The mitochondrial unfolded protein response (UPRmt) is triggered by cells to alleviate proteotoxicity in response to metabolic stress. The ability to anticipate and prime cells against mitochondrial stress, by sensing potentially toxic changes in the external or internal environment, would provide a survival advantage. Yet, whether and how animals anticipate mitochondrial stress remains unclear. Here, we show that the Caenorhabditis elegans receptor guanylyl cyclase GCY-9 regulates neuropeptide signaling from carbon dioxide-sensing neurons to govern a noncanonical mitochondrial stress response in the intestine. This noncell autonomous stress response induces atypical mitochondrial chaperone transcription, confers mitochondrial stress resistance, and increases mitochondrial membrane potential and respiration. We show that starvation decreases GCY-9 expression and propose that the resultant cytoprotective program is launched to offset metabolic and proteotoxic risks. Thus, environmental sensing by peripheral neurons can preemptively enhance systemic mitochondrial function in response to metabolic uncertainty.

Neuronal circuit formation is controlled by secreted guidance molecules and their receptors. However, the transcriptional mechanisms underlying neuronal circuit formation are not fully understood. In an unbiased genetic screen, we identified the Nuclear Factor-Y transcription factor (NF-Y) as a key regulator of neuron fate and axon guidance. NF-Y is a highly conserved and ubiquitous complex, composed of three subunits: NF-YA, NF-YB, and NF-YC. The NF-YA subunit directly binds to regulatory regions of target genes to control their expression. Here, we show that NFYA-1 controls neurite development of multiple neuronal subtypes in Caenorhabditis elegans Using the serotonergic neurosecretory-motor neurons (NSM) neurons as a model, we discovered that NFYA-1 acts cell-autonomously to limit NSM dorsal neurite length. Further, NSM neurite overextension caused by NFYA-1 loss is suppressed by overactivating DBL-1/BMP (Bone Morphogenetic Protein) signaling. A previous study found that in Drosophila melanogaster, NF-YC regulates axon targeting of photoreceptor neurons. Thus, NF-Y likely plays a broad role in shaping neuronal circuitry across species, albeit through distinct mechanisms.

Sphingomyelin is a critical sphingolipid found in plasma membranes of metazoa that provides structural and communicative functions. Sphingomyelin synthases are key enzymes that generate sphingomyelin but their precise functions in animal development and function are not fully understood. The Caenorhabditis elegans model encodes five sphingomyelin synthases (sms-1-5). Previously, egg-laying and locomotion phenotypes were observed in an sms-1(ok2399) deletion mutant. In this study, we attempted to replicate these findings to enable mechanistic dissection of sphingomyelin function. We indeed found that the sms-1(ok2399) mutant exhibited egg-laying and locomotion defects, however, we were unable to rescue this phenotype. Further, we generated two additional sms-1 deletion mutants (rp398 and rp399) and found that their egg-laying and locomotion behaviour is not different to wild-type animals. We suggest that the sms-1(ok2399) contains a background mutation that causes behavioural deficits, and that SMS-1 loss does not overtly affect C. elegans egg-laying or locomotion.

The ongoing health of our bodies is dependent on maintaining a balance between sympathetic and parasympathetic tone. These two branches of the autonomic nervous system work in concert to maintain homeostasis,protect the body against threats,and respond to internal and external stressors. As such,the appropriate and regulated activation of these systems is essential for maintaining health. The concept of balancing excitatory and inhibitory signals is conserved across species to the nematode Caenorhabditis elegans (C. elegans). Although C. elegans lack a distinct autonomic nervous system,they still rely on excitatory and inhibitory neurochemical signaling to mediate behavioral and biochemical processes. Two key players in this balancing act are the excitatory neurotransmitter,acetylcholine,and the inhibitory neurotransmitter,gamma-aminobutyric acid (GABA). In our recent work,we have shown that coordination of these systems is essential for maintaining mitochondrial health across the body (Cornell et al.,2024).

