David Raizen

University of Pennsylvania; Philadelphia PA, United States of America; Perelman School of Medicine

Raizen, David M. et al. (2011) International Worm Meeting "Is lethargus essential?"

Lamb, Annesia L., Driver, Robert J., Raizen, David M.

[International Worm Meeting, 2011]

Lethargus is a 2-3 hour period that occurs at each of the four larval stage transitions during C. elegans development from an embryo to an adult. During lethargus, the animal secretes and assembles a new cuticle and escapes from the old cuticle via a process of apolysis (separation of the old from the new cuticle) and ecdysis. Animals in lethargus show reduced movement and an absence of pharyngeal pumping. Quiescence of locomotion is fully reversible upon strong mechanical stimulation. However, pharyngeal pumping is not increased by such stimulation nor is it increased by exogenous serotonin, which is stimulatory to pumping outside of lethargus. Moreover, dissecting lethargus pharynxes and bathing them in an artificial saline containing serotonin does not stimulate pumping. In addition to quiescence, animals in lethargus show the sleep-like properties of an elevated sensory arousal threshold, and a homeostatic response to deprivation of lethargus quiescence. While testing for the homeostatic response, we noticed that following a 30-minute period of forced swimming during lethargus, 11% of wild-type animals did not recover. Microscopic observations of these worms showed that their adult cuticle was secreted and separated from their L4 cuticle, but they failed to escape from the L4 cuticle. We found that daf-16 mutants have a defective behavioral response to deprivation of lethargus quiescence and are hypersensitive to the lethal effects of deprivation. To deprive worms of lethargus quiescence genetically, we made strains carrying mutations in both egl-4 and kin-2, each of which is partially required for normal lethargus quiescence and elevation of arousal threshold. While 100% of egl-4 and 85% of kin-2 single mutants reached adulthood, 0% of egl-4; kin-2 double mutants reached adulthood. Instead, they arrested as early larvae with a molting-defective phenotype. We are currently testing whether this synthetic lethal phenotype can be explained by action of these genes in the nervous system. Given the tight association between lethargus and the molt, it might not be at all surprising that lethargus is required for the proper molt. But the precise function lethargus is serving remains a mystery. Given that total sleep deprivation in rats (Rechtschaffen et al, '83) and fruit flies (Shaw et al, '02) is also lethal, we are intrigued by the possibility that lethargus may serve a function similar to sleep.

Raizen, David M. (2009) International Worm Meeting "A role for the germline in the regulation of hermaphrodite quiescence?"

Raizen, David M.

[International Worm Meeting, 2009]

ad450, a gain of function mutant of the cGMP dependent kinase gene egl-4, stops movement as an adult and therefore forms few tracks on a lawn of bacteria. With the hope of identifying down stream signaling components of EGL-4/PKG, I screened 1200 F2 progeny of mutagenized egl-4(ad450) worms for mutants that form increased tracks on a lawn of bacteria. Surprisingly, most of the mutants I found, 24 mutants of a total of 32 isolated, were sterile. To test the possibility that there is an effect of the germline on behavioral quiescence, I compared behavior of glp-4(ts); egl-4(ad450) double mutants and of glp-4(ts) single mutants raised at the restrictive temperature to the same strains grown at the permissive temperature. Worms that lacked the germline due to the glp-4 mutation formed significantly more tracks than control worms of the same genotype, suggesting a role for the germline in the regulation of hermaphrodite behavioral quiescence. I will report the results of analyzing other ad450 double mutants. I will also reports the consequence of laser ablation of the germline precursors.

David Raizen (2007) International Worm Meeting "Suppressors of egl-4(ad450sd)."

