physical, genetic, and/or regulatory interactions, which enables researchers to more easily measure the confidence and biological relevance of their gene candidates of interest. For the C. elegans research community, Vennter can help compare and integrate the different types of interaction data available. It also provides hyperlinks to other useful WormBase tools in order to analyze the gene interactor list in a more efficient and automated manner.

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. The urgent need of the field is precise understanding of physiological mechanisms driving EV generation and function. Ciliary EVs act as signaling devices in Chlamydomonas and C. elegans [2–4]. Mammalian cilia shed EVs to eliminate unwanted receptors [5] or in the process of cilia retraction when cultured cells enter the cell cycle [6]. Here we used our established C. elegans model to study sensory-evoked ciliary EV release and targeting using a fluorescently labeled EV cargo polycystin-2 (PKD-2). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. We find that males deposit PKD-2::GFP-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2::GFP-carrying EVs from cilia. To our knowledge this is the first report of mechanoresponsive nature of the ciliary EV release and of ciliary EV directional transfer from one animal to another animal. Since the polycystins are evolutionary conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other animals.

One barrier to communication at large face-to-face meetings, especially those focusing on scientific or technical topics, is a rapid way to understand what a person knows and thinks about before initiating a conversation. We have devised and reduced to practice an effective way to accomplish this task, the wearable microPoster. In our experience, we observed that at most scientific meetings, especially once the number of participants passed 100, it is difficult to get productive conversations started with strangers. Thus, conversations tend to initiate among people that already know one another. This tends to exclude newer participants and those who do not have formal speaking slots. The difficulty increases at informal aspects of meetings: coffee breaks, lines for meals, social events, outdoor events in which a prominent speaker might have different clothes and sunglasses and is thus rendered unknown. Poster sessions have long provided a venue for rapid communication and finding participants. These are usually the most productive parts of meetings. Our thinking evolved from jokes about sandwich board posters, in which one walks around with large posters on the front and back, to shirts with printed information. The advent of printed cloth posters provides a practical solution in which a microPoster is cut from the larger poster (ideally cloth), or printed on one or more sheets of standard paper, which are then taped together. A microPoster comprises your name, affiliation (with appropriate granularity determined by the meeting), and a graphical abstract. The goal is to identify yourself in a much more informative way than a nametag. One convenient way to present the microPoster is pinned on your back like a bib number (e.g., as in marathons or dances). We carried out a pilot with ten microPosters at a meeting of 1500 people (Figure 1), and found them to be effective at attracting useful conversation. The addition of a machine readable QR code (e.g., Salt, 2019) is a possible useful addition but we note that the expense in time, money and carbon footprint of a face-to-face meeting is better spent on face-to-face conversations.

The GAL4-UAS system is a powerful tool for manipulating gene expression, but its application in Caenorhabditis elegans has not been described. Here we systematically optimize the system's three main components to develop a temperature-optimized GAL4-UAS system (cGAL) that robustly controls gene expression in C. elegans from 15 to 25 °C. We demonstrate this system's utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression; and we provide a basic driver and effector toolkit.

Genomic editing of the roundworm Caenorhabditis elegans using the CRISPR/Cas9 system has allowed for widespread creation of null mutants – vital for scientific understanding of this model organism. A closely-related nematode species, Caenorhabditis briggsae, is emerging as an alternative model organism to better understand how findings in C. elegans can broaden and develop the larger field of nematology (Gupta et al. 2007). To that end, we have developed an effective and efficient co-conversion CRISPR/Cas9 system for use in C. briggsae using the gene dpy-10. We modified the universal STOP-IN cassette method, as described by Wang et al. (2018), for use in C. briggsae (Wang et al. 2018). Using the wildtype AF16 strain, we tested the method by choosing the gene Cbr-dpy-10 because of its readily observed predicted phenotype. Cbr-dpy-10 is a predicted one-to-one ortholog of C. elegans dpy-10, which encodes a protein important for cuticle development and has a phenotype characterized by short, fat animals relative to wild type (Brenner 1974). A universal STOP-IN allele of Cbr-dpy-10, sy1387, was generated and confirmed by genotype sequencing. The expected phenotype was subsequently observed, consistent with the creation of a null allele (Figure 1). Surprisingly, sy1387 is dominant. Injections of 20 animals produced 15 successful injections; 247 animals from these 15 injections (F1) were singled out and allowed to self-fertilize to produce F2. 81% of the F1 Dumpy progeny were homozygous as evidenced by their segregation of only Dumpy progeny – one of these candidates became sy1387. 2% of the F1s did not produce progeny. 17% of the 247 F1 Dumpy progeny were heterozygotes and had a mixture of Dumpy and non-Dumpy progeny; these non-Dumpy F2s only produced non-Dumpy progeny. This discovery of a dominant mutation will allow for more effective use of this as a co-conversion marker when screening for other mutations. We used Cbr-dpy-10 as a potential co-CRISPR marker for a second target that will be described elsewhere. We used PCR to detect insertion of a STOP-IN cassette at this other locus; we screened 39 Dumpy strains to obtain 11 candidates for our target gene, from which we have three STOP-IN alleles.