Animals integrate environmental and internal cues to maintain homeostasis and health. The mitochondrial stress response is an essential cytoprotective mechanism, and priming its activation provides a survival advantage. Here, we show that the Caenorhabditis elegans receptor guanylyl cyclase GCY-9 regulates neuropeptide signalling from carbon dioxide sensing neurons to govern a non-canonical mitochondrial stress response in the intestine. This stress response induces atypical mitochondrial chaperone transcription, confers mitochondrial stress resistance, and increases mitochondrial membrane potential and respiration. GCY-9 loss disrupts pathogen avoidance, leading to indiscriminate feeding. We show that starvation decreases GCY-9 expression and propose that the resultant cytoprotective program is launched to offset risks associated with this behaviour. Thus, environmental sensing by peripheral neurons can pre-emptively enhance systemic mitochondrial function in response to metabolic uncertainty.

ABSTRACTMediator is an evolutionarily conserved multiprotein complex that acts as a critical coregulator of RNA polymerase II-mediated transcription. While core Mediator components are broadly required for transcription, others govern specific regulatory modules and signalling pathways. Here, we investigated the function of MDT-29/MED29 in theCaenorhabditis elegansgerm line. We found that endogenously-tagged MDT-29 is ubiquitously expressed and concentrated in discrete foci within germ cell nuclei. Functionally, depleting MDT-29 in the germ line during larval development boosted fecundity. We determined that the increase in progeny production was likely caused by a combination of an expanded germline stem cell pool and decreased germ cell apoptosis. Thus, MDT-29 may act to optimize specific gene expression programs to control distinct germ cell behaviors, providing flexibility to progeny production in certain environments.

Mediator is an evolutionarily conserved multiprotein complex that acts as a critical coregulator of RNA polymerase II-mediated transcription. While core Mediator components are broadly required for transcription, others govern specific regulatory modules and signaling pathways. Here, we investigated the function of MDT-29/MED29 in the Caenorhabditis elegans germ line. We found that endogenously tagged MDT-29 is ubiquitously expressed and concentrated in discrete foci within germ cell nuclei. Functionally, depleting MDT-29 in the germ line during larval development boosted fecundity. We determined that the increase in progeny production was likely caused by a combination of an expanded germline stem cell pool and decreased germ cell apoptosis. Thus, MDT-29 may act to optimize specific gene expression programs to control distinct germ cell behaviors, providing flexibility to progeny production in certain environments.

Mitochondria and the endoplasmic reticulum (ER) utilise unique unfolded protein response (UPR) mechanisms to maintain cellular proteostasis. Heat shock proteins (HSPs) are UPR chaperones induced by specific stressors to promote protein folding. Previous research has successfully employed transgenic reporters in Caenorhabditis elegans to report HSP induction. However, transgenic reporters are overexpressed and only show promoter regulation and not post-transcriptional regulation. To examine endogenous HSP regulation, we attempted to generate and validate endogenous reporters for mitochondrial ( HSP-60 ) and ER ( HSP-4 ) chaperones. Using CRISPR/Cas9 technology, F2A-GFP-H2B coding DNA was inserted downstream of each HSP gene and stress induction assays conducted to validate these tools. Endogenous reporters were successfully generated for hsp-4 and hsp-60 . However, GFP induction could not be detected with these endogenous reporters upon stress induction, likely due to low level expression.

Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. In response to neuronal mitochondrial stress, specific neuronal signals coordinate the systemic mitochondrial unfolded protein response (UPRmt) to promote organismal survival. Yet, whether chemical neurotransmitters are sufficient to control the UPRmt in physiological conditions is not well understood. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPRmt in the Caenorhabditis elegans intestine. GABA controls the UPRmt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPRmt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for non-autonomously regulating organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses.

