Deciphering the underlying mechanisms of the pharyngeal pumping motions in Caenorhabditis elegans.

The pharynx of the nematode Caenorhabditis elegans is a neuromuscular organ that exhibits typical pumping motions, which result in the intake of food particles from the environment. In-depth inspection reveals slightly different dynamics at the various pharyngeal areas, rather than synchronous pumping motions of the whole organ, which are important for its effective functioning. While the different pumping dynamics are well characterized, the underlying mechanisms that generate them are not known. In this study, the C. elegans pharynx was modeled in a bottom-up fashion, including all of the underlying biological processes that lead to, and including, its end function, food intake. The mathematical modeling of all processes allowed performing comprehensive, quantitative analyses of the system as a whole. Our analyses provided detailed explanations for the various pumping dynamics generated at the different pharyngeal areas; a fine-resolution description of muscle dynamics, both between and within different pharyngeal areas; a quantitative assessment of the values of many parameters of the system that are unavailable in the literature; and support for a functional role of the marginal cells, which are currently assumed to mainly have a structural role in the pharynx. In addition, our model predicted that in tiny organisms such as C. elegans, the generation of long-lasting action potentials must involve ions other than calcium. Our study exemplifies the power of mathematical models, which allow a more accurate, higher-resolution inspection of the studied system, and an easier and faster execution of in silico experiments than feasible in the lab.

Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans.

C. elegans is a bacteria-eating soil-dwelling nematode. Typical cultivation of laboratory-reared populations occurs on bacteria-covered solid media, where they move along with sinusoidal undulations. Nematodes decelerate when they encounter food. Dopaminergic and serotonergic neurotransmission regulate this behavior. Here, we describe the procedure for determining food-dependent locomotion rate of fed and fasting nematodes. We detail steps for assay plate preparation, C. elegans synchronization, and assessment of locomotion. The behaviors we describe provide information regarding the animal's physiological neuronal and muscular function. For complete details on the use and execution of this protocol, please refer to Petratou et al. (2023)1 and Sawin et al. (2000).2.

Anatomical restructuring of a lateralized neural circuit during associative learning by asymmetric insulin signaling.

Studies of neuronal connectivity in model organisms, i.e., of their connectomes, have been instrumental in dissecting the structure-function relationship of nervous systems. However, the limited sample size of these studies has impeded analyses into how variation of connectivity across populations may influence circuit architecture and behavior. Moreover, little is known about how experiences induce changes in circuit architecture. Here, we show that an asymmetric salt-sensing circuit in the nematode Caenorhabditis elegans exhibits variation that predicts the animals' salt preferences and undergoes restructuring during salt associative learning. Naive worms memorize and prefer the salt concentration they experience in the presence of food through a left-biased neural network architecture. However, animals conditioned at elevated salt concentrations change this left-biased network to a right-biased network. This change in circuit architecture occurs through the addition of new synapses in response to asymmetric, paracrine insulin signaling. Therefore, experience-dependent changes in an animal's neural connectome are induced by insulin signaling and are fundamental to learning and behavior.

Principles for coding associative memories in a compact neural network.

A major goal in neuroscience is to elucidate the principles by which memories are stored in a neural network. Here, we have systematically studied how four types of associative memories (short- and long-term memories, each as positive and negative associations) are encoded within the compact neural network of Caenorhabditis elegans worms. Interestingly, sensory neurons were primarily involved in coding short-term, but not long-term, memories, and individual sensory neurons could be assigned to coding either the conditioned stimulus or the experience valence (or both). Moreover, when considering the collective activity of the sensory neurons, the specific training experiences could be decoded. Interneurons integrated the modulated sensory inputs and a simple linear combination model identified the experience-specific modulated communication routes. The widely distributed memory suggests that integrated network plasticity, rather than changes to individual neurons, underlies the fine behavioral plasticity. This comprehensive study reveals basic memory-coding principles and highlights the central roles of sensory neurons in memory formation.

The longevity response to warm temperature is neurally controlled via the regulation of collagen genes.