We investigated the role of the low-density lipoprotein receptor lrp-2 in C. elegans vulval cell lineage polarity. We find that despite the high conservation of the Wnt signaling component, LRP5/6, in higher order organisms it appears to have evolved after the split of Nematoda due to its absence in all nematode genomes examined. C. elegans contains multiple low-density lipoprotein receptors within its genome, two of which are lrp-1 and lrp-2. Due to the position in the genome and high sequence similarity we believe that lrp-2 is the product of a recent duplication of lrp-1 within the Caenorhabditis lineage (Minor and Sternberg, 2019d). To understand the potential role of lrp-1 and lrp-2, we sought to score the vulval phenotypes of these mutants during L4 stage. lrp-1(ku156) mutants are sick and arrest during an early larval stage. For this reason we were not able to examine the role of lrp-1 in vulval formation. Despite high sequence similarity and proposed functional redundancy with lrp-1, lrp-2 mutants are remarkably wild-type. lrp-2 is expressed in the developing vulva at the same time as both cam-1 and vang-1. By examining double and triple mutant strains we find that lrp-2 is likely to function downstream of egl-20 along with transmembrane proteins cam-1 and vang-1 (Minor and Sternberg, 2019b; Minor and Sternberg, 2019c). All three genes antagonize the lin-17/Frizzled pathway by directing the aberrant localization of SYS-1 to the posterior daughter cell of P7.p, thereby leading to the posterior orientation of the P7.p lineage (Minor and Sternberg, 2019a). These observations suggest the simple model shown in Figure 1, in which LRP-2 cooperates with CAM-1 and VANG-1 to respond to EGL-20. This work provides evidence that despite lacking a true LRP5/6 ortholog, the formation of the C. elegans vulva is controlled by another member of the low-density lipoprotein superfamily, lrp-2. This data could potentially lead to insight into the evolution of both structure and function of the highly important Wnt pathway component, LRP5/6. Despite strong genetic evidence, this work does not describe the physical interaction between LRP-2 and CAM-1, VANG-1, EGL-20. Can LRP-2 bind with the other transmembrane proteins, CAM-1 and VANG-1, involved in this pathway? Can LRP-2 physically bind the Wnt ligand, EGL-20? Future work should focus on the biochemistry of this pathway. Answers to these questions could provide interesting insights into the evolution of low-density lipoprotein receptors, including LRP5/6, as well as how Wnt signaling has evolved within nematodes without the presence of one of the most important and highly conserved transmembrane proteins.

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. Specifically, in the absence of all Wnts, the VPCs display a randomized orientation, which is likely the default (Green et al., 2008; Minor et al. 2013). Two separate Wnts from the anchor cell, LIN-44 and MOM-2 acting through receptors LIN-17/Frizzled and LIN-18/Ryk, respectively, regulate P7.p orientation (Ferguson et al., 1987; Sternberg and Horvitz, 1988; Sawa et al., 1996; Inoue et al., 2004; Gleason et al., 2006). In the absence of these signals the orientation of the progeny of P7.p mimic those of P5.p and face toward the posterior of the worm, a phenotype referred to as posterior-reversed vulval lineage (P-Rvl). This posterior orientation is dependent on the instructive signal EGL-20, a Wnt expressed in the tail acting through CAM-1/ROR and VANG-1/Van Gogh, and is referred to as “ground polarity” (Green et al., 2008). Here we examine the role of a low-density lipoprotein receptor, lrp-2, and its role in controlling the orientation of P7.p daughter cells. To investigate this interaction double mutants were constructed with both alleles of lrp-2 and lin-17(n671) (Table 1). Much like cam-1(gm122) and vang-1(ok1142), both alleles of lrp-2 suppress the lin-17(n671) phenotype from 74 to approximately 50% P-Rvl leading us to hypothesize that lrp-2 functions in the same pathway as cam-1 and vang-1. Furthering this hypothesis we have shown that, like cam-1 and vang-1, lrp-2 controls the localization of SYS-1/b-catenin (Minor and Sternberg, 2019). To ensure that this phenotype was a result of loss of lrp-2 function as opposed to background effects we injected a fosmid (WRM0617cA02) containing the full-length sequence of lrp-2 and found that it does rescue the double mutant phenotype of lin-17(n671); lrp-2(gk272) from 55 to 73%. In order to better test this hypothesis a triple mutant was constructed between lin-17(n671), lrp-2(gk272), and cam-1(gm122) (Table 1). This triple mutant displays the same P-Rvl penetrance as both the lin-17(n671); lrp-2(gk272) and lin-17(n671); cam-1(gm122) double mutants confirming that lrp-2 functions in the same pathway as cam-1.