Establishing a functional nervous system is a complex process requiring tightly controlled gene expression programs to achieve the correct differentiation of distinct neuronal subtypes. The molecular programs required for neurons to acquire neuron-type-specific, and core pan-neuronal features mostly rely on sequence-specific transcription factors (TFs), which recognize and bind to cis-regulatory motifs present in the promoters of target genes. Recently, we investigated the role and mode of action of the NF-Y complex, a ubiquitously expressed transcriptional master regulator, in the Caenorhabditis elegans nervous system. We found that NFYA-1 is a pervasive regulator of neuron-specific and pan-neuronal gene batteries that are essential for neuronal development and function. Furthermore, we concluded that NFYA-1 acts cell autonomously by either directly binding to conserved motifs in target gene promoter regions or indirectly by regulating other transcriptional regulators to fine-tune gene expression. However, further studies are required to fully define the impact of the NF-Y complex on nervous system regulatory networks and how NF-Y coordinates with other TFs in this regard.

Nuclear factor Y is a ubiquitous heterotrimeric transcription factor complex conserved across eukaryotes that binds to CCAAT boxes,one of the most common motifs found in gene promoters and enhancers.Over the last 30 years,research has revealed that the nuclear factor Y complex controls many aspects of brain development,including differentiation,axon guidance,homeostasis,disease,and most recently regeneration.However,a complete understanding of transcriptional regulatory networks,including how the nuclear factor Y complex binds to specific CCAAT boxes to perform its function remains elusive.In this review,we explore the nuclear factor Y complex's role and mode of action during brain development,as well as how genomic technologies may expand understanding of this key regulator of gene expression.

Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Nucleic acid binding proteins (NBPs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire NBP repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed NBPs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 NBPs with discrete autonomous germline functions. By identifying NBPs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.

Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Transcription factors (TFs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire TF repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed TFs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 TFs with discrete autonomous germline functions. By identifying TFs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.

Parkinsons disease is a neurodegenerative disease, which is associated with accumulation of α-synuclein protein aggregates and Lewy Body formation. These neurotoxic inclusions are especially harmful for dopamine-producing neurons in the substantia nigra of the brain. The cellular degradation system autophagy can reduce neurotoxicity caused by accumulated α-synuclein, by targeting it for degradation. Previously, we demonstrated that human TBC1D15 inhibits autophagy in vitro, resulting in accumulation of neurotoxic protein aggregates. Conversely, lowering the TBC1D15 expression promotes autophagy and degradation of α-synuclein and huntingtin proteins in various cell models. Here we show that knockdown of murine Tbc1d15 in vivo activates autophagy, reduces α-synuclein-mediated neurotoxicity, and improves motor performance. Thus, targeting Tbc1d15 expression may be a therapeutic avenue for neurodegenerative diseases. ### Competing Interest Statement Declaration of interests D.C.R. is a consultant for Aladdin Healthcare Technologies Ltd., Mindrank AI, Nido Biosciences, Drishti Discoveries, Retro Biosciences, and PAQ Therapeutics and Alexion Pharma International Operations Limited. None of the other authors have competing interests.

The developmental fate of cells is regulated by intrinsic factors and the extracellular environment. The extracellular matrix (matrisome) delivers chemical and mechanical cues that can modify cellular development. However, comprehensive understanding of how matrisome factors control cells in vivo is lacking. Here we show that specific matrisome factors act individually and collectively to control germ cell development. Surveying development of undifferentiated germline stem cells through to mature oocytes in the Caenorhabditis elegans germ line enabled holistic functional analysis of 443 conserved matrisome-coding genes. Using high-content imaging, 3D reconstruction, and cell behavior analysis, we identify 321 matrisome genes that impact germ cell development, the majority of which (>80%) are undescribed. Our analysis identifies key matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior. Further, our results demonstrate that germ cell development requires continual remodeling of the matrisome landscape. Together, this study provides a comprehensive platform for deciphering how extracellular signaling controls cellular development and anticipate this will establish new opportunities for manipulating cell fates.

