The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.

The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress.

The conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, expression of O-GlcNAcylation-deficient forms of human or worm OGT rescue the hypertonic stress response phenotype. However, expression of an OGT protein lacking the tetracopeptide repeat (TPR) domain does not rescue. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins.

The C. elegans Hypertonic Stress Response: Big Insights from Shrinking Worms.

Cell volume is one of the most aggressively defended physiological set points in biology. Changes in intracellular ion and water concentrations, which are induced by changes in metabolism or environmental exposures, disrupt protein folding, enzymatic activity, and macromolecular assemblies. To counter these challenges, cells and organisms have evolved multifaceted, evolutionarily conserved molecular mechanisms to restore cell volume and repair stress induced damage. However, many unanswered questions remain regarding the nature of cell volume 'sensing' as well as the molecular signaling pathways involved in activating physiological response mechanisms. Unbiased genetic screening in the model organism C. elegans is providing new and unexpected insights into these questions, particularly questions relating to the hypertonic stress response (HTSR) pathway. One surprising characteristic of the HTSR pathway in C. elegans is that it is under strong negative regulation by proteins involved in protein homeostasis and the extracellular matrix (ECM). The role of the ECM in particular highlights the importance of studying the HTSR in the context of a live organism where native ECM-tissue associations are preserved. A second novel and recently discovered characteristic is that the HTSR is regulated at the post-transcriptional level. The goal of this review is to describe these discoveries, to provide context for their implications, and to raise outstanding questions to guide future research.

The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans.

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.

Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance.

Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration. SIGNIFICANCE STATEMENT: In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as "anti-chaperones" uncovers new and general targets for therapeutic intervention.

Stress and aging induce distinct polyQ protein aggregation states.

Many age-related diseases are known to elicit protein misfolding and aggregation. Whereas environmental stressors, such as temperature, oxidative stress, and osmotic stress, can also damage proteins, it is not known whether aging and the environment impact protein folding in the same or different ways. Using polyQ reporters of protein folding in both Caenorhabditis elegans and mammalian cell culture, we show that osmotic stress, but not other proteotoxic stressors, induces rapid (minutes) cytoplasmic polyQ aggregation. Osmotic stress-induced polyQ aggregates could be distinguished from aging-induced polyQ aggregates based on morphological, biophysical, cell biological, and biochemical criteria, suggesting that they are a unique misfolded-protein species. The insulin-like growth factor signaling mutant daf-2, which inhibits age-induced polyQ aggregation and protects C. elegans from stress, did not prevent the formation of stress-induced polyQ aggregates. However, osmotic stress resistance mutants, which genetically activate the osmotic stress response, strongly inhibited the formation of osmotic polyQ aggregates. Our findings show that in vivo, the same protein can adopt distinct aggregation states depending on the initiating stressor and that stress and aging impact the proteome in related but distinct ways.

CLHM-1 is a functionally conserved and conditionally toxic Ca2+-permeable ion channel in Caenorhabditis elegans.

Disruption of neuronal Ca(2+) homeostasis contributes to neurodegenerative diseases through mechanisms that are not fully understood. A polymorphism in CALHM1, a recently described ion channel that regulates intracellular Ca(2+) levels, is a possible risk factor for late-onset Alzheimer's disease. Since there are six potentially redundant CALHM family members in humans, the physiological and pathophysiological consequences of CALHM1 function in vivo remain unclear. The nematode Caenorhabditis elegans expresses a single CALHM1 homolog, CLHM-1. Here we find that CLHM-1 is expressed at the plasma membrane of sensory neurons and muscles. Like human CALHM1, C. elegans CLHM-1 is a Ca(2+)-permeable ion channel regulated by voltage and extracellular Ca(2+). Loss of clhm-1 in the body-wall muscles disrupts locomotory kinematics and biomechanics, demonstrating that CLHM-1 has a physiologically significant role in vivo. The motility defects observed in clhm-1 mutant animals can be rescued by muscle-specific expression of either C. elegans CLHM-1 or human CALHM1, suggesting that the function of these proteins is conserved in vivo. Overexpression of either C. elegans CLHM-1 or human CALHM1 in neurons is toxic, causing degeneration through a necrotic-like mechanism that is partially Ca(2+) dependent. Our data show that CLHM-1 is a functionally conserved ion channel that plays an important but potentially toxic role in excitable cell function.

