Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Systematic mapping of organism-scale gene-regulatory networks in aging using population asynchrony.

In aging, physiologic networks decline in function at rates that differ between individuals, producing a wide distribution of lifespan. Though 70% of human lifespan variance remains unexplained by heritable factors, little is known about the intrinsic sources of physiologic heterogeneity in aging. To understand how complex physiologic networks generate lifespan variation, new methods are needed. Here, we present Asynch-seq, an approach that uses gene-expression heterogeneity within isogenic populations to study the processes generating lifespan variation. By collecting thousands of single-individual transcriptomes, we capture the Caenorhabditis elegans "pan-transcriptome"-a highly resolved atlas of non-genetic variation. We use our atlas to guide a large-scale perturbation screen that identifies the decoupling of total mRNA content between germline and soma as the largest source of physiologic heterogeneity in aging, driven by pleiotropic genes whose knockdown dramatically reduces lifespan variance. Our work demonstrates how systematic mapping of physiologic heterogeneity can be applied to reduce inter-individual disparities in aging.

Dissecting the functional organization of the C. elegans serotonergic system at whole-brain scale.

Serotonin influences many aspects of animal behavior. But how serotonin acts on its diverse receptors across the brain to modulate global activity and behavior is unknown. Here, we examine how serotonin release in C. elegans alters brain-wide activity to induce foraging behaviors, like slow locomotion and increased feeding. Comprehensive genetic analyses identify three core serotonin receptors (MOD-1, SER-4, and LGC-50) that induce slow locomotion upon serotonin release and others (SER-1, SER-5, and SER-7) that interact with them to modulate this behavior. SER-4 induces behavioral responses to sudden increases in serotonin release, whereas MOD-1 induces responses to persistent release. Whole-brain imaging reveals widespread serotonin-associated brain dynamics, spanning many behavioral networks. We map all sites of serotonin receptor expression in the connectome, which, together with synaptic connectivity, helps predict which neurons show serotonin-associated activity. These results reveal how serotonin acts at defined sites across a connectome to modulate brain-wide activity and behavior.

Molecular topography of an entire nervous system.

We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses distinct codes of ∼23 neuropeptide genes and ∼36 neuropeptide receptors, delineating a complex and expansive "wireless" signaling network. To demonstrate the utility of this comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression and (2) reveal adhesion proteins with potential roles in process placement and synaptic specificity. Our expression data are available at https://cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity, and function throughout the C. elegans nervous system.

Three Rules Explain Transgenerational Small RNA Inheritance in C. elegans.

Experiences trigger transgenerational small RNA-based responses in C. elegans nematodes. Dedicated machinery ensures that heritable effects are reset, but how the responses segregate in the population is unknown. We show that isogenic individuals differ dramatically in the persistence of transgenerational responses. By examining lineages of more than 20,000 worms, three principles emerge: (1) The silencing each mother initiates is distributed evenly among her descendants; heritable RNAi dissipates but is uniform in every generation. (2) Differences between lineages arise because the mothers that initiate heritable responses stochastically assume different "inheritance states" that determine the progeny's fate. (3) The likelihood that an RNAi response would continue to be inherited increases the more generations it lasts. The inheritance states are determined by HSF-1, which regulates silencing factors and, accordingly, small RNA levels. We found that, based on the parents' inheritance state, the descendants' developmental rate in response to stress can be predicted.

Does Autophagy Promote Longevity? It Depends.

Zhou et al. challenge the well-known beneficial effect of autophagy in promoting longevity. Evidence presented demonstrate that autophagy induction coupled with increased mitochondrial permeability is detrimental to organismal health in both the nematode Caenorhabditis elegans and mammals.

Next-Gen Learning: The C. elegans Approach.

In this issue, Moore et al. and Posner et al., provide evidence for how the activity of the nervous system in C. elegans results in gene expression changes in the germline to pass on parental experiences and learned behavior to their progeny.

Piwi/PRG-1 Argonaute and TGF-ß Mediate Transgenerational Learned Pathogenic Avoidance.