The relatively simple nervous system and facile genetics of Caenorhabditis elegans makes it a potential model organism to study the genetic basis of complex neural diseases, such as autism spectrum disorder (ASD). Our previously published study identified conserved human ASD-missense variants in C. elegans orthologs that have a role on morphology, locomotion or fecundity, suggesting C. elegans as an efficient phenotypic model to study conserved ASD variants (Wong et al., 2019). One of the ASD-associated genes screened in this study is daf-18, an ortholog of PTEN. DAF-18/PTEN is a lipid phosphatase protein that dephosphorylates PIP3, a critical lipid second messenger that mediates downstream kinase cascade signaling of the insulin pathway, that in turn regulates expression of genes involved in C. elegans lifespan and dauer formation (Murphy and Hu, 2013). It is reported that the daf-18(e1375) strain, which has a 30–base pairs insertion in the fourth exon, has a dauer defective phenotype (Ogg and Ruvkun, 1998); however, the effect of daf-18/PTEN single amino acid substitution in dauer formation has not been evaluated before. Here, we investigate the dauer entry abilities of previously published ASD-associated daf-18/PTEN missense variants (Wong et al., 2019). We performed a dauer-entry assay using crude pheromone as a proxy for high population density and heat-killed bacteria as a limited food source. We first determined the crude pheromone concentration required to induce 50% of wild type (N2) strain to enter dauer stage (EC50). Using the calculated EC50value we evaluated each mutant strain in two independent experiments (Table 1). To control day-to-day variations caused by environmental conditions, N2 controls were used in every trail. Additionally, we used the previously reported daf-18 dauer-defective strain (e1375) as a control (Ogg and Ruvkun, 1998). To analyze the obtained data, we performed one-way ANOVA followed by multiple comparisons test (Dunnett test) (Figure 1A). Table 1 Evaluated ASD-associated daf-18/PTEN mutant strains. Strain name PS7439 PS7432 PS7436 PS7430 PS7434 C. elegans allele name daf-18(sy879) daf-18(sy887) daf-18(sy881) daf-18(sy885) daf-18(sy882) C. elegans protein change D66E L115V H138R H168Q T176I Human protein change D22E L70V H93R H123Q T131I As expected, we observed statistically significant difference between our wild type and dauer defective control (e1375). Our results show that H138R (PS7436) and T176I (PS7434) daf-18 mutant strains have a significant difference from the wild type control, and a non-significant difference from the dauer defective control, suggesting these missense alleles cause dauer defective phenotypes to the same level of the e1375 strain. In contrast, we found the D66E (PS7439), L115V (PS7432) and H168Q (PS7430) mutant strains are significantly different from the dauer defective control and not significantly different from the wild type control, suggesting these three missense alleles do not cause the same level of dauer defective phenotype as the defective control does. Altogether, our current data show that H138R and T176I are defective in dauer entry, while D66E, L115V and H168Q daf-18 mutant strains are not (Figure 1A). The Conserved Domain Database (Conserved Domain Search) of NCBI (ID: G5EE01) predicts that four of the five missense variations evaluated in this study are located in the phosphatase domain of DAF-18/PTEN(Figure 1B). It is predicted that amino acid Histidine 138 is located in the active site of the protein, suggesting that H138R (PS7436) amino acid change might have an impact on PIP3 substrate specificity, which could explain the dauer defect phenotype observed. Moreover, it is predicted that both amino acid Histidine 168 and Threonine 176 are located close to the catalytic site of the phosphatase domain. We found a dauer defective phenotype resulting from a T176I substitution (PS7434) but not from a H168Q amino acid substitution (PS7430). These data suggest that certain amino acids in the catalytic and active sites of DAF-18/PTEN decrease the function of the protein, in turn regulating C. elegans dauer development. Future evaluation of PIP3 phosphorylation levels could confirm the effect of these variations on the phosphatase activity. The results of this study contribute to the knowledge of the mechanisms controlling C. elegans dauer entry, and to the C. elegans phenotype-screening of conserved ASD variations.

