The apical extracellular matrix can form elaborate three-dimensional structures on animal surfaces. To better understand the mechanisms that pattern and shape these structures, we focus on development of collagen-rich cuticle ridges (alae) in adult C. elegans. Previous studies suggested that longitudinal actin filament bundles (AFBs) in the lateral seam epidermis specify alae position through a mechanism that involves post-secretory matrix delamination. Here we identify additional components of this highly organized cortical actin network and show that loss of the apical βH-spectrin SMA-1 specifically disrupts organization of the two AFBs that would normally flank the site where the middle alae ridge forms. Correspondingly, sma-1 loss, or mutation of its actin binding domains, also disrupts formation of the middle alae ridge. Ultrastructurally, sma-1 mutants have expanded regions of matrix delamination that can explain middle ridge loss. Together, these data highlight the importance of apical spectrin for organizing a patterned actin network within epithelia and show that, via its effects on actin organization, spectrin can also change the extracellular matrix and its patterns on animal surfaces.

Detecting and responding to pathogenic bacteria is an essential function of eukaryotic cells. As bacteria-derived organelles, mitochondria carry lipids, proteins, and other molecules such as iron which are required for bacterial growth, and thus are subject to pathogenic bacterial attack. In fact, mitochondrial morphology and function are often altered during early bacterial infection. However, the mechanism by which bacterial pathogen attack triggers mitochondrial responses is unknown. Here, we demonstrate that infection by the pathogenic bacterium Staphylococcus aureus or Pseudomonas aeruginosa or hypoxia leads to remodeling of the mitochondrial network in the host Caenorhabditis elegans. Our analysis discovers that lysosome-related organelle (LRO) genes are required for this remodeling and indicates that LROs also precipitate downstream infection-response gene expression.

The highly conserved body plan of nematodes makes members of this phylum excellent models to study cell type evolution. Early branching nematode lineages, mostly occupying aquatic habitats, usually contain caudal glands deployed for underwater attachment to a substrate, but have been thought to lack phasmid sensory organs, resulting in their historical classification as Aphasmidia. With the transition to a terrestrial environment, nematodes lost caudal glands and gained phasmid sensory neurons. The supposed mutually exclusive existence of caudal glands and phasmids has led to the suggestion that phasmid neurons may have evolved from caudal glands. Here, we rule out this possibility through light and electron microscopical analysis of Mononchus aquaticus, a member of the early branching Dorylaimia lineage, showing that phasmid sensory neurons and caudal glands do coexist. This observation not only argues against a proposed cell type evolution scenario accompanying aquatic-to-terrestrial transitions but also indicates that the presence of phasmid sensory organs may have been an ancestral trait of the nematode phylum.

Transcription factors are central to neuronal development, yet their functions beyond the cells in which they are expressed remain poorly understood. Here, we uncover unexpected non-cell-autonomous roles for UNC-3, a terminal selector of cholinergic motor neuron identity in C. elegans, whose human ortholog (EBF3) is linked to a neurodevelopmental syndrome. Single-cell RNA-sequencing reveals unc-3 loss in cholinergic motor neurons elicits pronounced transcriptional changes in GABAergic motor neurons that do not express unc-3, which can be rescued by cholinergic-specific UNC-3 restoration. Mechanistically, gene network analysis identifies the pro-regenerative bZIP factor CEBP-1/CEBPB as a key driver of these transcriptional changes. At the circuit level, unc-3 loss causes synaptic and axon pathfinding defects in GABA motor neurons alongside misregulation of neurite development genes. Finally, UNC-3 not only acts as a direct transcriptional activator but also suppresses inappropriate gene expression through indirect mechanisms. Together, these findings broaden terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, providing a mechanistic framework for understanding EBF3-associated neurodevelopmental disease.

Cilia are critical sensory organelles that project from the cell surface into the tissue environment, where they are surrounded by extracellular matrix (ECM). Abnormal ECM and fibrosis are two hallmarks of ciliopathies, yet the relationship between cilia and ECM is not well understood. Using the sense organs of C. elegans as a model, we found that a neomorphic mutation in the ECM gene mec-9 impacts sensory cilia function, ciliary protein localization, microtubule ultrastructure, and shedding of ciliary extracellular vesicles (EVs). We show that mec-9 is not expressed in EV releasing neurons, but rather by companion neurons in the sense organs, and may act cell non-autonomously. Our studies reveal pleiotropic roles for mec-9 in the C. elegans ciliated nervous system and provide an in vivo model to study the relationship between cilia and ECM.