David Raizen

[International Worm Meeting, 2007]

cGMP dependent protein kinases (PKGs) are key signaling molecules, yet their down stream signaling pathway(s) including their substrates remain poorly understood. I am using a genetic approach in worms to understand PKG signaling. Gain of function activity of the egl-4 gene, which encodes a worm PKG, results in a small body size, reduced intestinal darkness, increased intestinal nile red staining, reduced longevity, and sleep-like behavior during the adult stage. Loss of egl-4 function results in the opposite of these phenotypes as well as in sensory adaptation defects, Aldicarb resistance, and increased propensity to form dauers. Based on analysis of animals that are doubly mutant for known signaling pathway components and either an egl-4(gf) mutant or an egl-4(lf) mutant, the signaling pathways involved in some of these phenotypes have been identified. DAF-7 TGF-? signaling functions down stream of egl-4 in the control of intestinal darkness, DBL-1 TGF-? signaling functions down stream in the control the body size, insulin signaling is down stream in the control of longevity, and a cGMP-dependent cation channel is involved in the sensory adaptation pathway (Daniels et al ''00; Fujiwara et al 02, Hirose et al 04, lEtoile et al 02, Raizen et al, ‘06). To further our understanding of the PKG signaling pathway(s), I searched for mutants that suppressed the egl-4(gf) mutant ad450sd. I am particularly interested in those mutants that suppress most to all of the gf phenotypes since the genes affected in these mutants may act more proximally in egl-4 signaling pathways. I screened the progeny of ~6000 chemically mutagenized ad450 F1s for worms that appeared darker, more active, and/or larger than ad450 worms. The largest class of suppressors consists of loss of function mutations in egl-4, which occurred at a rate of approximately 1/1000 haploid genomes. A second class consists of 3 mutants that suppress the pale intestine phenotype but not other phenotypes of egl-4(gf). Other phenotypes of these suppressors suggest that they are likely to encode genes that function in the daf-7 TGF-? signaling pathway. Indeed, based on mapping, complementation, and sequencing, one of these, cs82, encodes a mutation in daf-8. A third class consists of 3 mutants that partially suppress more than one egl-4(gf) phenotypes, and do not map to the egl-4 locus. The genes affected in these mutants may function generally in PKG signaling. I am currently characterizing these mutants.

David M. Raizen et al. (2007) International Worm Meeting "Sleep-like behavioral properties of lethargus."

David M. Raizen, Uyen D. Ta

[International Worm Meeting, 2007]

We have been testing the hypothesis that lethargus is a sleep-like state. The impetus for this hypothesis comes from the observation that expression of the C. elegans PERIOD homologue LIN-42 cycles with a constant phase relationship to lethargus (Jeon et al, ‘99), just as PERIOD expression cycles with a constant phase relationship to sleep. A key feature of sleep, which distinguishes it from quiet wakefulness, is a reduced responsiveness to multiple sensory modalities. This reduced responsiveness has been explained as an increase in arousal threshold due to gating of the sensory information during sleep at the level of the central nervous system. To determine if during C. elegans lethargus arousal threshold is increased, we tested the animals responses to mechanical and olfactory stimuli, which are sensed in worms by distinct neurons. We find reduced responsiveness to both dish tap and dilute 1-octanol during lethargus. Following strong mechanical stimulation of the animal, the octanol response latency shortens to levels seen outside lethargus, indicating that this behavioral state is reversible. Following this strong stimulation, the animals octanol response latency lengthens again over a course of 10 minutes. When the animals are deprived of quiescence before the strong stimulation, the return to this sleep-like behavior is accelerated, showing homeostatic regulation of this behavior, as would be predicted for a sleep-like state. We used the egl-4 gf mutant ad450sd to dissociate this sleep-like behavior from the molting cycle. ad450sd adults have increased behavioral quiescence as well as an increased octanol response latency; both the quiescence and the long octanol response latencies are reversible following strong mechanical stimulation of these mutants, indicating that ad450sd mutants are not generally defective in locomotion or in their response to 1-octanol. Despite the fact that we spend one third of our lives sleeping and sleep-like states are ubiquitous among animals, the purpose of sleep remains unknown. Mammalian sleep is a time of increased biosynthetic activity and has recently been shown to be required for synaptic changes in the nervous system (Frank et al, Neuron 02). Lethargus too is a time of apparently high biosynthetic activity (Frand et al, 05). Furthermore, synaptic changes have been observed during at least the L1 lethargus (White et al 78). These intriguing similarities provide the basis for future hypothesis testing regarding the purpose of sleep-like states.