Protein interaction is an important data type to understand the biological function of proteins involved in the interaction, and helps researchers to deduce the biological nature of unknown proteins from the well-characterized functions of their interaction partners. High-throughput studies, coupled with the aggregation of individual experiments, provides a global 'snapshot' of the protein interactions occurring at all levels of biological processes or circumstances. This snapshot of the interaction network, the interactome, is important to understand the overall events up to the level of comparison between species or pathway simulation, or to find new factors yet undefined in the processes, or to add details to the biological processes and pathways. As of September 2018, WormBase (www.wormbase.org) (Lee et al. 2018) contains 28,279 physical protein-protein interactions for the roundworm Caenorhabditis elegans. Among these, 1500 protein-protein interactions have been curated by BioGRID as a collaboration with WormBase. Within the data set, 17,990 protein-protein interactions are unique, and 6,079 unique genes are involved in these interactions. In order to visualize the overall interaction map, a network diagram for all the unique interactions was generated by using the ‘Cytoscape’ program, version 3.6.1 (Shannon et al. 2003) (Figure 1A). These numbers represent a 108% increase in the number of interaction annotations since last year, 2017. These interaction data were curated from 1,251 peer-reviewed papers, which were selected from the literature by ‘Textpresso Central’ using automatic SVM (Support Vector Machine)-based text mining approaches (Fang et al. 2012; Müller et al. 2018) and manual verification. Compared to other databases providing C. elegans protein-protein interaction, WormBase now presents the largest data set, which has 1.72-fold more interaction annotations than IMEx (Orchard et al. 2012) and 4.51-fold more than BioGRID (Chatr-Aryamontri et al. 2017) (Figure 1B). Most significantly, WormBase now houses the complete protein interaction data from almost all of the C. elegans literature published from 1993 to 2018. The data sets presented at IMEx and BioGRID are annotated from 253 and 174 papers, respectively. All the physical interaction data in WormBase are supported by experimental evidence from original research papers. The statistics of the detection methods used as experimental evidence are shown in Figure 1C. The majority of the interaction data came from high throughput analysis such as large-scale yeast two-hybrid assays or mass-spectrometry, however, a significant portion of the data (13.1%) are supported by more direct detection methods using small-scale, low throughput methods such as co-immunoprecipitation or co-crystallography (Figure 1C). In WormBase, protein-protein interaction data can be found as a subclass of physical interaction data in the ‘Interactions widget’ on the gene report page. The Interactions widget provides all types of interaction data related to the gene of interest, such as physical, genetic, regulatory, and predicted interactions. All the interaction data are represented together in a graph created with ‘Cytoscape.js’ and a table. In the table, the gene names of interaction partners (bait-target) in the interaction are displayed along with the publication. The interaction details including the detection method are also captured in the summary and the remark field in the Interactions page. Users can query the data by using the search bar on the WormBase front page or download all the available data files from the WormBase FTP site (ftp://ftp.wormbase.org/pub/wormbase/releases/current-production-release/species/c_elegans/PRJNA13758 /annotation/c_elegans.PRJNA13758.WSXXX.interactions.txt.gz, where WSXXX is the database version release, like “WS267”). All the interaction data in WormBase will be available soon at the new information resource for multiple model organisms, the Alliance of Genome Resources (https://www.alliancegenome.org/). This site will integrate all the interaction data from human and from model organisms C. elegans, budding yeast (Saccharomyces cerevisiae), fruit fly (Drosophila melanogaster), zebrafish (Danio rerio), mouse (Mus musculus) and rat (Rattus norvegicus). Integrated views of interaction data from diverse model organisms will be extremely helpful to build interaction databases for species-to-species comparison, and to establish a disease model quickly based on the database. For the most efficient analysis of the interaction data in WormBase, we are now working on developing a new ‘Venn diagram tool’ and integrating the ‘Gene Set Enrichment Analysis tool’ (https://wormbase.org/tools/enrichment/tea/tea.cgi) into the Interactions widget. We will continue to curate other types of macro-molecular interactions including protein-DNA, protein-RNA and RNA-RNA interactions, as well as newly reported protein-protein interaction data to serve our research community.

