Mansfield, R. et al. (2015) International Worm Meeting "Disruption of cellular proteostasis drives sleep behavior in C. elegans."

Mansfield, R., Van Buskirk, C.

[International Worm Meeting, 2015]

Sleep is widely regarded to be a restorative state, and the physiological perturbations associated with sleep deprivation are extensive. Despite intensive study, none of these perturbations has in turn been shown to drive sleep. Recently our lab has shown that in C. elegans, environmental stressors such as heat, cold and toxins induce a sleep-like state (Hill et al., 2014). As noxious environmental stressors are known to interrupt normal proteostasis, the existence of a subsequent sleep state suggests that sleep serves to assist in the restoration of normal proteostasis. This idea is supported by our observation that sleepless animals have impaired survival following severe stress (Hill et al., 2014). Further, we have found that chaperone response defective mutants display exaggerated sleep responses. These mutants include animals lacking the stress-induced transcription factors hsf-1/HSF-1, daf-16/FOXO and a component of the endoplasmic reticulum stress-response pathway hsp-4/BiP. We are testing site-of-action for this effect by examining strains with tissue-specific rescue of HSF-1. In addition, we are using pharmacological inhibitors of the proteasome to test whether direct inhibition of the proteostasis machinery can trigger sleep. Last, we are performing RNAi against both positive and negative regulators of the heat shock transcriptional response, and screening for sleep phenotypes. The results of these efforts will be presented.Hill AJ, Mansfield R, Lopez JMG, Raizen DM, Van Buskirk C (2014) Cellular stress induces a protective sleep-like state in C. elegans. Curr Bio 24:1-7.Zimmerman JE, Naidoo N, Raizen DM, Pack AI (2008) Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 31:371-6.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Van Buskirk, Cheryl et al. (2013) International Worm Meeting "FIRE lab (Full Immersion Research Experience): Student-directed projects on ALA-dependent sleep in C. elegans."

Van Buskirk, Cheryl, Roman, Dany

[International Worm Meeting, 2013]

CSUN biology majors are eager for opportunities to work closely with faculty members and gain research experience. However, there are often not enough spaces in faculty labs to meet this need. The redesigned BIOL447/L, 'FIRE lab' helps to bridge that gap, allowing up to 18 students to participate in original, student-directed research. During the first half of the course, students are introduced to C. elegans and to a series of research articles that familiarize them with a specialized topic, in this case EGF-induced (ALA neuron dependent) sleep behavior1. During the second phase of the course, students design a series of experiments to address a unique question, and have the option of working individually or in groups. Prior to starting their project, each student applies for 'funding' by writing a grant proposal that undergoes rounds of instructor feedback and peer review. In the final week of the course, students present their results in a mock conference. The chosen projects fell into two main groups: 1. Half of the class chose to perform a group RNAi screen of predicted protease genes, in order to identify the putative sheddase responsible for release of soluble EGF ligand from its transmembrane precursor. This involved identification and prioritization of candidate genes, and design of an RNAi strategy that was consistent with the twice-per-week class schedule. 2. The remaining students worked as individuals, investigating potential roles of ALA neuron-dependent sleep. Students chose stressors that are known to inhibit feeding and locomotion, such as bacterial toxins and tissue injury, and determined whether the quiescent state was ALA-dependent. This involved literature searches, establishment of quiescence assays, and comparison of WT and ALA-defective (ceh-17 mutant) animals. This poster serves as an opportunity for a FIRE lab student and the instructor to share the experimental results and learning outcomes of this course. 1. Van Buskirk, C. & Sternberg, P. W. Epidermal growth factor signaling induces behavioral quiescence in Caenorhabditis elegans. Nat Neuro. 10, 1300-1307 (2007).

Van Buskirk, Cheryl L. et al. (2009) International Worm Meeting "Development and function of the sleep-inducing ALA neuron of C. elegans ."

Van Buskirk, Cheryl L., Sternberg, Paul W.