The Caenorhabditis elegans mucin-like protein OSM-8 negatively regulates osmosensitive physiology via the transmembrane protein PTR-23.

The molecular mechanisms of animal cell osmoregulation are poorly understood. Genetic studies of osmoregulation in yeast have identified mucin-like proteins as critical regulators of osmosensitive signaling and gene expression. Whether mucins play similar roles in higher organisms is not known. Here, we show that mutations in the Caenorhabditis elegans mucin-like gene osm-8 specifically disrupt osmoregulatory physiological processes. In osm-8 mutants, normal physiological responses to hypertonic stress, such as the accumulation of organic osmolytes and activation of osmoresponsive gene expression, are constitutively activated. As a result, osm-8 mutants exhibit resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. To identify genes required for Osm-8 phenotypes, we performed a genome-wide RNAi osm-8 suppressor screen. After screening ~18,000 gene knockdowns, we identified 27 suppressors that specifically affect the constitutive osmosensitive gene expression and Osr phenotypes of osm-8 mutants. We found that one suppressor, the transmembrane protein PTR-23, is co-expressed with osm-8 in the hypodermis and strongly suppresses several Osm-8 phenotypes, including the transcriptional activation of many osmosensitive mRNAs, constitutive glycerol accumulation, and osmotic stress resistance. Our studies are the first to show that an extracellular mucin-like protein plays an important role in animal osmoregulation in a manner that requires the activity of a novel transmembrane protein. Given that mucins and transmembrane proteins play similar roles in yeast osmoregulation, our findings suggest a possible evolutionarily conserved role for the mucin-plasma membrane interface in eukaryotic osmoregulation.

Endogenous gpdh-1 transcriptional reporters as new tools for the study of the osmotic stress response.

In vivo monitoring of gpdh-1 gene expression using standard transcriptional reporters is a powerful and commonly used tool for genetic dissection of the osmotic stress response in C. elegans . Like all transgene reporters, these gpdh-1 reporters have important limitations that restrict their utility. To overcome these limitations, we created three different gpdh-1 reporters using CRISPR/Cas9 methods to insert several variants of GFP into the endogenous gpdh-1 locus. These new strains provide a more powerful and accurate tool for the analysis of gpdh-1 regulatory pathways.

Regulation of the hypertonic stress response by the 3' mRNA cleavage and polyadenylation complex.

Maintenance of osmotic homeostasis is one of the most aggressively defended homeostatic set points in physiology. One major mechanism of osmotic homeostasis involves the upregulation of proteins that catalyze the accumulation of solutes called organic osmolytes. To better understand how osmolyte accumulation proteins are regulated, we conducted a forward genetic screen in Caenorhabditis elegans for mutants with no induction of osmolyte biosynthesis gene expression (Nio mutants). The nio-3 mutant encoded a missense mutation in cpf-2/CstF64, while the nio-7 mutant encoded a missense mutation in symk-1/Symplekin. Both cpf-2 and symk-1 are nuclear components of the highly conserved 3' mRNA cleavage and polyadenylation complex. cpf-2 and symk-1 block the hypertonic induction of gpdh-1 and other osmotically induced mRNAs, suggesting they act at the transcriptional level. We generated a functional auxin-inducible degron (AID) allele for symk-1 and found that acute, post-developmental degradation in the intestine and hypodermis was sufficient to cause the Nio phenotype. symk-1 and cpf-2 exhibit genetic interactions that strongly suggest they function through alterations in 3' mRNA cleavage and/or alternative polyadenylation. Consistent with this hypothesis, we find that inhibition of several other components of the mRNA cleavage complex also cause a Nio phenotype. cpf-2 and symk-1 specifically affect the osmotic stress response since heat shock-induced upregulation of a hsp-16.2::GFP reporter is normal in these mutants. Our data suggest a model in which alternative polyadenylation of 1 or more mRNAs is essential to regulate the hypertonic stress response.

Length-dependent RNA foci formation and Repeat Associated non-AUG dependent translation in a C. elegans G 4 C 2 model.