Channelrhodopsins are light-gated ion channels that serve as photoreceptors in photosynthetic microbes and have been applied as crucial optogenetic tools in genetic model organisms. When expressed in animals, they enable light-inducible control of ionic membrane permeability, which directly manipulates the activity of neurons expressing the protein. The application of channelrhodopsin-based optogenetics is particularly powerful when used in conjugation with the cGAL (GAL4-UAS) bipartite system (Wang, 2017). The mating of neuron-specific GAL4 driver lines to new channelrhodopsin effector lines could expand the genetic toolkit to perturb and manipulate neural circuits in the organism. Blue light-gated channelrhodopsins have been widely used in C. elegans neurobiology but often have to be performed in lite-1(ce314) mutant backgrounds because short-wavelength blue light is an aversive cue in wild-type animals and directly affects C. elegans neuronal physiology. Previously, a red light-gated variant of channelrhodopsin, termed Chrimson, has been successfully applied in Drosophila and mice, and has recently been codon-optimized for use in C. elegans (Klapoetke, 2014; Schild, 2015). Here, we constructed a Chrimson (15xUAS::chrimson::gfp) cGAL effector line. We introduced the UAS::chrimson::gfp effector DNA construct as an extrachromosomal array into a previously published cGAL pan-neuronal driver line (PS6961 syIs334) and generated integrants on chromosome II (PS8023, syIs503) and chromosome V (PS8024, syIs504) (Table 1) via standard X-ray irradiation. We showed Chrimson-GFP expression in the C. elegans head and tail neurons (Fig. 1A-1D). We also showed that red light could induce a seizure-like motility phenotype in C. elegans expressing Chrimson-GFP in a pan-neuronal manner (videos), while the negative controls expressing only the effector, or without light induction showed regular motility as expected (Table 2). The body curvature maps from normal and seizure-like motilities showed distinct patterns (Fig. 1E and 1F). We report the effector construct of red-light-gated channelrhodopsin Chrimson as an addition to our cGAL toolkit, which could be widely used in future research to overcome the technical restrictions of blue light-gated channelrhodopsins in C. elegans.

lov-1 and pkd-2, which encode the C. elegans orthologs of human polycystin-1 and -2, are necessary for three particular aspects of male mating behavior. In a screen for male mating defective mutants with similar spectrum of mating defects, we identified a mutation that apparently defines a new locus, lov-3. We isolated the sy682 mutation in an ethyl-methane sulphonate (EMS)-screen of Caenorhabditis elegans strain PS1395 [genotype: plg-1(e2001d); him-5(e1490)] for mutant males that do not mate efficiently and hence do not form plugs on hermaphrodites (Liu et al, 2017). sy682 is defective in the males’ response to contact with hermaphrodite and in vulval location (Table 1). The vulval location defect is failing to stop at the vulva. These two phenotypes are associated with lov-1 (Barr, 1999) and pkd-2 loss-of-function mutations (Barr et al. 2001; Whittaker et al., 2017). sy682 maps to the X chromosome and thus is distinct from lov-1 and pkd-2, so it defines a likely new locus, lov-3.

We describe an efficient screen for male mating defective mutants in Caenorhabditis elegans. We report the isolation of 20 alleles that confer specific mating defects. In a previously reported screen (Hajdu-Cronin et al, 2017), we isolated 19 Cod (for copulation defective) strains in which morphologically wild-type males fail to mate. Failure to mate could conceivably result from defects in any step of the mating process: response, turning, vulva location, spicule insertion, and sperm transfer. By observation, we identified mutants defective in each of these steps except for vulva location. We believe that this was due to both redundancy of sensory structures mediating this step and our stringent screening conditions. To address this, we modified the Cod screen, using the strain plg-1(e2001d); him-5(e1490), in which the presence of a copulatory "plug" over the hermaphrodite vulva provides a visible marker for successful mating (Hodgkin and Doniach, 1997). We backcrossed plg-1(e2001d) four times into him-5(e1490) to make strain PS1395, the parent of our initial screen (sy4xx series). We subsequently repeated our screen after re-isolating a plg-1(e2001d); him-5(e1490) strain PS3696 that had consistent mating behavior and brood size (PS3696 is the parental background for the sy6xx series). Since we select for whether males are able to mate with their moving siblings as opposed to paralyzed hermaphrodites, we expected to isolate more subtle Cod mutants (such as incompletely penetrant vulva location defects). The screen would also allow for the identification of plug formation defective and hermaphrodite-specific mating defective strains. In one PS3696 screen, of 1400 F2 clones, 5% were non-Plg, 280 were then examined for behavior; we kept 69 as candidates; eight had strong phenotypes and normal morphology and were given allele names (sy678, sy680, sy681, sy682, sy683, sy684, sy685, and sy678). sy681 turned out to have the same molecular lesion as sy680 and was discarded. Overall, we isolated 20 Cod mutants from several screens including several pilot screens (Table 1). sy671 was isolated in this screen and found to be an allele of unc-18 (Schindelman et al., 2006).