[International Worm Meeting, 2009]

The impact of sleep disorders on human health has underlined the need for a greater understanding of the regulation of sleep. Such studies have largely focused on mammalian systems, but recently sleep-like states have been described in invertebrates, including C. elegans (Raizen et al., 2008). Importantly, some of the factors regulating sleep behavior appear to be highly conserved, such as signaling through the Epidermal Growth Factor Receptor (EGFR) (Van Buskirk and Sternberg, 2007). In C. elegans, EGF-induced sleep is marked by a cessation of feeding and locomotion and is normally restricted to a short period preceding each larval molt. However, this sleep-like state can be induced at any time during development or adulthood through conditional overexpression of LIN-3/EGF. The effects of EGF expression in C. elegans are strikingly similar to those seen in response to EGF administration in mammals, pointing to an evolutionarily conserved role for EGF signaling in the regulation of behavior. We have found that EGF-induced sleep is triggered by activation of the EGF receptor (LET-23) within a single neuron called ALA, and we have identified several EGF-resistant mutants that define components of a signal transduction pathway mediating this effect. In addition, we have identified a class of mutations that render animals EGF-resistant due to defects in ALA-specific gene expression, including impaired let-23 transcription. Lastly, using a combination of genetic analyses and laser ablations, we have investigated the mechanism by which the ALA neuron signals to effect sleep behavior. We will present the results of these analyses on the development and function of the ALA neuron. Raizen, D.M. et al. (2008) Lethargus is a C. elegans sleep-like state. Nature 451, 569-72. Van Buskirk, C. and Sternberg, P.W. (2007) Epidermal Growth Factor signaling induces behavioral quiescence in C. elegans. Nat. Neurosci. 10, 1300-07.

Siddiqui, Shahid S. et al. (2011) International Worm Meeting "Fat Burning in Sleeping Worms: Regulation of Multiple Signaling Pathways by Kruppel Like Factors."

Gozal, David, Yang, Chen, Zhang, Jun, Siddiqui, Shahid S., Dhansingh, Immanuel, Hashmi, Sarwar

[International Worm Meeting, 2011]

Sleep is an important behavior across diverse species, regulated by conserved signaling pathways. OSAS (obstructive sleep apnea syndrome) is characterized by episodic hypoxia, affecting about 5% of adult American population and may lead to hypertension, if left untreated. In C. elegans post-embryonic development is accompanied by four larval stages [Sulston et al., 1983] that are interrupted by periods of inactivity called lethargus, and has been suggested to represent 'sleep' [Raizen et al., 2008]. The cAMP-CREB pathway has been shown to affect sleep-wake cycle in Drosophila, C. elegans and mammalian systems [Zimmerman et al., 2008], and cGMP dependent protein kinase expression is regulated by Rho and KLF4 [Zeng et al., 2006]. Genetic analysis has suggested the role of LIN-3 and LET-23 (the EGF like-ligand and EGF receptor, respectively) in sleep like behavior in C. elegans [Van Buskirk and Sternberg, 2007]. Overexpression of LIN-3 results in quiescent behavior during periods of normal activity, through the activity of ALA neuron that is regulated by paired (a family of Kruppel like transcription factors) and LIM class of transcription factors CEH17, CEH-14/Chx10, and CEH17/Phox2 through a feedback regulatory pathway [Van Buskirk and Sternberg, 2010]. We have shown the role of KLF-3 in beta oxidation of fatty acids in C. elegans that results in accumulation of fat in intestinal cells in klf-3(ok1975) homozygous loss of function mutants, arrested development and poor fecundity [Hashmi et al., 2008; Zhang et al. 2011 submitted], and may also affect sleep cycle by affecting the lethargus timings during larval molts. Interestingly, KLF4 in mammals not only mediates adipogenesis but also regulates kallistatin expression [Shen et al., 2009] that regulates kidney protein expression during episodic hypoxia (EH), by inhibiting vasodilation mediated by kallikrein-Kallistatin signaling pathway [Thonhboonkered et al., 2002]. The sleep regulating egl-4 gain of function mutation in cyclic GMP also results in the accumulation of fat in the intestine [Raizen et al., 2006]. Thus accumulating results suggest that klf-3 signaling pathway analysis in C. elegans may elucidate fat utilization (see the abstract by Hashmi et al., this meeting), sleep, and reproductive behavior.

