Sex-specific developmental gene expression atlas unveils dimorphic gene networks in C. elegans.

Sex-specific traits and behaviors emerge during development by the acquisition of unique properties in the nervous system of each sex. However, the genetic events responsible for introducing these sex-specific features remain poorly understood. In this study, we create a comprehensive gene expression atlas of pure populations of hermaphrodites and males of the nematode Caenorhabditis elegans across development. We discover numerous differentially expressed genes, including neuronal gene families like transcription factors, neuropeptides, and G protein-coupled receptors. We identify INS-39, an insulin-like peptide, as a prominent male-biased gene expressed specifically in ciliated sensory neurons. We show that INS-39 serves as an early-stage male marker, facilitating the effective isolation of males in high-throughput experiments. Through complex and sex-specific regulation, ins-39 plays pleiotropic sexually dimorphic roles in various behaviors, while also playing a shared, dimorphic role in early life stress. This study offers a comparative sexual and developmental gene expression database for C. elegans. Furthermore, it highlights conserved genes that may underlie the sexually dimorphic manifestation of different human diseases.

SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans.

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

Nematicidal Activity of 20-Deoxyingenol-3-angelate from Euphorbia peplus Latex Through Protein Kinase C Isotype TPA-1.

The latex of Euphorbia peplus and its major component 20-deoxyingenol-3-angelate (DI3A) displayed significant nematicidal activity against Caenorhabditis elegans and Panagrellus redivivus. DI3A treatment inhibited the growth and development of nematodes and caused significantly negative effects on locomotion behavior, reproduction, and accumulation of reactive oxygen species. Transcriptome analysis indicated that differential expression genes in DI3A-treated C. elegans were mainly associated with the metabolism, growth, and development process, which were further confirmed by RT-qPCR experiments. The expression level of TPA-1 gene encoding a protein kinase C isotype was obviously upregulated by DI3A treatment, and knockdown of TPA-1 by RNAi technology in the nematode could relieve the growth-inhibitory effect of DI3A. Metabolic analysis indicated that DI3A was hardly metabolized by C. elegans, but a glycosylated indole derivative was specifically accumulated likely due to the activation of detoxification. Overall, our findings suggested that DI3A from E. peplus latex exerted a potent nematicidal effect through the gene TPA-1, which provides a potential target for the control of nematodes and also suggests the potential application value of E. peplus latex and DI3A as botanical nematicides.

Dynamics of miRNA accumulation during C. elegans larval development.

Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.

PAR-4/LKB1 prevents intestinal hyperplasia by restricting endoderm specification in Caenorhabditis elegans embryos.

The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation.

The G protein alpha chaperone and guanine-nucleotide exchange factor RIC-8 regulates cilia morphogenesis in Caenorhabditis elegans sensory neurons.

Heterotrimeric G (αßγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset of Caenorhabditis elegans sensory neurons. Consistent with its role in ciliogenesis, C. elegans RIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite defects in AWC cilia morphology, ags-3 null mutants exhibit normal chemotaxis toward benzaldehyde unlike odr-3 mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction.

Individual cell types in C. elegans age differently and activate distinct cell-protective responses.

Aging is characterized by a global decline in physiological function. However, by constructing a complete single-cell gene expression atlas, we find that Caenorhabditis elegans aging is not random in nature but instead is characterized by coordinated changes in functionally related metabolic, proteostasis, and stress-response genes in a cell-type-specific fashion, with downregulation of energy metabolism being the only nearly universal change. Similarly, the rates at which cells age differ significantly between cell types. In some cell types, aging is characterized by an increase in cell-to-cell variance, whereas in others, variance actually decreases. Remarkably, multiple resilience-enhancing transcription factors known to extend lifespan are activated across many cell types with age; we discovered new longevity candidates, such as GEI-3, among these. Together, our findings suggest that cells do not age passively but instead react strongly, and individualistically, to events that occur during aging. This atlas can be queried through a public interface.

EAT-2 attenuates C. elegans development via metabolic remodeling in a chemically defined food environment.

Dietary intake and nutrient composition regulate animal growth and development; however, the underlying mechanisms remain elusive. Our previous study has shown that either the mammalian deafness homolog gene tmc-1 or its downstream acetylcholine receptor gene eat-2 attenuates Caenorhabditis elegans development in a chemically defined food CeMM (C. elegans maintenance medium) environment, but the underpinning mechanisms are not well-understood. Here, we found that, in CeMM food environment, for both eat-2 and tmc-1 fast-growing mutants, several fatty acid synthesis and elongation genes were highly expressed, while many fatty acid ß-oxidation genes were repressed. Accordingly, dietary supplementation of individual fatty acids, such as monomethyl branch chain fatty acid C17ISO, palmitic acid and stearic acid significantly promoted wild-type animal development on CeMM, and mutations in either C17ISO synthesis gene elo-5 or elo-6 slowed the rapid growth of eat-2 mutant. Tissue-specific rescue experiments showed that elo-6 promoted animal development mainly in the intestine. Furthermore, transcriptome and metabolome analyses revealed that elo-6/C17ISO regulation of C. elegans development may be correlated with up-regulating expression of cuticle synthetic and hedgehog signaling genes, as well as promoting biosynthesis of amino acids, amino acid derivatives and vitamins. Correspondingly, we found that amino acid derivative S-adenosylmethionine and its upstream metabolite methionine sulfoxide significantly promoted C. elegans development on CeMM. This study demonstrated that C17ISO, palmitic acid, stearic acid, S-adenosylmethionine and methionine sulfoxide inhibited or bypassed the TMC-1 and EAT-2-mediated attenuation of development via metabolic remodeling, and allowed the animals to adapt to the new nutritional niche.

pop-1/TCF, ref-2/ZIC and T-box factors regulate the development of anterior cells in the C. elegans embryo.