Environmental stress can remodel neural circuits, yet how such rewiring generates behavioral adaptation remains poorly understood. The dauer stage of Caenorhabditis elegans provides a unique model to address this question. Here, we investigated how dauer-specific circuit remodeling alters nociceptive behavior. We found that dauers exhibit markedly shorter avoidance durations than adults. Comparative connectomics revealed substantial expansion of gap junctions within the dauer nociceptive circuit. Calcium imaging further showed that dauer neurons exhibit faster and more transient activity dynamics throughout the circuit. Introducing synthetic dauer-specific gap junctions into adults was sufficient to recapitulate dauer-like neuronal activity patterns and significantly shorten avoidance duration. Despite extensive circuit rewiring, however, avoidance initiation remained preserved across developmental stages. Together, our findings demonstrate that the dauer connectome is selectively rewired through gap junction remodeling to tune behavioral persistence while robustly preserving behavioral initiation, revealing how developmental circuit reorganization balances flexibility and stability for survival under stress.

Neurons display unique shapes and establish intricate networks, which may differ between sexes. In complex organisms, studying sex differences in structure and function of individual neurons is difficult. The nematode Caenorhabditis elegans hermaphrodites and males present an exceptional model for studying neuronal morphogenesis in a simple, sexually dimorphic system. We focus on the polymodal sensory bilateral neuron pair PVD, which forms a complex but stereotypic dendritic tree composed of multiple subunits that resemble candelabra. PVD is well studied in hermaphrodites, but not in males. We show here that during larval development, male PVD extends a similar architecture to the hermaphrodite utilizing the sexually shared Menorin patterning mechanism. In early adulthood, however, male PVD develops a unique extension into the copulatory tail structure. Alongside established tail ray neurons RnA and RnB, we show PVD is a third, previously unrecognized, neuron within the tail rays. Unlike RnA and RnB, PVD extends anterogradely, branches and turns within the ray hypodermis, and is nonciliated. This PVD sexually dimorphic arborization is absent in mutant backgrounds which perturb the Menorin guidance complex. SAX-7/L1CAM, a hypodermal component of this complex, shows a male-specific expression pattern which precedes PVD extension, and its presence allows PVD to enter the tail rays. Further, our results reveal that genetically altered arborization or ablation of the PVD results in male mating behavioral defects, particularly as males turn around the hermaphrodite. These results uncover an adult-stage sexual dimorphism of dendritic branching and a function for PVD in male sexual behavior.

Experimental organisms such as the nematode Caenorhabditis elegans are fundamental to biological discovery. The success of C. elegans research has been greatly enabled by infrastructure that allows thousands of scientists to share and access research materials and unpublished information efficiently. Here, we celebrate the worm by interweaving vignettes describing four Nobel Prize-winning discoveries with descriptions of how the major NIH-supported research resources-the Caenorhabditis Genetics Center, WormBase, and WormAtlas-provide invaluable support for all C. elegans research. The synergy between investigation and the availability of shared resources for the C. elegans community is a paradigm for all model organism research, and the continued support of such community research resources will be essential for maximizing impactful discoveries in the future.

Discovery of new compounds that ameliorate the negative health impacts of aging promises to be of tremendous benefit across a number of age-based comorbidities. One method to prioritize a testable subset of the nearly infinite universe of potential compounds is to use computational prediction of their likely anti-aging capacity. Here, we present a survey of longevity effects for 16 compounds suggested by a previously published computational prediction set, capitalizing upon the comprehensive, multi-species approach utilized by the Caenorhabditis Intervention Testing Program. While 11 compounds (aldosterone, arecoline, bortezomib, dasatinib, decitabine, dexamethasone, erlotinib, everolimus, gefitinib, temsirolimus, and thalidomide) either had no effect on median lifespan or were toxic, 5 compounds (all-trans retinoic acid, berberine, fisetin, propranolol, and ritonavir) extended lifespan in Caenorhabditis elegans. These computer predictions yield a remarkable positive hit rate of 30%. Deeper genetic characterization of the longevity effects of one of the most efficacious compounds, the endogenous signaling ligand all-trans retinoic acid (atRA, designated tretinoin in medical products), demonstrated a requirement for the regulatory kinases AKT-1 and AKT-2. While the canonical Akt-target FOXO/DAF-16 was largely dispensable, other conserved Akt-targets (Nrf2/SKN-1 and HSF1/HSF-1), as well as the conserved catalytic subunit of AMPK AAK-2, were all necessary for longevity extension by atRA. Our results highlight the potential of combining computational prediction of longevity interventions with the power of nematode functional genetics and underscore that the manipulation of a conserved metabolic regulatory circuit by co-opting endogenous signaling molecules is a powerful approach for discovering aging interventions.