Cheryl L Van Buskirk et al. (2005) International Worm Meeting "A physiological role for LET-23/EGFR in C. elegans feeding behavior"

Cheryl L Van Buskirk, Paul W Sternberg

[International Worm Meeting, 2005]

The single EGF receptor homolog in C. elegans, LET-23, signals through the Ras/MAPK pathway to specify cell fates in the ventral cord, vulva, male tail, and excretory system. LET-23 also signals through a PLC-/IP3 pathway to regulate ovulation. We have observed that overexpression of the EGFR ligand LIN-3 from a heat shock inducible promoter, in addition to affecting the expected cell fates, also slows down growth of the animals as compared to heat-shocked controls. This growth defect appears to be due to a complete and reversible cessation of pharyngeal pumping that occurs in response to overexpression of LIN-3 at any developmental stage, including the adult, and is not suppressed by mutations in the Ras/MAPK pathway. The growth/feeding defect is suppressed by mutations in let-23 and PLC-, but not the IP3 receptor, suggesting that diacylglycerol (DAG) signaling may mediate this effect. In addition, mutants that cause selective neurodegeneration or compromise synaptic vesicle release act as potent suppressors, suggesting that LET-23 acts to control neurotransmission. This is the first evidence of a non-developmental role for EGFR in synaptic function. We will present the results of our suppression analysis as well as our progress in defining the site of action for LET-23 in the regulation of feeding behavior.

Wolf FW et al. (1998) West Coast Worm Meeting "Yes! you can suppress vab-8"

Wolf FW, Garriga G

[West Coast Worm Meeting, 1998]

vab-8 acts in posteriorly directed cell and axon growth cone migrations in a cell autonomous manner. vab-8 functions in posterior ALM cell migration but not in later anterior ALM axon migration, and continuous vab-8 expression in the ALM can reorient anterior ALM axon outgrowth posteriorly. This finding suggests that the direction of migration can be regulated at the level of vab-8 activity. vab-8 encodes two isoforms of a novel intracellular protein, and the opal null allele e1017 is predicted to remove both activities. Our analysis of vab-8 has led us to propose that there exists at least one mechanism for the global guidance of posterior migrations. To identify other genes that function in posterior guidance, we suppressed the highly penetrant CAN cell migration defect of vab-8(e1017) mutants. Extragenic suppressors could identify genes that act downstream of vab-8 as well as genes that act in parallel pathways. From screening over 20,000 genomes we isolated four partially penetrant van (variably abnormal normal) suppressors, each defining a distinct gene. van-1(gm128) is a semi-dominant suppressor of vab-8 cell and axon outgrowth defects that is allele non-specific but at least so far is gene specific. Alas, van-1 has no phenotype on its own. In dosage studies van-1(gm128) appears to alter gene function. We have mapped van-1 to LGI between stu-4 and unc-11 and transformation rescue experiments are underway.

van Schendel, R. et al. (2017) International Worm Meeting "Polymerase theta is a key driver of genome evolution, of CRISPR/Cas9-mediated mutagenesis and of mutagen-induced deletion allele formation."

Tijsterman, M., van Schendel, R.