Patterning of the anterior-posterior axis is fundamental to animal development. The Wnt pathway plays a major role in this process by activating the expression of posterior genes in animals from worms to humans. This observation raises the question of whether the Wnt pathway or other regulators control the expression of the many anterior-expressed genes. We found that the expression of five anterior-specific genes in Caenorhabditis elegans embryos depends on the Wnt pathway effectors pop-1/TCF and sys-1/ß-catenin. We focused further on one of these anterior genes, ref-2/ZIC, a conserved transcription factor expressed in multiple anterior lineages. Live imaging of ref-2 mutant embryos identified defects in cell division timing and position in anterior lineages. Cis-regulatory dissection identified three ref-2 transcriptional enhancers, one of which is necessary and sufficient for anterior-specific expression. This enhancer is activated by the T-box transcription factors TBX-37 and TBX-38, and surprisingly, concatemerized TBX-37/38 binding sites are sufficient to drive anterior-biased expression alone, despite the broad expression of TBX-37 and TBX-38. Taken together, our results highlight the diverse mechanisms used to regulate anterior expression patterns in the embryo.

A male pheromone that improves the quality of the oogenic germline.

Pheromones exchanged by conspecifics are a major class of chemical signals that can alter behavior, physiology, and development. In particular, males and females communicate with potential mating partners via sex pheromones to promote reproductive success. Physiological and developmental mechanisms by which pheromones facilitate progeny production remain largely enigmatic. Here, we describe how a Caenorhabditis elegans male pheromone, ascr#10, improves the oogenic germline. Before most signs of aging become evident, C. elegans hermaphrodites start producing lower-quality gametes characterized by abnormal morphology, increased rates of chromosomal nondisjunction, and higher penetrance of deleterious alleles. We show that exposure to the male pheromone substantially ameliorates these defects and reduces embryonic lethality. ascr#10 stimulates proliferation of germline precursor cells in adult hermaphrodites. Coupled to the greater precursor supply is increased physiological germline cell death, which is required to improve oocyte quality in older mothers. The hermaphrodite germline is sensitive to the pheromone only during a time window, comparable in duration to a larval stage, in early adulthood. During this period, prereproductive adults assess the suitability of the environment for reproduction. Our results identify developmental events that occur in the oogenic germline in response to a male pheromone. They also suggest that the opposite effects of the pheromone on gamete quality and maternal longevity arise from competition over resource allocation between soma and the germline.

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.

SETDB1-like MET-2 promotes transcriptional silencing and development independently of its H3K9me-associated catalytic activity.

Transcriptionally silenced heterochromatin bearing methylation of histone H3 on lysine 9 (H3K9me) is critical for maintaining organismal viability and tissue integrity. Here we show that in addition to ensuring H3K9me, MET-2, the Caenorhabditis elegans homolog of the SETDB1 histone methyltransferase, has a noncatalytic function that contributes to gene repression. Subnuclear foci of MET-2 coincide with H3K9me deposition, yet these foci also form when MET-2 is catalytically deficient and H3K9me is compromised. Whereas met-2 deletion triggers a loss of silencing and increased histone acetylation, foci of catalytically deficient MET-2 maintain silencing of a subset of genes, blocking acetylation on H3K9 and H3K27. In normal development, this noncatalytic MET-2 activity helps to maintain fertility. Under heat stress MET-2 foci disperse, coinciding with increased acetylation and transcriptional derepression. Our study suggests that the noncatalytic, focus-forming function of this SETDB1-like protein and its intrinsically disordered cofactor LIN-61 is physiologically relevant.

Dietary S. maltophilia induces supersized lipid droplets by enhancing lipogenesis and ER-LD contacts in C. elegans.

Dietary and symbiotic bacteria can exert powerful influence on metazoan lipid metabolism. Recent studies have emerged that microbiota have a role in animal obesity and related health disorders, but the mechanisms by which bacteria influence lipid storage in their host are unknown. To reduce the complexity of the relationship between gut microbiota and the host, Caenorhabditis elegans (C. elegans) has been chosen as a model organism to study interspecies interaction. Here, we demonstrate that feeding C. elegans with an opportunistic pathogenic bacterium Stenotrophomonas maltophilia (S. maltophilia) retards growth and promotes excessive neutral lipid storage. Gene expression analysis reveals that dietary S. maltophilia induces a lipogenic transcriptional response that includes the SREBP ortholog SBP-1, and fatty acid desaturases FAT-6 and FAT-7. Live imaging and ultrastructural analysis suggest that excess neutral lipid is stored in greatly expanded lipid droplets (LDs), as a result of enhanced endoplasmic reticulum (ER)-LD interaction. We also report that loss of function mutations in dpy-9 in C. elegans confers resistance to S. maltophilia. Dietary S. maltophilia induces supersized LDs by enhancing lipogenesis and ER-LD contacts in C. elegans. This work delineates a new model for understanding microbial regulation of metazoan physiology.

