The epidermal growth factor receptor (EGFR)/ErbB receptor tyrosine kinases regulate several aspects of development, including the development of the mammalian nervous system. ErbB signaling also has physiological effects on neuronal function, with influences on synaptic plasticity and daily cycles of activity. However, little is known about the effectors of EGFR activation in neurons. Here we show that EGF signaling has a nondevelopmental effect on behavior in Caenorhabditis elegans. Ectopic expression of the EGF-like ligand LIN-3 at any stage induces a reversible cessation of feeding and locomotion. These effects are mediated by neuronal EGFR (also called LET-23) and phospholipase C-gamma (PLC-gamma), diacylglycerol-binding proteins, and regulators of synaptic vesicle release. Activation of EGFR within a single neuron, ALA, is sufficient to induce a quiescent state. This pathway modulates the cessation of pharyngeal pumping and locomotion that normally occurs during the lethargus period that precedes larval molting. Our results reveal an evolutionarily conserved role for EGF signaling in the regulation of behavioral quiescence.

Summary Sleep is recognized to be ancient in origin, with vertebrates and invertebrates experiencing behaviorally quiescent states that are regulated by conserved genetic mechanisms[1, 2]. Despite its conservation throughout phylogeny the function of sleep remains debated. Hypotheses for the purpose of sleep include nervous system-specific functions such as modulation of synaptic strength and clearance of metabolites from the brain[3, 4], and more generalized cellular functions such as energy conservation and macromolecule biosynthesis[5]. These models are supported by the identification of synaptic and metabolic processes that are perturbed during prolonged wakefulness. It remains to be seen whether perturbations of cellular homeostasis in turn drive sleep. Here we show that under conditions of cellular stress, including noxious heat, cold, hypertonicity, and tissue damage, the nematode Caenorhabditis elegans engages a behavioral quiescence program. The stress-induced quiescent state displays properties of sleep and is dependent on the ALA neuron, which mediates the conserved soporific effect of Epidermal Growth Factor (EGF) ligand overexpression. We characterize heat-induced quiescence in detail and show that it is indeed dependent on components of EGF signaling, providing physiological relevance to the behavioral effects of EGF family ligands. We find that following noxious heat exposure, quiescence-defective animals show elevated expression of cellular stress reporter genes and are impaired for survival, demonstrating the benefit of stress-induced behavioral quiescence. These data provide evidence that cellular stress can induce a protective sleep-like state in C. elegans and suggest that a deeply conserved function of sleep is to mitigate disruptions of cellular homeostasis.

Our results suggest that rising and falling levels of LIN-42A allow the start and completion, respectively, of larval molts. We propose that LIN-42A and affiliated factors regulate molting cycles in much the same way that PER-based oscillators drive rhythmic behaviors and metabolic processes in mature mammals. Further, the combination of reiterative and stage-specific functions of LIN-42 may coordinate periodic molts with successive development of the epidermis.

Summary Among the most important decisions an animal makes is whether to engage in active movement and feeding behavior or to become quiescent. The molecular signaling mechanisms underlying this decision remain largely unknown. The nematode Caenorhabditis elegans displays sleep-like quiescence following exposures that result in cellular stress [1]. The neurosecretory ALA neuron is required for this stress-induced recovery quiescence [1] but the mechanisms by which ALA induces quiescence have been unknown. We report here that quiescence induced by heat stress requires ALA depolarization and release of FMRFamide-like neuropeptides encoded by the flp-13 gene. Optogenetic activation of ALA reduces feeding and locomotion in a FLP-13-dependent manner. Over expression of flp-13 is sufficient to induce quiescent behavior during normally active periods. We have here identified a major biological role for FMRFamide-like neuropeptides in nematodes, and suggest that they may function in a similar capacity in other organisms.

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Here we describe a factor, Half pint (Hfp), that regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. We find that hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp.

During Drosophila oogenesis, localization of the transforming growth factor alpha (TGFalpha)-like signaling molecule Gurken to the oocyte membrane is required for polarity establishment of the egg and embryo. To test Gurken domain functions, full-length and truncated forms of Gurken were expressed ectopically using the UAS/Gal4 expression system, or in the germline using the endogenous promoter. GrkDeltaC, a deletion of the cytoplasmic domain, localizes to the oocyte membrane and can signal. GrkDeltaTC, which lacks the transmembrane and cytoplasmic domains, retains signaling ability when ectopically expressed in somatic cells. However, in the germline, the GrkDeltaTC protein accumulates throughout the oocyte cytoplasm and cannot signal. In addition, we found that several strong gurken alleles contain point mutations in the transmembrane region. We conclude that secretion of Gurken requires its transmembrane region, and propose a model in which the gene cornichon mediates this process.

SUMMARYThe ancient origin of sleep is evidenced by deeply conserved signaling pathways regulating sleep-like behavior, such as signaling through the Epidermal growth factor receptor (EGFR). In Caenorhabditis elegans, a sleep-like state can be induced at any time during development or adulthood through conditional expression of LIN-3/EGF. The behavioral response to EGF is mediated by EGFR activity within a single cell, the ALA neuron, and mutations that impair ALA differentiation are expected to confer EGFresistance. Here we describe three such EGF-resistant mutants. One of these corresponds to the LIM class homeodomain (HD) protein CEH-14/Lhx3, and the other two correspond to Paired-like HD proteins CEH-10/Chx10 and CEH-17/Phox2. Whereas CEH-14 is required for ALA-specific gene expression throughout development, the Prd-like proteins display complementary temporal contributions to gene expression, with the requirement for CEH-10 decreasing as that of CEH-17 increases. We present evidence that CEH-17 participates in a positive autoregulatory loop with CEH-14 in ALA, and that CEH-10, in addition to its role in ALA differentiation, functions in the generation of the ALA neuron. Similarly to CEH-17, CEH-10 is required for the posterior migration of the ALA axons, but CEH-14 appears to regulate an aspect of ALA axon outgrowth that is distinct from that of the Prd-like proteins. Our findings reveal partial modularity among the features of a neuronal differentiation program and their coordination by Prd and LIM class HD proteins.

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