David M Raizen et al. (2005) International Worm Meeting "Characterization of behavioral quiescence in wild-type and egl-4"

David M Raizen, Meera Sundaram, Allan I Pack, Matthew Maycock

[International Worm Meeting, 2005]

In mammals, periods of quiescence usually correspond to sleep, and are controlled by circadian and homeostatic processes. Behavioral quiescence in Drosophila is also controlled by these two processes and by some of the neurochemicals that function in mammalian sleep, suggesting that the evolution of sleep is ancient. The timing of sleep maintains a constant phase relationship with the timing of expression of the circadian protein PERIOD. The purpose of sleep is unknown. Locomotion of C. elegans has been noted to stop only during the lethargus periods, which precede ecdysis. The timing of lethargus maintains a constant phase relationship with expression of the PERIOD orthologue LIN-42 (Jeon et al '99). We have been measuring the quiescence associated with lethargus by observing single worms from the late L3 until adulthood. Using a digital imaging approach, we define quiescence as a body movement of less than 10 microns in 10 seconds. We found that N2 worms have periods of quiescence only during the lethargus periods. When mechanically stimulated at various times after the onset of lethargus, the animal is able to move normally, indicating that this quiescent state is reversible, and therefore under nervous system regulation. When the animal is kept moving continuously for over 30 minutes during lethargus but not outside of lethargus, it becomes increasingly difficult to arouse, suggesting that the quiescence is important for the animal and cannot be bypassed. We are currently testing for homeostatic regulation of this behavior to address the idea that lethargus is a sleep-like state. The gene defined by eat-7(ad450sd) appears to regulate quiescent behavior. In ad450sd mutants, the intervals between lethargus periods are unchanged but there is an increase in quiescence during lethargus. ad450sd also has periods of quiescence outside lethargus. When mechanically stimulated or when starved, ad450sd mutants move normally, indicating that the increased behavioral quiescence is not explained by an inability to move well. Body size, life span, roaming behaviors, and intestinal darkness are all decreased in ad450sd. We found that ad450sd is a hypermorphic allele of the gene egl-4, which encodes a cGMP-dependent protein kinase. We will describe our characterization of 6 mutants that suppress some or all of the ad450sd phenotypes.

David M Raizen et al. (2003) International Worm Meeting "Long-term observations of L1 rest/activity cycles"

Joan C Hendricks, Allan I Pack, David M Raizen, Meera Sundaram

[International Worm Meeting, 2003]

Diurnal environmental cues consisting of light and temperature changes play an important role in control of animal behavior. Behavior of food-deprived L1s, a long-lasting developmental state, may be particularly adaptive to daily changes in conditions, and may therefore be a useful system to study diurnal changes in behavior. Using time-lapse video analysis of L1s in the absence of food, we have been observing movements of L1 for several days. We transfer ~3000 worms in solution onto an agar surface of a 3 cm plate. We videotape the worms at 30-minute intervals for 2 minutes under red light, and then analyze the videos in time-lapse mode. For some experiments, the worms are exposed to white light for 12 hours alternating with darkness for 12 hours. We have observed two distinct behavioral states. In one state, the worms are swimming rapidly across the agar surface with few changes in direction. This state is reminiscent of the roam state defined for adult worms in the presence of food by Fujiwara et al (Neuron 02). In the other state, which is reminiscent of the dwell state defined by Fujiwara et al, the worms make little to no net movement over the two minutes of observation. We define worms that had made at least one net body length movement in the two-minute observation bin as active and other worms as resting. L1s populations remain active continuously for the first day after transferring from solution onto an agar surface. After approximately one day of continuous activity, there is decay in worm activity over the next several days. This decay in activity is unlikely to be explained simply by fatigue or sickness of the worms for the following reasons: (1) the rest state is not permanent. (2) Survival of L1s maintained on an agar surface in the absence of food does not drop significantly until 7-10 days after plating, yet activity drops significantly by the second day after plating. (3) After spending a week on one agar plate, the fraction of active worms drops to approximately 0.3. If these worms are then washed off this plate and transferred to a fresh plate, the fraction active increases immediately back to nearly 1.0. We suggest that this activity decay process reflects habituation to a novel environment. Our second observation of activity relates to the rest/activity patterns within each day. We have found the when the day is divided into 12 hours of light and 12 hours of darkness, worm activity is significantly higher during the light (L) phase than during the dark (D) phase. FFT analysis of rest/activity cycles reveals a peak at a period of 24.5 hours, indicating that there is a diurnal rhythm of rest/activity under L:D conditions.

