Pathogenic bacteria modulate pheromone response to promote mating.

Pathogens generate ubiquitous selective pressures and host-pathogen interactions alter social behaviours in many animals1-4. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal5-13. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

Global regulatory features of alternative splicing across tissues and within the nervous system of C. elegans.

Alternative splicing plays a major role in shaping tissue-specific transcriptomes. Among the broad tissue types present in metazoans, the central nervous system contains some of the highest levels of alternative splicing. Although many documented examples of splicing differences between broad tissue types exist, there remains much to be understood about the splicing factors and the cis sequence elements controlling tissue and neuron subtype-specific splicing patterns. By using translating ribosome affinity purification coupled with deep-sequencing (TRAP-seq) in Caenorhabditis elegans, we have obtained high coverage profiles of ribosome-associated mRNA for three broad tissue classes (nervous system, muscle, and intestine) and two neuronal subtypes (dopaminergic and serotonergic neurons). We have identified hundreds of splice junctions that exhibit distinct splicing patterns between tissue types or within the nervous system. Alternative splicing events differentially regulated between tissues are more often frame-preserving, are more highly conserved across Caenorhabditis species, and are enriched in specific cis regulatory motifs, when compared with other types of exons. By using this information, we have identified a likely mechanism of splicing repression by the RNA-binding protein UNC-75/CELF via interactions with cis elements that overlap a 5' splice site. Alternatively spliced exons also overlap more frequently with intrinsically disordered peptide regions than constitutive exons. Moreover, regulated exons are often shorter than constitutive exons but are flanked by longer intron sequences. Among these tissue-regulated exons are several highly conserved microexons <27 nt in length. Collectively, our results indicate a rich layer of tissue-specific gene regulation at the level of alternative splicing in C. elegans that parallels the evolutionary forces and constraints observed across metazoa.

C. elegans aversive olfactory learning generates diverse intergenerational effects.

Parental experience can modulate the behavior of their progeny. While the molecular mechanisms underlying parental effects or inheritance of behavioral traits have been studied under several environmental conditions, it remains largely unexplored how the nature of parental experience affects the information transferred to the next generation. To address this question, we used C. elegans, a nematode that feeds on bacteria in its habitat. Some of these bacteria are pathogenic and the worm learns to avoid them after a brief exposure. We found, unexpectedly, that a short parental experience increased the preference for the pathogen in the progeny. Furthermore, increasing the duration of parental exposure switched the response of the progeny from attraction to avoidance. To characterize the underlying molecular mechanisms, we found that the RNA-dependent RNA Polymerase (RdRP) RRF-3, required for the biogenesis of 26 G endo-siRNAs, regulated both types of intergenerational effects. Together, we show that different parental experiences with the same environmental stimulus generate different effects on the behavior of the progeny through small RNA-mediated regulation of gene expression.

An Elongin-Cullin-SOCS Box Complex Regulates Stress-Induced Serotonergic Neuromodulation.

Neuromodulatory cells transduce environmental information into long-lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the translating ribosome affinity purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing amphid neuron dual ciliated sensory ending (ADF) serotonergic sensory neurons, and we found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress. We demonstrate that ELC-2 and the von Hippel-Lindau protein VHL-1, components of an Elongin-Cullin-SOCS box (ECS) E3 ubiquitin ligase, modulate this behavior after experiencing stress. Also, heat stress induces a transient redistribution of ELC-2, becoming more nuclearly enriched. Together, our results demonstrate dynamic regulation of an E3 ligase and a role for an ECS complex in neuromodulation and control of lasting behavioral states.

CRISPR-mediated genetic interaction profiling identifies RNA binding proteins controlling metazoan fitness.

Genetic interaction screens have aided our understanding of complex genetic traits, diseases, and biological pathways. However, approaches for synthetic genetic analysis with null-alleles in metazoans have not been feasible. Here, we present a CRISPR/Cas9-based Synthetic Genetic Interaction (CRISPR-SGI) approach enabling systematic double-mutant generation. Applying this technique in Caenorhabditis elegans, we comprehensively screened interactions within a set of 14 conserved RNA binding protein genes, generating all possible single and double mutants. Many double mutants displayed fitness defects, revealing synthetic interactions. For one interaction between the MBNL1/2 ortholog mbl-1 and the ELAVL ortholog exc-7, double mutants displayed a severely shortened lifespan. Both genes are required for regulating hundreds of transcripts and isoforms, and both may play a critical role in lifespan extension through insulin signaling. Thus, CRISPR-SGI reveals a rich genetic interaction landscape between RNA binding proteins in maintaining organismal health, and will serve as a paradigm applicable to other biological questions.

Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique.

Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods. Here, we describe the use of the Translating Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression patterns at the level of translating mRNAs. In TRAP, a ribosomal protein is fused to a tag (GFP) and is expressed under cell type-specific promoters to mark genetically defined cell types in vivo. Affinity purification of lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNAs are used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide profiles of transcripts from the targeted cell type. The ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to enable the discovery of molecular components in response to external or genetic perturbations.

Regulation of Tissue-Specific Alternative Splicing: C. elegans as a Model System.

Alternative pre-mRNA splicing serves as an elegant mechanism for generating transcriptomic and proteomic diversity between cell and tissue types. In this chapter, we highlight key concepts and outstanding goals in studies of tissue and cell-specific alternative splicing. We place particular emphasis on the use of C. elegans as a tractable model organism for in vivo studies of alternative splicing between tissues and also at single cell resolution. We describe our current understanding of tissue and cell-specific regulation in the animal, and emerging techniques that will allow for future mechanistic studies as well as systems level investigations of spatio-temporal splicing under laboratory conditions and in response to environmental stimuli.

Fertility and germline stem cell maintenance under different diets requires nhr-114/HNF4 in C. elegans.

Animals can thrive on variable food resources as a result of autonomous processes and beneficial relationships with their gut microbes [1]. Food intake elicits major physiological changes, which are counteracted by transient systemic responses that maintain homeostasis in the organism. This integration of external information occurs through cellular sensory elements, such as nuclear receptors, which modulate gene expression in response to specific cues [2]. Given the importance of germline stem cells (GSCs) for the development of the germline and the continuity of species, it is reasonable to assume that GSCs might be shielded from the negative influence of environmental perturbations. To our knowledge, however, there are no mechanisms reported that protect GSCs from harmful dietary metabolites. Using Caenorhabditis elegans as a model, we report that the somatic activity of the conserved nuclear receptor nhr-114/HNF4 protects GSC integrity from dietary metabolites. In the absence of nhr-114 and on certain bacterial diets, otherwise somatically normal animals accumulate germ cell division defects during development and become sterile. We found that, in nhr-114(-) animals, the induction of germline defects and sterility depend on bacterial metabolic status, with respect to the essential amino acid tryptophan. This illustrates an animal-microbe interaction in which somatic nuclear receptor activity preserves the germline by buffering against dietary metabolites, most likely through a somatic detoxifying response. Overall, our findings uncover an unprecedented, and presumably evolutionarily conserved, soma-to-germline axis of communication that maintains reproductive robustness on variable food resources.

Mind the gut: Dietary impact on germline stem cells and fertility.

Animals thrive in environments where food resources are abundant. While this correlation between population growth and food abundance is well established, much less is known about the influence of diet quality on physiological and developmental programs that support animal reproduction. Here we discuss dietary impact on fertility, and highlight a recent report on the activity of a nuclear receptor that protects against dietary metabolites to maintain germline stem cell integrity and reproduction.

The nuclear receptor NR4A1 induces a form of cell death dependent on autophagy in mammalian cells.

The control of cell death is a biological process essential for proper development, and for preventing devastating pathologies like cancer and neurodegeneration. On the other hand, autophagy regulation is essential for protein and organelle degradation, and its dysfunction is associated with overlapping pathologies like cancer and neurodegeneration, but also for microbial infection and aging. In the present report we show that two evolutionarily unrelated receptors--Neurokinin 1 Receptor (NK(1)R,) a G-protein coupled receptor, and Insulin-like Growth Factor 1 Receptor (IGF1R), a tyrosine kinase receptor--both induce non-apoptotic cell death with autophagic features and requiring the activity of the autophagic core machinery proteins PI3K-III, Beclin-1 and Atg7. Remarkably, this form of cell death occurs in apoptosis-competent cells. The signal transduction pathways engaged by these receptors both converged on the activation of the nuclear receptor NR4A1, which has previously been shown to play a critical role in some paradigms of apoptosis and in NK(1)R-induced cell death. The activity of NR4A1 was necessary for IGF1R-induced cell death, as well as for a canonical model of cell death by autophagy induced by the presence of a pan-caspase inhibitor, suggesting that NR4A1 is a general modulator of this kind of cell death. During cell death by autophagy, NR4A1 was transcriptionally competent, even though a fraction of it was present in the cytoplasm. Interestingly, NR4A1 interacts with the tumor suppressor p53 but not with Beclin-1 complex. Therefore the mechanism to promote cell death by autophagy might involve regulation of gene expression, as well as protein interactions. Understanding the molecular basis of autophagy and cell death mediation by NR4A1, should provide novel insights and targets for therapeutic intervention.