The ability to inherit learned information from parents could be evolutionarily beneficial, enabling progeny to better survive dangerous conditions. We discovered that, after C. elegans have learned to avoid the pathogenic bacteria Pseudomonas aeruginosa (PA14), they pass this learned behavior on to their progeny, through either the male or female germline, persisting through the fourth generation. Expression of the TGF-ß ligand DAF-7 in the ASI sensory neurons correlates with and is required for this transgenerational avoidance behavior. Additionally, the Piwi Argonaute homolog PRG-1 and its downstream molecular components are required for transgenerational inheritance of both avoidance behavior and ASI daf-7 expression. Animals whose parents have learned to avoid PA14 display a PA14 avoidance-based survival advantage that is also prg-1 dependent, suggesting an adaptive response. Transgenerational epigenetic inheritance of pathogenic learning may optimize progeny decisions to increase survival in fluctuating environmental conditions.

Neuronal Small RNAs Control Behavior Transgenerationally.

It is unknown whether the activity of the nervous system can be inherited. In Caenorhabditis elegans nematodes, parental responses can transmit heritable small RNAs that regulate gene expression transgenerationally. In this study, we show that a neuronal process can impact the next generations. Neurons-specific synthesis of RDE-4-dependent small RNAs regulates germline amplified endogenous small interfering RNAs (siRNAs) and germline gene expression for multiple generations. Further, the production of small RNAs in neurons controls the chemotaxis behavior of the progeny for at least three generations via the germline Argonaute HRDE-1. Among the targets of these small RNAs, we identified the conserved gene saeg-2, which is transgenerationally downregulated in the germline. Silencing of saeg-2 following neuronal small RNA biogenesis is required for chemotaxis under stress. Thus, we propose a small-RNA-based mechanism for communication of neuronal processes transgenerationally.

A Cold-Sensing Receptor Encoded by a Glutamate Receptor Gene.

In search of the molecular identities of cold-sensing receptors, we carried out an unbiased genetic screen for cold-sensing mutants in C. elegans and isolated a mutant allele of glr-3 gene that encodes a kainate-type glutamate receptor. While glutamate receptors are best known to transmit chemical synaptic signals in the CNS, we show that GLR-3 senses cold in the peripheral sensory neuron ASER to trigger cold-avoidance behavior. GLR-3 transmits cold signals via G protein signaling independently of its glutamate-gated channel function, suggesting GLR-3 as a metabotropic cold receptor. The vertebrate GLR-3 homolog GluK2 from zebrafish, mouse, and human can all function as a cold receptor in heterologous systems. Mouse DRG sensory neurons express GluK2, and GluK2 knockdown in these neurons suppresses their sensitivity to cold but not cool temperatures. Our study identifies an evolutionarily conserved cold receptor, revealing that a central chemical receptor unexpectedly functions as a thermal receptor in the periphery.

Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Protein Sequence Editing of SKN-1A/Nrf1 by Peptide:N-Glycanase Controls Proteasome Gene Expression.

The proteasome mediates selective protein degradation and is dynamically regulated in response to proteotoxic challenges. SKN-1A/Nrf1, an endoplasmic reticulum (ER)-associated transcription factor that undergoes N-linked glycosylation, serves as a sensor of proteasome dysfunction and triggers compensatory upregulation of proteasome subunit genes. Here, we show that the PNG-1/NGLY1 peptide:N-glycanase edits the sequence of SKN-1A protein by converting particular N-glycosylated asparagine residues to aspartic acid. Genetically introducing aspartates at these N-glycosylation sites bypasses the requirement for PNG-1/NGLY1, showing that protein sequence editing rather than deglycosylation is key to SKN-1A function. This pathway is required to maintain sufficient proteasome expression and activity, and SKN-1A hyperactivation confers resistance to the proteotoxicity of human amyloid beta peptide. Deglycosylation-dependent protein sequence editing explains how ER-associated and cytosolic isoforms of SKN-1 perform distinct cytoprotective functions corresponding to those of mammalian Nrf1 and Nrf2. Thus, we uncover an unexpected mechanism by which N-linked glycosylation regulates protein function and proteostasis.

Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation.

Bioactive molecules can pass between microbiota and host to influence host cellular functions. However, general principles of interspecies communication have not been discovered. We show here in C. elegans that nitric oxide derived from resident bacteria promotes widespread S-nitrosylation of the host proteome. We further show that microbiota-dependent S-nitrosylation of C. elegans Argonaute protein (ALG-1)-at a site conserved and S-nitrosylated in mammalian Argonaute 2 (AGO2)-alters its function in controlling gene expression via microRNAs. By selectively eliminating nitric oxide generation by the microbiota or S-nitrosylation in ALG-1, we reveal unforeseen effects on host development. Thus, the microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development. Our findings suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.