RP3439

Caenorhabditis elegans

David M Raizen et al. (2004) East Coast Worm Meeting "Behavioral quiescence during the L1 stage and its alteration in eat-7 mutants"

Meera Sundaram, Allan I Pack, David M Raizen

[East Coast Worm Meeting, 2004]

Animals display periods of behavioral quiescence, when locomotion stops. In mammals, these periods of quiescence usually correspond to sleep, and are controlled by circadian and homeostatic processes. Behavioral quiescence in Drosophila is also controlled by these two processes and by some of the same neurochemicals that function in mammalian sleep(1,2), suggesting that the genetic control of behavioral quiescence is phylogenetically ancient. Locomotion of C. elegans has been observed to stop for prolonged periods during the lethargus periods, immediately prior to the molts(3). We have been measuring the quiescence associated with the L1 lethargus by videotaping single worms from the time of hatching. We digitize images at one-minute intervals and then track the path of the worm by sending the location of the developing gonad to a spreadsheet. We have found that in N2 worms grown at 22 deg, the longest consolidated quiescent period begins at 11.5+/-0.2, consistent with the reported time of onset of the L1 lethargus. When mechanically stimulated during its predicted consolidated quiescent period, the animal is able to move normally, indicating that this quiescent state is reversible. In eat-7 , a mutant that was isolated based on small size, and decreased movement and eating when not stimulated(4), the duration of the L1 quiescent period is significantly prolonged while the time of onset of this quiescent period is unchanged. The fraction of quiescent periods in eat-7 prior to the onset of its longest quiescent period is also slightly prolonged though this difference does not reach statistical significance. Hence, eat-7 mutants display increased behavioral quiescence during the L1 stage. In addition to this L1 quiescent phenotype, we have found that adult eat-7 mutants, when left unperturbed, make fewer tracks than N2 worms on a lawn of bacteria. When mechanically stimulated however, eat-7 mutants are capable of rapid and coordinated movement, indicating that the increased behavioral quiescence cannot be explained simply by an inability to move well. eat-7 is defined by a single dominant allele, ad450sd , which maps between ced-2 and lin-1 (4), a genetic interval in which the gene egl-4 is found. Body size, life span, and roaming behaviors are increased in egl-4 mutants while these three phenotypes are decreased in eat-7 mutants, suggesting that ad450sd may be a hypermorphic egl-4 allele. This suggestion is supported by the finding that two egl-4 recessive alleles, n477 and n479 , dominantly suppress the small body phenotype of eat-7(ad450sd)/+ . We sequenced the EGL-4a cDNA in eat-7 mutants and have found a Glycine to Arginine mutation. This Glycine, located in the second cGMP binding domain, is conserved in all proteins containing a cGMP or cAMP binding domain. We will report the results of experiments testing the effect of expressing the EGL-4a cDNA with the G->R mutation in wild-type worms. (1)Hendricks et al, Neuron 2000. (2) Shaw et al, Science 2000. (3)Singh and Sulston, Nematologica 1978. (4)Avery, Genetics 1993.

DA1055

Caenorhabditis elegans