Grainyhead 1 acts as a drug-inducible conserved transcriptional regulator linked to insulin signaling and lifespan.

Aging is impacted by interventions across species, often converging on metabolic pathways. Transcription factors regulate longevity yet approaches for their pharmacological modulation to exert geroprotection remain sparse. We show that increased expression of the transcription factor Grainyhead 1 (GRH-1) promotes lifespan and pathogen resistance in Caenorhabditis elegans. A compound screen identifies FDA-approved drugs able to activate human GRHL1 and promote nematodal GRH-1-dependent longevity. GRHL1 activity is regulated by post-translational lysine methylation and the phosphoinositide (PI) 3-kinase C2A. Consistently, nematodal longevity following impairment of the PI 3-kinase or insulin/IGF-1 receptor requires grh-1. In BXD mice, Grhl1 expression is positively correlated with lifespan and insulin sensitivity. In humans, GRHL1 expression positively correlates with insulin receptor signaling and also with lifespan. Fasting blood glucose levels, including in individuals with type 2 diabetes, are negatively correlated with GRHL1 expression. Thereby, GRH-1/GRHL1 is identified as a pharmacologically malleable transcription factor impacting insulin signaling and lifespan.

Principles of microtubule polarity in linear cells.

The microtubule cytoskeleton is critical for maintenance of long and long-lived neurons. The overlapping array of microtubules extends from the major site of synthesis in the cell body to the far reaches of axons and dendrites. New materials are transported from the cell body along these neuronal roads by motor proteins, and building blocks and information about the state of affairs in other parts of the cell are returned by motors moving in the opposite direction. As motor proteins walk only in one direction along microtubules, the combination of correct motor and correctly oriented microtubules is essential for moving cargoes in the right direction. In this review, we focus on how microtubule polarity is established and maintained in neurons. At first thought, it seems that figuring out how microtubules are organized in neurons should be simple. After all, microtubules are essentially sticks with a slow-growing minus end and faster-growing plus end, and arranging sticks within the constrained narrow tubes of axons and dendrites should be straightforward. It is therefore quite surprising how many mechanisms contribute to making sure they are arranged in the correct polarity. Some of these mechanisms operate to generate plus-end-out polarity of axons, and others control mixed or minus-end-out dendrites.

Phosphoglycolate phosphatase homologs act as glycerol-3-phosphate phosphatase to control stress and healthspan in C. elegans.

Metabolic stress due to nutrient excess and lipid accumulation is at the root of many age-associated disorders and the identification of therapeutic targets that mimic the beneficial effects of calorie restriction has clinical importance. Here, using C. elegans as a model organism, we study the roles of a recently discovered enzyme at the heart of metabolism in mammalian cells, glycerol-3-phosphate phosphatase (G3PP) (gene name Pgp) that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol. We identify three Pgp homologues in C. elegans (pgph) and demonstrate in vivo that their protein products have G3PP activity, essential for glycerol synthesis. We demonstrate that PGPH/G3PP regulates the adaptation to various stresses, in particular hyperosmolarity and glucotoxicity. Enhanced G3PP activity reduces fat accumulation, promotes healthy aging and acts as a calorie restriction mimetic at normal food intake without altering fertility. Thus, PGP/G3PP can be considered as a target for age-related metabolic disorders.

The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function.

Tetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in metazoans. While much is known about their biochemical properties, the in vivo functions and distribution patterns of different tetraspanin proteins are less understood. Previous studies have shown that two paralogous tetraspanins that belong to the TspanC8 subfamily, TSP-12 and TSP-14, function redundantly to promote both Notch signaling and bone morphogenetic protein (BMP) signaling in C. elegans. TSP-14 has two isoforms, TSP-14A and TSP-14B, where TSP-14B has an additional 24 amino acids at its N-terminus compared to TSP-14A. By generating isoform specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B share distinct as well as overlapping expression patterns and functions. While TSP-14A functions redundantly with TSP-12 to regulate body size and embryonic and vulva development, TSP-14B primarily functions redundantly with TSP-12 to regulate postembryonic mesoderm development. Importantly, TSP-14A and TSP-14B exhibit distinct subcellular localization patterns. TSP-14A is localized apically and on early and late endosomes. TSP-14B is localized to the basolateral cell membrane. We further identified a di-leucine motif within the N-terminal 24 amino acids of TSP-14B that serves as a basolateral membrane targeting sequence, and showed that the basolateral membrane localization of TSP-14B is important for its function. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in C. elegans, and demonstrates the importance of dissecting the functions of these important proteins in an intact living organism.