Biology subjects

Sternberg, P. W.

Publications and source records attributed to Sternberg, P. W..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematode Steinernema hermaphroditum

The entomopathogenic nematode Steinernema hermaphroditum was recently rediscovered and is being developed as a genetically tractable experimental system for the study of previously unexplored biology, including parasitism of its insect hosts and mutualism with its bacterial endosymbiont Xenorhabdus griffiniae. Through whole-genome re-sequencing and genetic mapping we have for the first time molecularly identified the gene responsible for a mutationally defined phenotypic locus in an entomopathogenic nematode. In the process we observed an unexpected mutational spectrum following EMS mutagenesis in this species. We find that the ortholog of the essential C. elegans peroxidase gene skpo-2 controls body size and shape in S. hermaphroditum. We confirmed this identification by inactivating the gene using CRISPR-Cas9. We propose that the identification of skpo-2 will accelerate gene targeting in other Steinernema entomopathogenic nematodes used commercially in pest control, as skpo-2 is X-linked and males hemizygous for loss of its function can mate, making skpo-2 an easily recognized and maintained marker for use in co-CRISPR.

genetics↗

bioRxiv2023

Fluorescence dynamics of lysosomal-related organelle flashing in the intestinal cells of Caenorhabditis elegans

The lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules displays a "flash" phenomenon in which fluorescence decreases are preceded by sharp increases in fluorescence intensity that expand into the surrounding area when the granules are stimulated with blue light. Autofluorescent granules are present in the intestinal cells of all six nematode species examined, with differences in morphology and distribution pattern. Five species exhibit the flash phenomenon: Panagrellus redivivus (Clade IV), Steinernema hermaphroditum (Clade IV), C. elegans (Clade V), Oscheius tipulae (Clade V), and Pristionchus pacificus (Clade V). The reaction of the granules to blue light stimulation greatly differs among different developmental stages and may also be dependent on physiological conditions. In addition, even within the same animal, the sensitivity of individual granules differs, with some of the variation associated with other characteristics of the granules, such as their overall location within the intestine. We hypothesize that the differences in response to blue light indicate the existence of different sub-populations of gut granules in nematode intestines, and the visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on their eventual characterization.

cell biology↗

bioRxiv2023

Semantic Representation of Neural Circuit Knowledge in Caenorhabditis elegans

In modern biology, new knowledge is generated quickly, making it challenging for researchers to efficiently acquire and synthesise new information from the large volume of primary publications. To address this problem, computational approaches that generate machine-readable representations of scientific findings in the form of knowledge graphs have been developed. These representations can integrate different types of experimental data from multiple papers and biological knowledge bases in a unifying data model, providing a complementary method to manual review for interacting with published knowledge. The Gene Ontology Consortium (GOC) has created a semantic modelling framework that extends individual functional gene annotations to structured descriptions of causal networks representing biological processes (Gene Ontology Causal Activity Modelling, or GO-CAM). In this study, we explored whether the GO-CAM framework could represent knowledge of the causal relationships between environmental inputs, neural circuits and behavior in the model nematode C. elegans (C. elegans Neural Circuit Causal Activity Modelling (CeN- CAM)). We found that, given extensions to several relevant ontologies, a wide variety of author statements from the literature about the neural circuit basis of egg-laying and carbon dioxide (CO2) avoidance behaviors could be faithfully represented with CeN-CAM. Through this process, we were able to generate generic data models for several categories of experimental results. We also discuss how semantic modelling may be used to functionally annotate the C. elegans connectome. Thus, Gene Ontology-based semantic modelling has the potential to support various machine-readable representations of neurobiological knowledge. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/538760v4_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@15ca6deorg.highwire.dtl.DTLVardef@1cf3b92org.highwire.dtl.DTLVardef@4ff138org.highwire.dtl.DTLVardef@65c908_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

LINKIN-associated proteins necessary for tissue integrity during collective cell migration