​Description To identify genes involved in male copulatory behavior, we carried out an F2 clonal screen in a him-5 mutant background. We identified 20 mutations that affect male mating behavior without causing gross defects in morphology. ​ Male mating in C. elegans comprises at least five steps (Liu and Sternberg, 1995). (l) The male responds to the hermaphrodite by backing his tail along the length of the hermaphrodite, (2) he turns over or under her body before reaching the head or tail, (3) he locates the vulva with his tail, at which point he stops backing, (4) he inserts his spicules into the vulva, and (5) he transfers sperm. To study the genetic basis for male mating behavior, we are isolating and characterizing Copulation Defective (Cod) mutations. We screened for mutant strains defective in this behavior using the screen described by Hodgkin (1983). him-5(e1490) worms are mutagenized with ethyl methane sulfonate (EMS); 20 P0 L4 hermaphrodites are picked singly to Petri plates; ten F1 worms are picked per mutagenized P0; and ten F2 L4 hermaphrodites are singled per P0 and their male progeny tested for mating efficiency via a qualitative mating test (six males crossed with six unc-52(e444) hermaphrodites, which are paralyzed at adulthood (Brenner, 1974). Mutants with phenotypes that are likely to reduce mating efficiency in a non-specific manner (such as those causing an Unc, Dpy, or Lon phenotype) were discarded. Those strains that appear morphologically normal under the dissecting microscope yet fail to mate or mate at a very low efficiency (1-5% cross progeny compared to wild type) were screened under Nomarski optics for defects in male reproductive structures. We screened over 3000 haploid genomes, and picked over 100 strains with reproduction defects. Nineteen strains were successfully backcrossed, which represents about 25% of the total strains attempted. This result suggests that most strains harbor two or more mutations that contribute to the mating-deficiency defect. Preliminary analysis of behavior suggests that every major step in the wild-type mating pathway has at least one corresponding Cod mutation blocking the behavior, with several mutations blocking at the spicule insertion step. The screen also yielded morphological mutants, whose phenotypes include crumpled spicules, abnormal rays, and a gonad migration defect; some of these will be described elsewhere (Chamberlin & Sternberg; micropublication in preparation).

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.—Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm.

A new allele of pkd-2 was isolated in a behavioral genetic screen for male mating defects, and found to result in a substitution of Arginine for Glycine in the equivalent of human PKD2 alanine 615. The C. elegans ortholog of polcystin-2 is encoded by pkd-2 (Barr et al., 2001). From an EMS screen of a plg-1; him-5 strain for male mating defective mutants and a secondary behavioral screen for defects in discrete steps of male mating behavior, namely response to contact to hermaphrodites and vulval location (described in Schindelman et al., 2006), we identified a new allele of pkd-2 based on mapping and complementation. sy680 fails to complement pkd-2(sy606) for defects in response to contact with hermaphrodite and vulval location. Here we report the sequence of this allele. PCR amplification and sequencing of pkd-2 exons indicated that there was a c-->t transition in the transcribed DNA strand (g-->a in the pkd-2 sense strand; Figure 1A). This change leads to an altered codon, a Glycine to Arginine substitution the PKD-2 protein. This position corresponds to A615 of the human protein (Figure 1B).

Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division (Kapitein and Hoogenraad, 2015). They are comprised of αβ‐tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms (Gogonea et al., 1999), and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code (Fulton and Simpson, 1976). Here, we use the well‐characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question, through genetic dissection of process outgrowth both in vivo and in vitro. With single‐cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons, and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba‐1, tba‐2, tbb‐1, and tbb‐2) function partially or fully redundantly, while two others (mec‐7 and mec‐12) perform specialized, context‐dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies between cell types and in vivo and in vitro contexts.

ina-1 is expressed throughout uterine toroids. At mid L4, expression is seen in uterine toroid 1 and 4 (ut1, ut4) and in the anterior uterine toroids 2 and 3 (ut2, ut3). In addition, ina-1::GFP is detected in the spermathecal-uterine junction and spermatheca. At the L4 lethargus stage, bright expression is observed in uterine toroids 1 and 4, the spermathecal-uterine junction, and spermatheca. Dimmer expression is present in uterine toroid 2 and posterior toroid 3. Fluorescence is also detected in vulE. Reagents: gmIs5

Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin–proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema 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::Tn5, 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::Tn5 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.

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus.

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC–MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles.

The study of nematode genomes over the last three decades has relied heavily on the model organism Caenorhabditis elegans, which remains the best-assembled and annotated metazoan genome. This is now changing as a rapidly expanding number of nematodes of medical and economic importance have been sequenced in recent years. The advent of sequencing technologies to achieve the equivalent of the $1000 human genome promises that every nematode genome of interest will eventually be sequenced at a reasonable cost. As the sequencing of species spanning the nematode phylum becomes a routine part of characterizing nematodes, the comparative approach and the increasing use of ecological context will help us to further understand the evolution and functional specializations of any given species by comparing its genome to that of other closely and more distantly related nematodes. We review the current state of nematode genomics and discuss some of the highlights that these genomes have revealed and the trend and benefits of ecological genomics, emphasizing the potential for new genomes and the exciting opportunities this provides for nematological studies.