[International Worm Meeting, 2017]

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. We have recently demonstrated for two of the most widely used mutagens in C. elegans, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs) 1, 2. This discovery allowed us to survey many thousands of available deletion alleles, which reveals a step-wise and versatile model for the in vivo mechanism of TMEJ, explaining the molecular nature of mutagen-induced deletion alleles 3. We also found that CRISPR/Cas9-induced genomic changes are exclusively generated through POLQ action, refuting a previously assumed requirement for NHEJ in their formation; Unlike somatic cells, which use NHEJ to repair DNA breaks, germ cells use an alternative route 4. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification. New insights into the mechanism of genome engineering, genome maintenance and evolution will be discussed. 1. Koole,W. et al. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 5, 3216 (2014). 2. Roerink,S.F., van Schendel,R., & Tijsterman,M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954-962 (2014). 3. van Schendel R., van Heteren J., Welten,R., & Tijsterman,M. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining. PLoS. Genet. 12, e1006368 (2016). 4. van Schendel,R., Roerink,S.F., Portegijs,V., van den Heuvel,S., & Tijsterman,M. Polymerase Theta is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat. Commun. 6, 7394 (2015).

Fry, Amanda et al. (2011) International Worm Meeting "Function of VAV-1 in nervous system control of locomotion."

Fry, Amanda, Norman, Kenneth

[International Worm Meeting, 2011]

C. elegans VAV-1 is a guanine nucleotide exchange factor (GEF) for Rho/Rac family GTPases and is homologous to the mammalian Vav proto-oncogenes. We found previously that VAV-1 regulates a variety of rhythmic activities in C. elegans, including pharyngeal pumping, ovulation and fertilization, and the defecation cycle, but its role in locomotion has remained unexplored. Interestingly, we have found that vav-1 null animals display an elevated rate of locomotion and are hypersensitive to aldicarb (an acetylcholine esterase inhibitor), indicating heightened nervous system activity. The aldicarb hypersensitivity observed in vav-1 mutants is rescued by pan-neural expression of VAV-1, and using further pharmacological assays, we have shown that this hypersensitivity is not due to altered response of vav-1 mutant muscle cells to acetylcholine. Together, these data indicate that VAV-1 inhibits locomotion through a nervous system mechanism. Remarkably, Vav3 knockout mice are known to exhibit sympathetic nervous system hyperactivity, suggesting a conserved role of Vav proteins in negatively regulating nervous system activity, but the mechanism by which this regulation occurs is not fully understood (1-3). Since VAV-1 is known to be upstream of regulators of nervous system development (e.g. Rho/Rac GTPases), and impaired GABAergic neuron function can lead to aldicarb hypersensitivity, we examined the structure and function of GABA neurons by a combination of cell biological and pharmacological approaches. These analyses showed that both the development and function of GABA-releasing neurons is normal. To identify the cells that express vav-1 and gain insight into the neural circuit that regulates locomotion, we have analyzed vav-1 reporter expression in vivo and found that VAV-1 is expressed in a subset of neurons in the head ganglia of the worm, and highly expressed in the ALA neuron. Interestingly, the only known function of the ALA neuron is to regulate behavioral quiescence, the quantification of which is the rate of locomotion (4). We intend to elucidate the mechanism responsible for the control of locomotion by VAV-1 by determining the requirement of VAV-1 in the ALA neuron, as well as investigating the participation of VAV-1 in signaling pathways active in this cell. 1. Sauzeau et al. (2006). Nat. Med. 12, 841-845 2. Sauzeau et al. (2010). Mol Biol Cell. 21, 4251-63 3. Quevedo C. (2010). 15, 1125-39 4. Van Buskirk, C. and Sternberg, P.W. (2007) Nat. Neurosci. 10, 1300-07.

van Luenen HGAM et al. (1995) International C. elegans Meeting "Mechanism of Tc3 transposition"

van Luenen HGAM, Plasterk RHA

[International C. elegans Meeting, 1995]

The strain Bristol N2 has no endogenous Tc3 transposition activity, but forced expression of Tc3 transposase results in somatic transposition of endogenous Tc3 elements as well as Tc3 elements located on a transgene (Van Luenen et al. 1993 EMBO J. 12:2513-2520). This provides us with an assay for the requirements of Tc3 transposition in vivo.