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Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the Caenorhabditis elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells, most notably in gonadal cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel sites of monoaminergic neurotransmitter uptake. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.

Summary: The auxin-inducible degron (AID) system is a broadly used tool for spatiotemporal and reversible control of protein depletion in multiple experimental model systems. AID2 technology relies on a synthetic ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA), for improved specificity and efficiency of protein degradation. Here, we provide a protocol for cost-effective 5-Ph-IAA synthesis utilizing the Suzuki coupling of 5-chloroindole and phenylboronic acid. We describe steps for evaluating the quality of lab-synthesized 5-Ph-IAA using a C. elegans AID2 tester strain. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Heat shock factor 1 (HSF-1) is a component of the heat shock response pathway that is induced by cytoplasmic proteotoxic stress. In addition to its role in stress response, HSF-1 also acts as a key regulator of the rate of organismal aging. Overexpression of HSF-1 promotes longevity in C. elegans via mechanisms that remain less understood. Moreover, genetic ablation of a negative regulator of HSF-1, termed as heat shock factor binding protein 1 (HSB-1), results in hsf-1-dependent life span extension in animals. Here we show that in the absence of HSB-1, HSF-1 acquires increased DNA binding activity to its genomic target sequence. Using RNA-Seq to compare the gene expression profiles of the hsb-1 mutant and hsf-1 overexpression strains, we found that while more than 1,500 transcripts show ≥1.5-fold upregulation due to HSF-1 overexpression, HSB-1 inhibition alters the expression of less than 500 genes in C. elegans. Roughly half of the differentially regulated transcripts in the hsb-1 mutant have altered expression also in hsf-1 overexpressing animals, with a strongly correlated fold-expression pattern between the two strains. In addition, genes that are upregulated via both HSB-1 inhibition and HSF-1 overexpression include numerous DAF-16 targets that have known functions in longevity regulation. This study identifies how HSB-1 acts as a specific regulator of the transactivation potential of HSF-1 in non-stressed conditions, thus providing a detailed understanding of the role of HSB-1/HSF-1 signaling pathway in transcriptional regulation and longevity in C. elegans.

Many cell types display the remarkable ability to alter their cellular phenotype in response to specific external or internal signals. Such phenotypic plasticity is apparent in the nematode Caenorhabditis elegans when adverse environmental conditions trigger entry into the dauer diapause stage. This entry is accompanied by structural, molecular, and functional remodeling of a number of distinct tissue types of the animal, including its nervous system. The transcription factor (TF) effectors of 3 different hormonal signaling systems, the insulin-responsive DAF-16/FoxO TF, the TGFβ-responsive DAF-3/SMAD TF, and the steroid nuclear hormone receptor, DAF-12/VDR, a homolog of the vitamin D receptor (VDR), were previously shown to be required for entering the dauer arrest stage, but their cellular and temporal focus of action for the underlying cellular remodeling processes remained incompletely understood. Through the generation of conditional alleles that allowed us to spatially and temporally control gene activity, we show here that all 3 TFs are not only required to initiate tissue remodeling upon entry into the dauer stage, as shown before, but are also continuously required to maintain the remodeled state. We show that DAF-3/SMAD is required in sensory neurons to promote and then maintain animal-wide tissue remodeling events. In contrast, DAF-16/FoxO or DAF-12/VDR act cell-autonomously to control anatomical, molecular, and behavioral remodeling events in specific cell types. Intriguingly, we also uncover non-cell autonomous function of DAF-16/FoxO and DAF-12/VDR in nervous system remodeling, indicating the presence of several insulin-dependent interorgan signaling axes. Our findings provide novel perspectives into how hormonal systems control tissue remodeling.

Temperature potently modulates various physiologic processes including organismal motility, growth rate, reproduction, and ageing. In ectotherms, longevity varies inversely with temperature, with animals living shorter at higher temperatures. Thermal effects on lifespan and other processes are ascribed to passive changes in metabolic rate, but recent evidence also suggests a regulated process. Here, we demonstrate that in response to temperature, daf-41/ZC395.10, the C. elegans homolog of p23 co-chaperone/prostaglandin E synthase-3, governs entry into the long-lived dauer diapause and regulates adult lifespan. daf-41 deletion triggers constitutive entry into the dauer diapause at elevated temperature dependent on neurosensory machinery (daf-10/IFT122), insulin/IGF-1 signaling (daf-16/FOXO), and steroidal signaling (daf-12/FXR). Surprisingly, daf-41 mutation alters the longevity response to temperature, living longer than wild-type at 25°C but shorter than wild-type at 15°C. Longevity phenotypes at 25°C work through daf-16/FOXO and heat shock factor hsf-1, while short lived phenotypes converge on daf-16/FOXO and depend on the daf-12/FXR steroid receptor. Correlatively daf-41 affected expression of DAF-16 and HSF-1 target genes at high temperature, and nuclear extracts from daf-41 animals showed increased occupancy of the heat shock response element. Our studies suggest that daf-41/p23 modulates key transcriptional changes in longevity pathways in response to temperature.