As an animal matures, its neural circuit undergoes alterations, yet the developmental changes in intracellular organelles to facilitate these changes is less understood. Using 3D electron microscopy and deep learning, the study develops semi-automated methods for reconstructing mitochondria in C. elegans and collected mitochondria reconstructions from normal reproductive stages and dauer, enabling comparative study on mitochondria structure within the neuromuscular system. It is found that various mitochondria structural properties in neurons correlate with synaptic connections and these properties are preserved across development in different neural circuits. To test the necessity of these universal mitochondria properties, the study examines the behavior in drp-1 mutants with impaired mitochondria fission and discovers that it causes behavioral deficits. Moreover, it is observed that dauer neurons display distinctive mitochondrial features, and mitochondria in dauer muscles exhibit unique reticulum-like structure. It is proposed that these specialized mitochondria structures may serve as an adaptive mechanism to support stage-specific behavioral and physiological needs.

Transcription factors (TFs) are essential for neuronal identity, yet their potential non-cell-autonomous functions remain largely unexplored. Here, we uncover both cell- and non-cell-autonomous roles for the conserved terminal selector UNC-3 in C. elegans motor neurons (MNs). UNC-3 is an ortholog of human EBF3, mutations in which cause a severe neurodevelopmental syndrome. Single-cell RNA sequencing of cholinergic MNs, which express unc-3, and downstream GABA MNs, which do not, revealed that unc-3 loss disrupts neuronal identity in distinct ways across MN classes. Four cholinergic MN classes lose their molecular identity entirely, whereas the AS class retains it partially, illuminating terminal selector-driven neuronal diversification processes. Integrated transcriptomic and genomic analyses uncovered a dual cell-autonomous role for UNC-3 as both a direct activator and repressor of neuron-type-specific genes in cholinergic MNs, including repression of alternate neurotransmitter programs. Unexpectedly, unc-3 loss also caused widespread transcriptional, morphological, and connectivity defects in downstream GABA MNs. Mechanistically, these non-cell-autonomous effects are mediated by cholinergic neurotransmission and include activation of the pro-regenerative bZIP TF CEBP-1 (C/EBP) and dysregulation of UNC-6/Netrin signaling. These findings redefine terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, offering a mechanistic framework for understanding EBF3 syndrome pathogenesis.

Ciliopathies are often caused by defects in the ciliary microtubule core. Glutamylation is abundant in cilia, and its dysregulation may contribute to ciliopathies and neurodegeneration. Mutation of the deglutamylase CCP1 causes infantile-onset neurodegeneration. In C. elegans, ccpp-1 loss causes age-related ciliary degradation that is suppressed by a mutation in the conserved NEK10 homolog nekl-4. NEKL-4 is absent from cilia, yet it negatively regulates ciliary stability via an unknown, glutamylation-independent mechanism. We show that NEKL-4 was mitochondria-associated. Additionally, nekl-4 mutants had longer mitochondria, a higher baseline mitochondrial oxidation state, and suppressed ccpp-1∆ mutant lifespan extension in response to oxidative stress. A kinase-dead nekl-4(KD) mutant ectopically localized to ccpp-1∆ cilia and rescued degenerating microtubule doublet B-tubules. A nondegradable nekl-4(PEST∆) mutant resembled the ccpp-1∆ mutant with dye-filling defects and B-tubule breaks. The nekl-4(PEST∆) Dyf phenotype was suppressed by mutation in the depolymerizing kinesin-8 KLP-13/KIF19A. We conclude that NEKL-4 influences ciliary stability by activating ciliary kinesins and promoting mitochondrial homeostasis.