Cell adhesion plays essential roles in almost every aspect of metazoan biology. LINKIN (Human: ITFG1, Caenorhabditis elegans: lnkn-1) is a conserved transmembrane protein that has been identified to be necessary for tissue integrity during migration. In C. elegans, loss of lnkn-1 results in the detachment of the lead migratory cell from the rest of the developing male gonad. Previously, three interactors of ITFG1/lnkn-1 - RUVBL1/ruvb-1, RUVBL2/ruvb-2, and alpha-tubulin - were identified by immunoprecipitation-mass spectrometry (IP-MS) analysis using human HEK293T cells and then validated in the nematode male gonad. The ITFG1-RUVBL1 interaction has since been independently validated in a breast cancer cell line model that also implicates the involvement of the pair in metastasis. Here, we showed that epitope-tagged ITFG1 localized to the cell surface of MDA-MB-231 breast cancer cells. Using IP-MS analysis, we identified a new list of potential interactors of ITFG1. Loss-of-function analysis of their C. elegans orthologs found that three of the interactors - ATP9A/tat-5, NME1/ndk-1, and ANAPC2/apc-2 - displayed migratory detachment phenotypes similar to that of lnkn-1. Taken together with the other genes whose reduction-of-function phenotype is similar to that of lnkn-1 (notably cohesion and condensin), suggests the involvement of membrane remodeling and chromosome biology in LINKIN-dependent cell adhesion and supports the hypothesis for a structural role of chromosomes in post-mitotic cells.

developmental biology↗

bioRxiv2022

PEZO-1 and TRP-4 mechanosensors are involved in mating behavior in Caenorhabditis elegans

Male mating in Caenorhabditis elegans is a complex behavior with a strong mechanosensory component. C. elegans has several characterized mechanotransducer proteins, but few have been shown to contribute to mating. Here, we investigated the roles of PEZO-1, a piezo channel, and TRP-4, a mechanotransducing TRPN channel, in male mating behavior. We show that pezo-1 is expressed in several male-specific neurons with known roles in mating. We show that, among other neurons, trp-4 is expressed in the PCA sensory neuron, which monitors relative sliding between the male and the hermaphrodite and inhibits neurons involved in vulva detection. Mutations in both genes compromise many steps of mating, including initial response to the hermaphrodite, scanning, turning, and vulva detection. We performed pan-neuronal imaging during mating between freely-moving mutant males and hermaphrodites. Both pezo-1 and trp-4 mutants showed spurious activation of the sensory neurons involved in vulva detection. In trp-4 mutants, this spurious activation might be caused by PCA failure to inhibit vulva-detecting neurons during scanning. Indeed, we show that without functional TRP-4, PCA fails to detect the relative sliding between the male and hermaphrodite. Cell-specific TRP-4 expression restores PCAs mechanosensory function. Our results demonstrate new roles for both PEZO-1 and TRP-4 mechanotransducers in C. elegans mating behavior.

neuroscience↗

bioRxiv2022

Single-nucleus resolution mapping of the adult C. elegans and its application to elucidate inter- and trans-generational response to alcohol.

Single-cell RNA transcriptomic platforms have significantly contributed to our understanding of tissue heterogeneity as well as of developmental and cellular differentiation trajectories. They also provide an opportunity to map an organisms response to environmental cues with high resolution and unbiasedly identify the cell types that are the most transcriptionally sensitive to exposures. Here, we applied single nucleus RNA-seq experimental and computational approaches to C. elegans to establish the transcriptome of the adult nematode and comprehensively characterize the transcriptional impact of ethanol as a model environmental exposure on the entire organism at cell type-resolution over several generations. Clustering, tissue and phenotype enrichment, and gene ontology analyses identified 31 clusters representing a diverse number of adult cell types, including those from syncytial and multi-nucleated tissues which are difficult to assess by single cell RNA-seq, such as the mitotic and meiotic germline, hypodermal cells, and the intestine. We applied this method to identify the impact of inter- and trans-generational exposure to two human-relevant doses of alcohol. Cell type proportions were not significantly altered by ethanol. However, Euclidean distance analysis identified several germline, striated muscle, and neuronal clusters as being major transcriptional targets of ethanol at both the F1 and F3 generations although the relative order of clusters changed between generations. The impact on germline clusters was further confirmed by phenotypic enrichment analysis as well as functional validation, namely a remarkable inter- and trans-generational increase in germline apoptosis, aneuploidy, and embryonic lethality. Together, snRNA-seq of the adult C. elegans represents a powerful approach for the detailed examination of an adult organisms response to environmental cues.