Nervous system function relies on the establishment of complex gene expression programs that provide neuron-type-specific and core pan-neuronal features. These complementary regulatory paradigms are controlled by terminal selector and parallel-acting transcription factors (TFs), respectively. Here, we identify the nuclear factor Y (NF-Y) TF as a pervasive direct and indirect regulator of both neuron-type-specific and pan-neuronal gene expression. Mapping global NF-Y targets reveals direct binding to the cis-regulatory regions of pan-neuronal genes and terminal selector TFs. We show that NFYA-1 controls pan-neuronal gene expression directly through binding to CCAAT boxes in target gene promoters and indirectly by regulating the expression of terminal selector TFs. Further, we find that NFYA-1 regulation of neuronal gene expression is important for neuronal activity and motor function. Thus, our research sheds light on how global neuronal gene expression programs are buffered through direct and indirect regulatory mechanisms.

The extracellular matrix (matrisome) provides chemical and mechanical cues to control the structure and function of cells and tissues. Yet, comprehensive understanding of how matrisome factors individually and collectively control cell and tissue behavior in vivo is lacking. Here, we systematically investigate the function of 443 conserved matrisome-coding genes in controlling germ cell behavior within a complex tissue - the Caenorhabditis elegans germline. Using high-content imaging, 3D reconstruction and cell behavior analysis of >3500 germlines and >7 million germ cells, we identify specific matrisome factors that regulate germline structure, protein distribution, germ cell cycle and fate, apoptosis, and oocyte health. These findings reveal matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior, providing new avenues to study and manipulate cell fates.

Maternal diet and environment can influence the neuronal health of offspring. Here, we report that diet-induced intestinal sphingolipid biosynthesis reduces adult-onset neurodegeneration intergenerationally in Caenorhabditis elegans . Feeding C. elegans with ursolic acid (UA), a natural plant product, provides neuroprotection by enhancing maternal provisioning of sphingosine-1-phosphate (S1P) - a bioactive sphingolipid. S1P promotes neuronal health across generations by upregulating transcription of the acid ceramidase-1 ( asah-1 ) gene in the intestine. Intergenerational intestine-to-oocyte S1P transfer is essential for promoting neuronal health and is dependent on the lipoprotein yolk receptor RME-2 (Receptor-Mediated Endocytosis-2). Spatially regulating sphingolipid biosynthesis is critical, as inappropriate asah-1 neuronal expression causes developmental axon outgrowth defects. Our results reveal that sphingolipid homeostasis impacts the development and intergenerational health of the nervous system. One-Sentence Summary An intestinal lipid prevents neurodegeneration across generations.

DMD-9 is a Caenorhabditis elegans Doublesex/MAB-3 Domain transcription factor (TF) of unknown function. Single-cell transcriptomics has revealed that dmd-9 is highly expressed in specific head sensory neurons, with lower levels detected in non-neuronal tissues (uterine cells and sperm). Here, we characterized endogenous dmd-9 expression and function in hermaphrodites and males to identify potential sexually dimorphic roles. In addition, we dissected the trans- and cis-regulatory mechanisms that control DMD-9 expression in neurons. Our results show that of the 22 neuronal cell fate reporters we assessed in DMD-9-expressing neurons, only the neuropeptide-encoding flp-19 gene is cell-autonomously regulated by DMD-9. Further, we did not identify defects in behaviors mediated by DMD-9 expressing neurons in dmd-9 mutants. We found that dmd-9 expression in neurons is regulated by 4 neuronal fate regulatory TFs: ETS-5, EGL-13, CHE-1, and TTX-1. In conclusion, our study characterized the DMD-9 expression pattern and regulatory logic for its control. The lack of detectable phenotypes in dmd-9 mutant animals suggests that other proteins compensate for its loss.

Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. Specific neuropeptidergic signals coordinate the systemic mitochondrial stress response (UPR mt ), but whether chemical neurotransmitters also influence this process is unclear. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPR mt in the Caenorhabditis elegans intestine. GABA controls the UPR mt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPR mt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for nonautonomously regulating mitochondrial dynamics and organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses. One-Sentence Summary Regulation of mitochondrial health with neurotransmission.