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Hydrogen sulfide (H₂S) acts as an energy source, a toxin, and a gasotransmitter across diverse biological contexts. We use the robust locomotory responses of Caenorhabditis elegans to high levels of H₂S to elucidate the molecular mechanisms underlying its acute and adaptive responses. We find that the H₂S-evoked behavioral response is shaped by multiple environmental factors including oxygen (O₂) levels and nutritional state and is modulated by various pathways such as insulin, TGF-β, and HIF-1 signaling, as well as by input from O₂-sensing neurons. Prolonged exposure to H₂S activates HIF-1 signaling, leading to the upregulation of stress-responsive genes, including those involved in H₂S detoxification. This promotes an adaptive state in which locomotory speed is reduced in H₂S, while responsiveness to other stimuli is preserved. In mutants deficient in HIF-1 signaling, iron storage, and detoxification mechanisms, animals display a robust initial response but rapidly enter a sleep-like behavior characterized by reduced mobility and diminished responsiveness to subsequent sensory stimuli. Furthermore, while acute production of mitochondria-derived reactive O₂ species (ROS) appears to initiate the avoidance response to H₂S, persistently high ROS promotes an adaptive state, likely by activating various stress-response pathways, without substantially compromising cellular H₂S detoxification capacity. Taken together, our study provides comprehensive molecular insights into the mechanisms through which C. elegans modulates and adapts its response to H₂S exposure.

Urochordate Ciona spp. are ideal marine model organisms for studying embryogenesis and developmental and evolutionary biology. However, the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging. This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp. The destination vector sets contained two Gateway cassettes flanked by Minos sites, allowing the N- or C-terminal tagging of a protein of interest with various fluorescent markers. In addition, we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry, a fluorescent indicator for Cas9 expression at cellular resolution. We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs. Furthermore, we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues. The genetic tools developed in this study offer a robust method for gene expression, cell tracking, and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.

The Shaker/Kv1 subfamily of voltage-gated potassium (K+) channels is essential for modulating membrane excitability. Their loss results in prolonged depolarization and excessive calcium influx. These channels have also been implicated in a variety of other cellular processes, but the underlying mechanisms remain poorly understood. Through comprehensive screening of K+ channel mutants in C. elegans, we discovered that shk-1 mutants are highly susceptible to bacterial pathogen infection and oxidative stress. This vulnerability is associated with reduced glycogen levels and substantial mitochondrial dysfunction, including decreased ATP production and dysregulated mitochondrial membrane potential under stress conditions. SHK-1 is predominantly expressed and functions in body wall muscle to maintain glycogen storage and mitochondrial homeostasis. RNA-sequencing data reveal that shk-1 mutants have decreased expression of a set of cation-transporting ATPases (CATP), which are crucial for maintaining electrochemical gradients. Intriguingly, overexpressing catp-3, but not other catp genes, restores the depolarization of mitochondrial membrane potential under stress and enhances stress tolerance in shk-1 mutants. This finding suggests that increased catp-3 levels may help restore electrochemical gradients disrupted by shk-1 deficiency, thereby rescuing the phenotypes observed in shk-1 mutants. Overall, our findings highlight a critical role for SHK-1 in maintaining stress tolerance by regulating glycogen storage, mitochondrial homeostasis, and gene expression. They also provide insights into how Shaker/Kv1 channels participate in a broad range of cellular processes.

Sensation provides a pivotal ability, allowing animals to survive in complex environments. The cues sensed by animals are represented by external stimuli and internal signals. However, the mechanisms mediating sensations in molecular and cellular level are still not well-studied. In this thesis, by using free-living nematodes C. elegans with relatively simple nerve system, we are trying to get better understandings of molecular mechanisms by which animals sense and interpret external cues and internal signals.