Neurons rely on mitochondria for an efficient supply of ATP and other metabolites. However, while neurons are highly elongated, mitochondria are discrete and limited in number. Due to the slow rates of diffusion over long distances it follows that neurons would benefit from an ability to control the distribution of mitochondria to sites of high metabolic activity, such as synapses. It is assumed that neurons' possess this capacity, but ultrastructural data over substantial portions of a neuron's extent that would allow for tests of such hypotheses are scarce. Here, we mined the Caenorhabditis elegans electron micrographs of John White and Sydney Brenner and found systematic differences in average mitochondrial length (ranging from 1.3 to 2.4 μm), volume density (3.7% to 6.5%) and diameter (0.18 to 0.24 μm) between neurons of different neurotransmitter type and function, but found limited differences in mitochondrial morphometrics between axons and dendrites of the same neurons. Analyses of distance intervals found mitochondria to be distributed randomly with respect to presynaptic specializations, and an indication that mitochondria were displaced from postsynaptic specializations. Presynaptic specializations were primarily localized to varicosities, but mitochondria were no more likely to be found in synaptic varicosities than non-synaptic varicosities. Consistently, mitochondrial volume density was no greater in varicosities with synapses. Therefore, beyond the capacity to disperse mitochondria throughout their length, at least in C. elegans, fine caliber neurons manifest limited sub-cellular control of mitochondrial size and distribution.

Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate the extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here, we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.

Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in Caenorhabditis elegans Posterior Lateral Mechanosensory neurons. The synaptic vesicle anterograde kinesin motor UNC-104/KIF1A regulates this vesicle behavior at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM-4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behavior at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight toward the asynaptic end. This suggests that the neuron optimizes UNC-104 levels on the cargo surface to maximize the fraction of vesicles entering the branch and minimize the fraction going to the asynaptic end.

As an animal matures, its neural circuit undergoes alterations, leading to changes in both neuronal morphology and the connectivity between neurons. However, less is known about the mitochondrial structure changes across development to facilitate these changes, while mitochondria are highly dynamic organelles related to neuronal development. Here, we attempt to answer this question with a model organism C . elegans using 3D electron microscopy (EM). We developed semi-automated methods for reconstructing mitochondria in C . elegans EM images using deep learning. Consequently, we collected mitochondria reconstructions from normal reproductive stages and dauer, enabling comparative study on mitochondrial morphology and spatial organization within the neuromuscular system across different stages. We have identified that the mitochondria structural properties in neurons are correlated with synaptic properties. Neuronal compartments have distinct roles, leading axonal mitochondria to differ morphologically from dendritic mitochondria. We tested this by analyzing behavior in animals with a mutation in drp-1 , required for proper mitochondrial fission, confirming that compartment-specific mitochondrial morphology is vital for effective functioning of synapses. Given that these functions are essential throughout development, the structural properties of mitochondria are preserved across development. We report that dauer inter- and motor neurons, predominantly cholinergic and glutamatergic, show distinctive mitochondrial structure and increased mitochondria density. In addition, mitochondria in dauer body wall muscles exhibit distinctive reticulum-like structure. We propose that the stage-specific mitochondrial structure observed in C . elegans dauer may constitute an adaptive mechanism to support stage-specific behavioral and physiological characteristics. ### Competing Interest Statement The authors have declared no competing interest.

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.1,2 Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.3,4 Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans5,6 and in EVs in the mouse embryonic node and isolated from human urine.7,8 In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.6,9 Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.9,10 Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Aging is characterized by declining health that results in decreased cellular resilience and neuromuscular function. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of pharmacological anti -aging interventions. Here we assessed swimming performance as well as survival under thermal and oxidative stress across a nematode genetic diversity test panel to evaluate health effects for three compounds previously studied in the Caenorhabditis Intervention Testing Program and thought to promote longevity in different ways - NP1 (nitrophenyl piperazine-containing compound 1), propyl gallate, and resveratrol. Overall, we find the relationships among median lifespan, oxidative stress resistance, thermotolerance, and mobility vigor to be complex. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species -specific. In this study, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Notably, some compounds exert strong impact on some health measures without an equally strong impact on lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible anti -aging interventions, with data underscoring how personalized treatments might be required to optimize health benefits.

A fundamental question in neurodevelopmental biology is how flexibly the nervous system changes during development. To address this, we reconstructed the chemical connectome of dauer, an alternative developmental stage of nematodes with distinct behavioral characteristics, by volumetric reconstruction and automated synapse detection using deep learning. With the basic architecture of the nervous system preserved, structural changes in neurons, large or small, were closely associated with connectivity changes, which in turn evoked dauer-specific behaviors such as nictation. Graph theoretical analyses revealed significant dauer-specific rewiring of sensory neuron connectivity and increased clustering within motor neurons in the dauer connectome. We suggest that the nervous system in the nematode has evolved to respond to harsh environments by developing a quantitatively and qualitatively differentiated connectome.