genetics↗

bioRxiv2021

Interneuron Control of C. elegans Developmental Decision-making

Animals integrate external stimuli to shape their physiological responses throughout development. In adverse environments, Caenorhabditis elegans larvae can enter a stress-resistant diapause state with arrested metabolism and reproductive physiology. Amphid sensory neurons feed into both rapid chemotactic and short-term foraging mode decisions, mediated by amphid and premotor interneurons, as well as the long-term diapause decision. We identify amphid interneurons that integrate pheromone cues and propagate this information via a neuropeptidergic pathways to influence larval developmental fate, bypassing the pre-motor system. AIA interneuron-derived FLP-2 neuropeptide signaling promotes reproductive growth and AIA activity is suppressed by pheromone. FLP-2 acts antagonistically to the insulin-like INS-1. FLP-2s growth promoting effects are inhibited by upstream metabotropic glutamatergic signaling and mediated by the broadly-expressed neuropeptide receptor NPR-30. Conversely, the AIB interneurons and their neuropeptide receptor NPR-9/GALR2 promote diapause entry. These neuropeptidergic outputs allow reuse of parts of a sensory system for a decision with a distinct timescale.

neuroscience↗

bioRxiv2021

Transcriptomic profiling of sex-specific olfactory neurons reveals subset-specific receptor expression in C. elegans

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

neuroscience↗

bioRxiv2021

Gene identification and genome annotation in Caenorhabditis briggsae by high throughput 5' RNA end determination

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying the 5 ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 5 end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 493 operons, of which two-thirds are fully supported by tags. In addition, two SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that four of the 12 novel operons are conserved in C. nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species.

evolutionary biology↗

bioRxiv2021

Entry to and exit from diapause arrest in Caenorhabditis elegans are both regulated by a steroid hormone pathway

Diapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. While a steroid hormone pathway regulates the entry decision into Caenorhabditis elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions, and we establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit decision. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling. Summary StatementIn animals such as Caenorhabditis elegans, a steroid hormone pathway controls both the entry and exit decisions into and out of the developmentally arrested dauer state in response to environmental signaling.

developmental biology↗

bioRxiv2021

The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis

Entomopathogenic nematodes, including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as first-generation hermaphroditic and second-generation gonochoristic. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.

genetics↗

bioRxiv2021

Distinct neural circuits establish the same chemosensory behavior in C. elegans

Animals frequently exhibit the same behavior under different environmental or physiological conditions. To what extent these behaviors are generated by similar vs. distinct mechanisms is unclear. Moreover, the circumstances under which divergent neural mechanisms establish the same behavior, and the molecular signals that regulate the same behavior across conditions, are poorly understood. We show that in C. elegans, distinct neural mechanisms mediate the same chemosensory behavior at two different life stages. Both dauer larvae and starved adults are attracted to carbon dioxide (CO2), but CO2 attraction is mediated by distinct sets of interneurons at the two life stages. Some interneurons mediate CO2 response only in dauers, some show CO2-evoked activity in adults and dauers but contribute to CO2 response only in adults, and some show CO2-evoked activity that opposes CO2 attraction in adults but promotes CO2 attraction in dauers. We also identify a novel role for insulin signaling in establishing life-stage-specific CO2 responses by modulating interneuron activity. Further, we show that a combinatorial code of both shared and life-stage-specific molecular signals regulate CO2 attraction. Our results identify a mechanism by which the same chemosensory behavior can be generated by distinct neural circuits, revealing an unexpected complexity to chemosensory processing.