Studies in diverse species have associated higher temperatures with shorter lifespan and lower temperatures with longer lifespan. These inverse effects of temperature on longevity are traditionally explained using the rate of living theory, which posits that higher temperatures increase chemical reaction rates, thus speeding up the aging process. Recent studies have identified specific molecules and cells that affect the longevity response to temperature, indicating that this response is regulated, not simply thermodynamic. Here, we demonstrate that in Caenorhabditis elegans, functional loss of NPR-8, a G protein-coupled receptor related to mammalian neuropeptide Y receptors, increases worm lifespan at 25°C but not at 20°C or 15°C, and that the lifespan extension at 25°C is regulated by the NPR-8-expressing AWB and AWC chemosensory neurons as well as AFD thermosensory neurons. Integrative transcriptomic analyses revealed that both warm temperature and old age profoundly alter gene expression and that genes involved in the metabolic and biosynthetic processes increase expression at 25°C relative to 20°C, indicating elevated metabolism at warm temperature. These data demonstrate that the temperature-induced longevity response is neurally regulated and also provide a partial molecular basis for the rate of living theory, suggesting that these two views are not mutually exclusive. Genetic manipulation and functional assays further uncovered that the NPR-8-dependent longevity response to warm temperature is achieved by regulating the expression of a subset of collagen genes. As increased collagen expression is a common feature of many lifespan-extending interventions and enhanced stress resistance, collagen expression could be critical for healthy aging.

Reprogramming the topology of the nociceptive circuit in C. elegans reshapes sexual behavior.

The effect of the detailed connectivity of a neural circuit on its function and the resulting behavior of the organism is a key question in many neural systems. Here, we study the circuit for nociception in C. elegans, which is composed of the same neurons in the two sexes that are wired differently. We show that the nociceptive sensory neurons respond similarly in the two sexes, yet the animals display sexually dimorphic behaviors to the same aversive stimuli. To uncover the role of the downstream network topology in shaping behavior, we learn and simulate network models that replicate the observed dimorphic behaviors and use them to predict simple network rewirings that would switch behavior between the sexes. We then show experimentally that these subtle synaptic rewirings indeed flip behavior. Interestingly, when presented with aversive cues, rewired males were compromised in finding mating partners, suggesting that network topologies that enable efficient avoidance of noxious cues have a reproductive "cost." Our results present a deconstruction of the design of a neural circuit that controls sexual behavior and how to reprogram it.

Caenorhabditis elegans sine oculis/SIX-type homeobox genes act as homeotic switches to define neuronal subtype identities.

The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.

Stereotyped behavioral maturation and rhythmic quiescence in C. elegans embryos.

Systematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here, we apply this approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were built using a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom tracking software. Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping nor the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in diverse animals and may play an important role in promoting normal developmental outcomes.

The physiological cargo adaptor of kinesin-2 functions as an evolutionary conserved lockpick.

Specific recognition of cellular cargo and efficient transport to its correct intracellular destination is an infrastructural challenge faced by most eukaryotic cells. This remarkable deed is accomplished by processive motor proteins that are subject to robust regulatory mechanisms. The first level of regulation entails the ability of the motor to suppress its own activity. This autoinhibition is eventually relieved by specific cargo binding. To better understand the role of the cargo during motor activation, we dissected the activation mechanism of the ciliary homodimeric kinesin-2 from Caenorhabditis elegans by its physiological cargo. In functional reconstitution assays, we identified two cargo adaptor proteins that together are necessary and sufficient to allosterically activate the autoinhibited motor. Surprisingly, the orthologous adaptor proteins from the unicellular green algae Chlamydomonas reinhardtii also fully activated the kinesin-2 from worm, even though C. reinhardtii itself lacks a homodimeric kinesin-2 motor. The latter suggested that a motor activation mechanism similar to the C. elegans model existed already well before metazoans evolved, and prompted us to scrutinize predicted homodimeric kinesin-2 orthologs in other evolutionarily distant eukaryotes. We show that the ciliate Tetrahymena thermophila not only possesses a homodimeric kinesin-2 but that it also shares the same allosteric activation mechanism that we delineated in the C. elegans model. Our results point to a much more fundamental role of homodimeric kinesin-2 in intraflagellar transport (IFT) than previously thought and warrant further scrutiny of distantly related organisms toward a comprehensive picture of the IFT process and its evolution.