The fluorescence microscope is one of the most important tools in modern clinical diagnosis and biological science. However, its expense, size and limited field-of-view (FOV) are becoming bottlenecks in key applications such as large-scale phenotyping and low-resource-setting diagnostics. Here we report a low-cost, compact chip-scale fluorescence-imaging platform, termed the Fluorescence Talbot Microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly-scalable FOV. The FTM prototype has a resolution of 1.2 microns and an FOV of 3.9 mm x 3.5 mm. We demonstrate the imaging capability of FTM on fluorescently labeled breast cancer cells (SK-BR-3) and HEK cells expressing green fluorescent protein.

Background: The blood-feeding hookworm Necator americanus infects hundreds of millions of people worldwide. In order to elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of the adult stage of Necator americanus was explored using next-generation sequencing and bioinformatic analyses. Methodology/PrincipalFindings:A total of 19,997 contigs were assembled from the sequence data; 6,771 of these contigs had known orthologues in the free-living nematode Caenorhabditis elegans, and most of them encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that the C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%); most of these molecules were predicted to be involved in more than one biological pathway. Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. For instance, proteinase inhibitors were inferred to be highly represented in the former species, whereas SCP/Tpx-1/Ag5/PR-1/Sc7 proteins ( = SCP/TAPS or Ancylostoma-secreted proteins) were predominant in the latter. In N. americanus, essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have homologues in the human host. These candidate targets were inferred to be linked to mitochondrial (e.g., processing proteins) or amino acid metabolism (e.g., asparagine t-RNA synthetase). Conclusions: This study has provided detailed insights into the transcriptome of the adult stage of N. americanus and examines similarities and differences between this species and A. caninum. Future efforts should focus on comparative transcriptomic and proteomic investigations of the other predominant human hookworm, A. duodenale, for both fundamental and applied purposes, including the prevalidation of anti-hookworm drug targets.

Cell migration is a common event during organogenesis, yet little is known about how migration is temporally coordinated with organ development. We are investigating stage-specific programs of cell migration using the linker cell (LC), a migratory cell crucial for male gonadogenesis of C. elegans. During the L3 and L4 larval stages of wild-type males, the LC undergoes changes in its position along the migratory route, in transcriptional regulation of the unc-5 netrin receptor and zmp-1 zinc matrix metalloprotease, and in cell morphology. We have identified the tailless homolog nhr-67 as a cell-autonomous, stage-specific regulator of timing in LC migration programs. In nhr-67-deficient animals, each of the L3 and L4 stage changes is either severely delayed or never occurs, yet LC development before the early L3 stage or after the mid-L4 stage occurs with normal timing. We propose that there is a basal migration program utilized throughout LC migration that is modified by stage-specific regulators such as nhr-67.

The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35(-) embryonic arrest phenotype was variable. We report here that the NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and that CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. Our data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, our results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm.

WormBase (www.wormbase.org) is the major publicly available database of information about Caenorhabditis elegans, an important system for basic biological and biomedical research. Derived from the initial ACeDB database of C. elegans genetic and sequence information, WormBase now includes the genomic, anatomical and functional information about C. elegans, other Caenorhabditis species and other nematodes. As such, it is a crucial resource not only for C. elegans biologists but the larger biomedical and bioinformatics communities. Coverage of core areas of C. elegans biology will allow the biomedical community to make full use of the results of intensive molecular genetic analysis and functional genomic studies of this organism. Improved search and display tools, wider cross-species comparisons and extended ontologies are some of the features that will help scientists extend their research and take advantage of other nematode species genome sequences.

We developed an information retrieval and extraction system that processes the full text of biological papers. The system, called Textpresso, separates text into sentences, labels words and phrases according to an ontology (an organized lexicon), and allows queries to be performed on a database of labeled sentences. The current ontology comprises approximately one hundred categories of terms, such as "gene", "regulation", "human disease", "brain area" etc., and also contains main Gene Ontology (GO) categories. Extraction of particular biological facts, such as gene-���������gene interactions, or the curation of GO cellular components, can be accelerated significantly by ontologies, with Textpresso automatically performing nearly as well as expert curators to identify sentences. Search engine for four literatures, C. elegans, Drosophila, Arabidopsis and Neuroscience have been established by us, and thirteen systems for other literatures have been developed by other groups around the world. Currently, our four systems contain 112,000 papers with 40 million sentences, all systems worldwide contain 190,000 papers with approximately 65 million sentences.

Protein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs); RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite. A full-length cDNA (Tv-rio-1) encoding a RIO1 protein kinase (Tv-RIO1) was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida). The uninterrupted open reading frame (ORF) of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3), and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1). This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes.