neuroscience↗

bioRxiv2021

Single cell tools for WormBase

We present two web apps for interactively performing common tasks with single cell RNA sequencing data: scdefg for interactive differential expression and wormcells-viz for visualization of gene expression. We made these tools available with public C. elegans datasets curated by WormBase at single-cell.wormbase.org. They can also be readily deployed for use with any other datasets using the source code available at github.com/WormBase/scdefg and at github.com/WormBase/wormcells-viz.

bioinformatics↗

bioRxiv2020

Modular metabolite assembly in C. elegans lysosome-related organelles

Signaling molecules derived from attachment of diverse metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, many aspects of their biogenesis remain unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of most modular ascarosides as well as previously undescribed modular glucosides. Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism, suggesting that modular glucosides, like the ascarosides, may serve signaling functions. We further show that carboxylesterases that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides via ester and amide linkages. Further exploration of LRO function and carboxylesterase homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/262956v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@6e83cborg.highwire.dtl.DTLVardef@18e9302org.highwire.dtl.DTLVardef@1557670org.highwire.dtl.DTLVardef@1d2ca50_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Sensory-evoked extracellular vesicle release and targeting

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. An urgent need in the field is a fundamental 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 to retract cilia before entering 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 cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [7]. We find that males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2-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 evolutionarily conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other systems and biological contexts.

cell biology↗

bioRxiv2019

Caenorhabditis elegans PIEZO Channel Coordinates Multiple Reproductive Tissues to Govern Ovulation

The PIEZO proteins are involved in a wide range of developmental and physiological processes. Human PIEZO1 and PIEZO2 are newly identified excitatory mechano-sensitive proteins; they are non-selective ion channels that exhibit a preference for calcium in response to mechanical stimuli. To further understand the function of these proteins, we investigated the roles of pezo-1, the sole PIEZO ortholog in C. elegans. pezo-1 is expressed throughout development in C. elegans, with strong expression in reproductive tissues. A number of deletion alleles as well as a putative gain-of-function mutant caused severe defects in reproduction. A reduced brood size was observed in the strains depleted of PEZO-1. In vivo observations show that oocytes undergo a variety of transit defects as they enter and exit the spermatheca during ovulation. Post ovulation oocytes were frequently damaged during spermathecal contraction. Calcium signaling in the spermatheca is normal during ovulation in pezo-1 mutants, however, pezo-1 interacts genetically with known regulators of calcium signaling. Lastly, loss of PEZO-1 caused defective sperm navigation after being pushed out of the spermatheca during ovulation. Mating with males rescued these reproductive deficiencies in our pezo-1 mutants. These findings suggest that PEZO-1 may act in different reproductive tissues to promote proper ovulation and fertilization in C. elegans.

developmental biology↗

bioRxiv2019

RNA Pol II Length and Disorder Enable Cooperative Scaling of Transcriptional Bursting

RNA Polymerase II contains a disordered C-terminal domain (CTD) whose length enigmatically correlates with genome size. The CTD is crucial to eukaryotic transcription, yet the functional and evolutionary relevance of this variation remains unclear. Here, we use smFISH, live imaging, and RNA-seq to investigate how CTD length and disorder influence transcription. We find that length modulates the size and frequency of transcriptional bursting. Disorder is highly conserved and mediates CTD-CTD interactions, an ability we show is separable from protein sequence and necessary for efficient transcription. We build a data-driven quantitative model, simulations of which recapitulate experiments and support CTD length promotes initial polymerase recruitment to the promoter but slows down its release from it, and that CTD-CTD interactions enable promoter recruitment of multiple polymerases. Our results reveal how these tunable parameters provide access to a range of transcriptional activity, offering a new perspective for the mechanistic significance of CTD length and disorder in transcription across eukaryotes.

molecular biology↗

bioRxiv2019

Caenorhabditis elegans AF4/FMR2 family homolog affl-2 is required for heat shock induced gene expression

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

genetics↗