Changes in body shape implicate cuticle stretch in C. elegans growth control.

Growth control establishes organism size, requiring mechanisms to sense and adjust growth during development. Studies of single cells revealed that size homeostasis uses distinct control methods. In multicellular organisms, mechanisms that regulate single cell growth must integrate control across organs and tissues during development to generate adult size and shape. We leveraged the roundworm Caenorhabditis elegans as a scalable and tractable model to collect precise growth measurements of thousands of individuals, measure feeding behavior, and quantify changes in animal size and shape during a densely sampled developmental time course. As animals transitioned from one developmental stage to the next, we observed changes in body aspect ratio while body volume remained constant. Then, we modeled a physical mechanism by which constraints on cuticle stretch could cause changes in C. elegans body shape. The model-predicted shape changes are consistent with those observed in the data. Theoretically, cuticle stretch could be sensed by the animal to initiate larval-stage transitions, providing a means for physical constraints to influence developmental timing and growth rate in C. elegans.

Temporal scaling in C. elegans larval development.

SignificanceAn enduring mystery of development is how its timing is controlled, particularly for development after birth, where timing is highly flexible and depends on environmental conditions, such as food availability and diet. We followed timing of cell- and organism-level events in individual Caenorhabditis elegans larvae developing from hatching to adulthood, uncovering widespread variations in event timing, both between isogenic individuals in the same environment and when changing conditions and genotypes. However, in almost all cases, we found that events occurred at the same time, when time was rescaled by the duration of development measured in each individual. This observation of "temporal scaling" poses strong constraints on models to explain timing of larval development.

NHR-80 senses the mitochondrial UPR to rewire citrate metabolism for lipid accumulation in Caenorhabditis elegans.

Mitochondria are known as the powerhouse of the cell. Dysfunction of mitochondria homeostasis induces the mitochondrial unfolded protein response (UPRmt), altering cellular metabolism. How cells sense the UPRmt to rewire metabolism is largely unknown. Here, we show that inactivation of either the citric/tricarboxylic acid (TCA) cycle enzymes aco-2 or idha-1, which encode aconitase and isocitrate dehydrogenase respectively, leads to citrate accumulation. In Caenorhabditis elegans, both in vitro and in vivo, citrate accumulation consequently triggers the UPRmt and also promotes lipid accumulation. The transcription factor DVE-1 binds to the promoter of the nuclear hormone receptor nhr-80 to transactivate its expression. NHR-80 then upregulates lipogenesis and lipid accumulation, shifting excess citrate for use in lipogenesis and for storage as triacylglycerol in lipid droplets. Inactivation of DVE-1 or NHR-80 fully abolishes the citrate-induced lipid accumulation. Therefore, our work uncovers a DVE-1-NHR-80-lipogenesis axis linking the transmission of the mitochondrial stress signal to lipid metabolism.

O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 modulate seizure susceptibility in C. elegans.

Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to be used as a behavioral readout of the locomotor circuit and neuronal function. C. elegans possess conserved nervous system features such as gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (Ach) and acetylcholine receptors in excitatory neurotransmission. Our previously published data has shown that decreasing inhibition in the motor circuit, via GABAergic manipulation, will extend the time of locomotor recovery following electroshock. Similarly, mutations in a HECT E3 ubiquitin ligase called EEL-1 leads to impaired GABAergic transmission, E/I imbalance and altered sensitivity to electroshock. Mutations in the human ortholog of EEL-1, called HUWE1, are associated with both syndromic and non-syndromic intellectual disability. Both EEL-1 and its previously established binding protein, OGT-1, are expressed in GABAergic motor neurons, localize to GABAergic presynaptic terminals, and function in parallel to regulate GABA neuron function. In this study, we tested behavioral responses to electroshock in wildtype, ogt-1, eel-1 and ogt-1; eel-1 double mutants. Both ogt-1 and eel-1 null mutants have decreased inhibitory GABAergic neuron function and increased electroshock sensitivity. Consistent with EEL-1 and OGT-1 functioning in parallel pathways, ogt-1; eel-1 double mutants showed enhanced electroshock susceptibility. Expression of OGT-1 in the C. elegans nervous system rescued enhanced electroshock defects in ogt-1; eel-1 double mutants. Application of a GABA agonist, Baclofen, decreased electroshock susceptibility in all animals. Our C. elegans electroconvulsive seizure assay was the first to model a human X-linked Intellectual Disability (XLID) associated with epilepsy and suggests a potential novel role for the OGT-1/EEL-1 complex in seizure susceptibility.