The order Strongylida (clade V) represents parasites most closely related to C. elegans, and the group most likely to benefit from the comparative value of its genome sequence. C. elegans has already served as an essential guide for exploring the genomes of other nematode species. However, transcriptomic data from parasitic nematode species, generated by EST approaches, have already served as a boon for C. elegans biology, as they confirm gene predictions and add depth to analyses of structure-function. Here, we propose sequencing the genomes of 20 species, members of the order Strongylida (‘strongylids’). Strongylids, including human hookworms and many intestinal parasites of livestock, are relatively closely related to one another and may have resulted from a recent radiation. To advance and facilitate molecular studies of strongylid parasites, an expressed sequence tag (EST)-based gene discovery program has been carried out, as a part of a broad study of the transcriptomes of members of the phylum Nematoda. More than 60,000 ESTs have been generated for key parasites of human and domestic animals, and for model parasitic species belonging to order Strongylida (comprising 83% of all clade V non-Caenorhabditis ESTs), and deposited in the public databases. Although the transcriptomic data hold promise for the development of novel control strategies for these important pathogens, emerging whole genome data will be pivotal in a more comprehensive approach to identify alternative control strategies, which is increasingly pressing as levels of parasite resistance to available drugs is rising, making some treatment strategies that worked just 5 years ago, ineffective. Also, some drug treatments and environmental nematocideecological contamination.

During C. elegans vulval development, the anchor cell (AC) in the somatic gonad expresses lin-3, activating the EGF receptor signaling pathway in vulval precursor cells (VPCs) and thereby inducing and patterning VPCs. Previous studies with lin-3 mutants and transgene expression have revealed that the level of LIN-3 in the AC must be precisely regulated for proper vulval development. To understand how lin-3 expression is achieved in the AC, we identified a 59 bp lin-3 enhancer sufficient to activate lin-3 transcription solely in the AC. The enhancer contains two E-box elements, and one FTZ-F1 nuclear hormone receptor (NHR) binding site that is mutated in a vulvaless mutant, lin-3(e1417). Mutagenesis studies show that both E-boxes and the NHR binding site are necessary to express lin-3 in the AC. In vitro DNA-binding studies and in vivo functional assays indicate that distinct trans-acting factors, including the E-protein/Daughterless homolog HLH-2 and unidentified nuclear hormone receptor(s), are necessary for lin-3 transcription in the AC and thus are involved in vulval development.

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.

Caenorhabditis elegans polycystins LOV-1 and PKD-2 are expressed in the male-specific HOB neuron, and are necessary for sensation of the hermaphrodite vulva during mating. We demonstrate that male vulva location behavior and expression of lov-1 and pkd-2 in the ciliated sensory neuron HOB require the activities of transcription factor EGL-46 and to some extent also EGL-44. This EGL-46- regulated program is specific to HOB and is distinct from a general ciliogenic pathway functioning in all ciliated neurons. The ciliogenic pathway regulator DAF-19 affects downstream components of the HOB-specific program indirectly and is independent of EGL-46 activity. The sensory function of HOB requires the combined action of these two distinct regulatory pathways.

LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lin-11 have previously been shown to alter the cell division pattern of a subset of the 2° lineage vulval cells. We demonstrate multiple functions of lin-11 during vulval development. We examined the fate of vulval cells in lin-11 mutant animals using five cellular markers and found that lin-11 is necessary for the patterning of both 1° and 2° lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, we show that lin-11 is initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2° lineage cells. Using a conditional RNAi approach, we show that lin-11 regulates vulval morphogenesis. Finally, we show that LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning.

Negative regulation of receptor tyrosine kinase (RTK)/RAS signaling pathways is important for normal development and the prevention of disease in humans. We have used a genetic screen in C. elegans to identify genes that antagonize the activity of activated LET-23, a member of the EGFR family of RTKs. We identified two loss-of-function mutations in dpy-22, previously cloned as sop-1, that promote the ability of activated LET-23 to induce ectopic vulval fates. DPY-22 is a glutamine-rich protein that is most similar to human TRAP230, a component of a transcriptional mediator complex. DPY-22 has previously been shown to regulate WNT responses through inhibition of the ß-catenin-like protein BAR-1. We provide evidence that DPY-22 also inhibits RAS-dependent vulval fate specification independently of BAR-1, and probably regulates the activities of multiple transcription factors during development. Furthermore, we demonstrate that although inhibition of BAR-1-dependent gene expression has been shown to require the C-terminal glutamine-rich region, this region is dispensable for inhibition of RAS-dependent cell differentiation. Thus, the glutamine-rich region contributes to specificity of this class of mediator protein.