GC-rich repeat expansion mutations are implicated in several neurodegenerative diseases and can lead to repeat associated non-AUG-dependent (RAN) translation and concentrations of nuclear RNA foci. To model C9orf72 ALS/FTD, we engineered C. elegans to express pure GGGGCC (G 4 C 2 ) repeats of varying lengths and observed RAN translation and nuclear RNA foci. RNA foci were observed in animals expressing ≥20 G 4 C 2 repeats while RAN translation occured in animals expressing ≥33 G 4 C 2 repeats. These findings show that in C. elegans , RAN translation can occur even in the absence of C9orf72 intronic sequence normally surrounding the repeat. Given that the currently accepted repeat threshold for C9 disease is >30 repeats, our data are consistent with a model in which RAN peptides are key drivers of C9orf72 disease pathology.

PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.

Measuring RAN Peptide Toxicity in C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington's disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

C. elegans dysferlin homolog fer-1 is expressed in muscle, and fer-1 mutations initiate altered gene expression of muscle enriched genes.

Mutations in the human dysferlin gene cause Limb Girdle Muscular Dystrophy 2B (LGMD2B). The Caenorhabditis elegans dysferlin homolog, fer-1, affects sperms development but is not known to be expressed in or have a functional roles outside of the male germline. Using several approaches, we show that fer-1 mRNA is present in C. elegans muscle cells but is absent from neurons. In mammals, loss of muscle-expressed dysferlin causes transcriptional deregulation of muscle expressed genes. To determine if similar alterations in gene expression are initiated in C. elegans due to loss of muscle-expressed fer-1, we performed whole genome Affymetrix microarray analysis of two loss-of-function fer-1 mutants. Both mutants gave rise to highly similar changes in gene expression and altered the expression of 337 genes. Using multiple analysis methods, we show that this gene set is enriched for genes known to regulate the structure and function of muscle. However, these transcriptional changes do not appear to be in response to gross sarcomeric damage, since genetically sensitized fer-1 mutants exhibit normal thin filament organization. Our data suggest that processes other than sarcomere stability may be affected by loss of fer-1 in C. elegans muscle. Therefore, C. elegans may be an attractive model system in which to explore new muscle-specific functions of the dysferlin protein and gain insights into the molecular pathogenesis of LGMD2B.

Models and mechanisms of repeat expansion disorders: a worm's eye view.

The inappropriate genetic expansion of various repetitive DNA sequences underlies over 20 distinct inherited diseases. The genetic context of these repeats in exons, introns and untranslated regions has played a major role in thinking about the mechanisms by which various repeat expansions might cause disease. Repeat expansions in exons are thought to give rise to expanded toxic protein repeats (i.e. polyQ). Repeat expansions in introns and UTRs (i.e. FXTAS) are thought to produce aberrant repeat-bearing RNAs that interact with and sequester a wide variety of essential proteins, resulting in cellular toxicity. However, a new phenomenon termed 'repeat-associated nonAUG dependent (RAN) translation' paints a new and unifying picture of how distinct repeat expansion-bearing RNAs might act as substrates for this noncanonical form of translation, leading to the production of a wide range of repeat sequence-specific-encoded toxic proteins. Here, we review how the model system Caenorhabditis elegans has been utilized to model many repeat disorders and discuss how RAN translation could be a previously unappreciated contributor to the toxicity associated with these different models.

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis.

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening.

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.

Functional genomic approaches in C. elegans.

The nematode Caenorhabditis elegans is an extraordinarily powerful model organism for the application of functional genomic approaches. Two such approaches, whole genome microarray analysis and genome-wide RNA interference (RNAi)-mediated phenotypic screening, are highly advanced and can be used by virtually any laboratory to study biological processes of interest. Using studies of the osmotic stress response in C. elegans as an example, this chapter describes methods for conducting whole genome microarray experiments and for carrying out genome-wide reverse-genetic screens using a commercially available C. elegans bacterial RNAi feeding library. Both approaches are complimentary and can be used to rapidly gain genome-wide insights into the genes and gene networks controlling specific physiological processes.

Stress signaling: serotonin spreads systemic stress.

Cells respond to elevated temperatures through a well-characterized heat-shock response that enables short-term survival, long-term adaptation and mitigation of macromolecular damage. New work reveals a cell non-autonomous layer of stress-response regulation between neurons and the gonad involving serotonin.