A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior.

Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.

daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71.

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans.

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential "prion-like" domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59's localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation.

Investigating the molecular mechanisms of learning and memory using Caenorhabditis elegans.

Learning is an essential biological process for survival since it facilitates behavioural plasticity in response to environmental changes. This process is mediated by a wide variety of genes, mostly expressed in the nervous system. Many studies have extensively explored the molecular and cellular mechanisms underlying learning and memory. This review will focus on the advances gained through the study of the nematode Caenorhabditis elegans. C. elegans provides an excellent system to study learning because of its genetic tractability, in addition to its invariant, compact nervous system (~300 neurons) that is well-characterised at the structural level. Importantly, despite its compact nature, the nematode nervous system possesses a high level of conservation with mammalian systems. These features allow the study of genes within specific sensory-, inter- and motor neurons, facilitating the interrogation of signalling pathways that mediate learning via defined neural circuits. This review will detail how learning and memory can be studied in C. elegans through behavioural paradigms that target distinct sensory modalities. We will also summarise recent studies describing mechanisms through which key molecular and cellular pathways are proposed to affect associative and non-associative forms of learning.

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division.

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell-stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

The nematode C. elegans senses airborne sound.

Unlike olfaction, taste, touch, vision, and proprioception, which are widespread across animal phyla, hearing is found only in vertebrates and some arthropods. The vast majority of invertebrate species are thus considered insensitive to sound. Here, we challenge this conventional view by showing that the earless nematode C. elegans senses airborne sound at frequencies reaching the kHz range. Sound vibrates C. elegans skin, which acts as a pressure-to-displacement transducer similar to vertebrate eardrum, activates sound-sensitive FLP/PVD neurons attached to the skin, and evokes phonotaxis behavior. We identified two nAChRs that transduce sound signals independently of ACh, revealing an unexpected function of nAChRs in mechanosensation. Thus, the ability to sense airborne sound is not restricted to vertebrates and arthropods as previously thought, and might have evolved multiple times independently in the animal kingdom, suggesting convergent evolution. Our studies also demonstrate that animals without ears may not be presumed to be sound insensitive.

SLC-30A9 is required for Zn2+ homeostasis, Zn2+ mobilization, and mitochondrial health.

The trace element zinc is essential for many aspects of physiology. The mitochondrion is a major Zn2+ store, and excessive mitochondrial Zn2+ is linked to neurodegeneration. How mitochondria maintain their Zn2+ homeostasis is unknown. Here, we find that the SLC-30A9 transporter localizes on mitochondria and is required for export of Zn2+ from mitochondria in both Caenorhabditis elegans and human cells. Loss of slc-30a9 leads to elevated Zn2+ levels in mitochondria, a severely swollen mitochondrial matrix in many tissues, compromised mitochondrial metabolic function, reductive stress, and induction of the mitochondrial stress response. SLC-30A9 is also essential for organismal fertility and sperm activation in C. elegans, during which Zn2+ exits from mitochondria and acts as an activation signal. In slc-30a9-deficient neurons, misshapen mitochondria show reduced distribution in axons and dendrites, providing a potential mechanism for the Birk-Landau-Perez cerebrorenal syndrome where an SLC30A9 mutation was found.