G protein-coupled receptors (GPCRs), as one of the major families of transmembrane proteins, participate in a variety of physiological responses to both external stimuli and internal cues. Previous studies have shown that GPCR signals are broadly involved in many processes in C. elegans, such as olfactory sensing, nociceptive responses, social behavior, pathogen responses, and mating. However, the complexity and diversity of GPCRs pose significant challenges to systematic dissection of their function as well as identification of receptor-ligand pairs which play crucial roles for animals´ sensory behaviors. Interestingly, the genome of C. elegans encodes one of the largest GPCR repertoires among any sequenced organisms, indicating a dramatical expansion and high degree of gene redundancy. To comprehensively dissect GPCR signaling in C. elegans and gain more insights into their roles in sensations, we developed an approach by employing CRISPR/Cas9-based gene editing to mutate closely related GPCRs and neuropeptide genes (internal signals) in a single strain on a genome-wide scale, resulting in disrupting nearly all the GPCR and neuropeptide genes (more than 1800 genes in total) and eliminating high degree of gene redundancy as well. Then using these two genetic libraries, we successfully identified neuropeptide (FLP -1) and cognate receptors (DMSR-4, DMSR-7 and DMSR-8) required for hypoxia-evoked locomotory responses, obtained a set of novel regulators of the pathogen-induced immune response including FMI-1 and DOP-6, and especially identified receptors (SRX-64) in AWA neurons for the volatile odorant pyrazine and redundant receptors (SRX-1, SRX-2 and SRX-3) in AWCOFF neuron for 2,3-pentanedione.

In nature, animals often experience and sense constantly changing gas environments. And human bodies also generate internal gas as gasotransmitters for signal transduction, such as CO, NO and H2S. For the mechanism governing sensory and adaptive responses to different gaseous cues, extensive studies are still needed. Here, taking advantage of the robust locomotory responses to H2S in C. elegans, we delineated the molecular mechanisms of H2S sensation and adaptation. We found that C. elegans exhibited transiently increased locomotory and turning activity as a strategy to escape the noxious H2S. The behavioral responses to H2S were modulated by a complex network of signaling pathways, ranging from cyclic GMP signaling in ciliated sensory neurons, calcineurin, nuclear hormone receptors, to the major starvation regulators such as insulin and TGF-β signaling. Prolonged exposure to H2S robustly evoked H2S detoxification and reprogrammed gene expression, where genes involved in iron homeostasis, including ftn-1 and smf-3, were robustly modified, implying that labile iron levels are affected by H2S. In addition, the roles of labile iron for modulating H2S response were further investigated by using genetic studies and chemical applications. Interestingly, the response to H2S was substantially affected by the ambient O2 levels and their prior experience in low O2 environments, suggesting an intricate interplay between O2 and H2S sensing. The crosstalk is often seen between different experiences and sensations. In addition to the interplay between O2 and H2S sensing, we found hypoxia challenge could induce food leaving behavior in C. elegans. The alteration of food behavior by hypoxia experience was independent of the known mechanisms involved in O2 response, including pathways in acute hypoxia and HIF-1 signaling for chronic hypoxia response. The robust failure of induced food avoidance in egl-3 and egl-21 mutants suggested that neuropeptidergic signaling was required for this response. And future work is needed for comprehensively understanding the roles of neuropeptide signaling in the crosstalk between hypoxia experience and food leaving behavior.

In summary, our studies shed light on the molecular and cellular mechanisms of how animals sense and interpret the signals, allowing them to survive in a complex environment niche. More specifically, 1) we demonstrated the dissection of genetic landscape of GPCR signaling through phenotypic profiling in C. elegans. And as a powerful genetic resource, our libraries can greatly expedite the analyses of GPCR signaling in multiple additional contexts. 2) we provided molecular insights into how C. elegans detects and adapts its response to H2S and modulates behaviors through ambient environment and experience. 

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

Hypoxia alters eating behavior in different animals. In C. elegans, hypoxia induces a strong food leaving response. We found that this behavior was independent of the known O 2 response mechanisms including acute O₂ sensation and HIF-1 signaling of chronic hypoxia response. Mutating egl-3 and egl-21, encoding the neuropeptide pro-protein convertase and carboxypeptidase, led to defects in hypoxia induced food leaving, suggesting that neuropeptidergic signaling was required for this response. However, we failed to identify any neuropeptide mutants that were severely defective in hypoxia induced food leaving, suggesting that multiple neuropeptides act redundantly to modulate this behavior.

Certain sets of genes are derived from gene duplication and share substantial sequence similarity in C. elegans, presenting a significant challenge in determining the specific roles of each gene and their collective impact on cellular processes. Here, we show that a collection of genes can be disrupted in a single animal via multiple rounds of CRISPR/Cas9 mediated genome editing. We found that up to three genes can be simultaneously disrupted in a single editing event with high efficiency. Our approach offers an opportunity to explore the genetic interaction and molecular underpinning of gene clusters with redundant function.