Plasticity of the Electrical Connectome of C. elegans.

The specific patterns and functional properties of electrical synapses of a nervous system are defined by the neuron-specific complement of electrical synapse constituents. We systematically examined the molecular composition of the electrical connectome of the nematode C. elegans through a genome- and nervous-system-wide analysis of the expression patterns of the invertebrate electrical synapse constituents, the innexins. We observe highly complex combinatorial expression patterns throughout the nervous system and found that these patterns change in a strikingly neuron-type-specific manner throughout the nervous system when animals enter an insulin-controlled diapause arrest stage under harsh environmental conditions, the dauer stage. By analyzing several individual synapses, we demonstrate that dauer-specific electrical synapse remodeling is responsible for specific aspects of the altered locomotory and chemosensory behavior of dauers. We describe an intersectional gene regulatory mechanism involving terminal selector and FoxO transcription factors mediating dynamic innexin expression plasticity in a neuron-type- and environment-specific manner.

ASICs Mediate Food Responses in an Enteric Serotonergic Neuron that Controls Foraging Behaviors.

Animals must respond to the ingestion of food by generating adaptive behaviors, but the role of gut-brain signaling in behavioral regulation is poorly understood. Here, we identify conserved ion channels in an enteric serotonergic neuron that mediate its responses to food ingestion and decipher how these responses drive changes in foraging behavior. We show that the C. elegans serotonergic neuron NSM acts as an enteric sensory neuron that acutely detects food ingestion. We identify the novel and conserved acid-sensing ion channels (ASICs) DEL-7 and DEL-3 as NSM-enriched channels required for feeding-dependent NSM activity, which in turn drives slow locomotion while animals feed. Point mutations that alter the DEL-7 channel change NSM dynamics and associated behavioral dynamics of the organism. This study provides causal links between food ingestion, molecular and physiological properties of an enteric serotonergic neuron, and adaptive feeding behaviors, yielding a new view of how enteric neurons control behavior.

Intestinal immunity in C. elegans is activated by pathogen effector-triggered aggregation of the guard protein TIR-1 on lysosome-related organelles.

Toll/interleukin-1/resistance (TIR)-domain proteins with enzymatic activity are essential for immunity in plants, animals, and bacteria. However, it is not known how these proteins function in pathogen sensing in animals. We discovered that the lone enzymatic TIR-domain protein in the nematode C. elegans (TIR-1, homolog of mammalian sterile alpha and TIR motif-containing 1 [SARM1]) was strategically expressed on the membranes of a specific intracellular compartment called lysosome-related organelles. The positioning of TIR-1 on lysosome-related organelles enables intestinal epithelial cells in the nematode C. elegans to survey for pathogen effector-triggered host damage. A virulence effector secreted by the bacterial pathogen Pseudomonas aeruginosa alkalinized and condensed lysosome-related organelles. This pathogen-induced morphological change in lysosome-related organelles triggered TIR-1 multimerization, which engaged its intrinsic NAD+ hydrolase (NADase) activity to activate the p38 innate immune pathway and protect the host against microbial intoxication. Thus, TIR-1 is a guard protein in an effector-triggered immune response, which enables intestinal epithelial cells to survey for pathogen-induced host damage.

Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.

The THO Complex Coordinates Transcripts for Synapse Development and Dopamine Neuron Survival.

Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C. elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.

mTOR Regulates Phase Separation of PGL Granules to Modulate Their Autophagic Degradation.

The assembly of phase-separated structures is thought to play an important role in development and disease, but little is known about the regulation and function of phase separation under physiological conditions. We showed that during C. elegans embryogenesis, PGL granules assemble via liquid-liquid phase separation (LLPS), and their size and biophysical properties determine their susceptibility to autophagic degradation. The receptor SEPA-1 promotes LLPS of PGL-1/-3, while the scaffold protein EPG-2 controls the size of PGL-1/-3 compartments and converts them into less dynamic gel-like structures. Under heat-stress conditions, mTORC1-mediated phosphorylation of PGL-1/-3 is elevated and PGL-1/-3 undergo accelerated phase separation, forming PGL granules that are resistant to autophagic degradation. Significantly, accumulation of PGL granules is an adaptive response to maintain embryonic viability during heat stress. We revealed that mTORC1-mediated LLPS of PGL-1/-3 acts as a switch-like stress sensor, coupling phase separation to autophagic degradation and adaptation to stress during development.