"Medicine, Faculty of"@en . "Medical Genetics, Department of"@en . "DSpace"@en . "UBCV"@en . "Ardiel, Evan"@en . "2009-04-27T15:33:45Z"@en . "2008"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Many organisms change their life history, size, and shape in response to environmental\nsignals. Although touted as a \u00E2\u0080\u0098developmentally hardwired\u00E2\u0080\u0099 system, the soil-dwelling nematode\nC. elegans is no exception. Previous research has shown that sensory perception mutants are\nsmaller than wild-type worms (Fujiwara et al., 2005). This suggests that sensory input from the\nenvironment can regulate the neuroendocrine functions controlling adult body size. Based on this\nthesis and the work in Rose et al. (2005), I propose that cues from conspecifics are one source of\nsensory input capable of affecting body size. Rose et al. (2005) found that worms reared in\nisolation have a decreased response to mechanical stimulation, a down-regulation of a pre- (snb\n1) and post-synaptic (gir-1) marker in the mechanosensory neural circuit, and delayed physical\ndevelopment compared to worms reared in groups (colony worms). In this thesis I propose that\ncolony worms integrate mechanosensory and chemosensory information to modulate growth in\nresponse to the presence of another worm. Using several sensory perception mutants I\u00E2\u0080\u0099ve\nidentified the sensory neurons that are required for colony worms to grow bigger than isolated\nworms."@en . "https://circle.library.ubc.ca/rest/handle/2429/7557?expand=metadata"@en . "1237614 bytes"@en . "application/pdf"@en . "CONSPECIFIC CUES MODULATE BODY SIZE TN AENORHABDITJS ELEGANS by Evan Ardiel A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Genetics THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2008 \u00C2\u00A9 Evan Ardiel, 2008 ABSTRACT Many organisms change their life history, size, and shape in response to environmental signals. Although touted as a \u00E2\u0080\u0098developmentally hardwired\u00E2\u0080\u0099 system, the soil-dwelling nematode C. elegans is no exception. Previous research has shown that sensory perception mutants are smaller than wild-type worms (Fujiwara et al., 2005). This suggests that sensory input from the environment can regulate the neuroendocrine functions controlling adult body size. Based on this thesis and the work in Rose et al. (2005), I propose that cues from conspecifics are one source of sensory input capable of affecting body size. Rose et al. (2005) found that worms reared in isolation have a decreased response to mechanical stimulation, a down-regulation of a pre- (snb 1) and post-synaptic (gir-]) marker in the mechanosensory neural circuit, and delayed physical development compared to worms reared in groups (colony worms). In this thesis I propose that colony worms integrate mechanosensory and chemosensory information to modulate growth in response to the presence of another worm. Using several sensory perception mutants I\u00E2\u0080\u0099ve identified the sensory neurons that are required for colony worms to grow bigger than isolated worms. 11 TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables iv List of Figures v Acknowledgments vi 1 Introduction 1 1.1 Role of conspecifics in development 1 1.2 Caenorhabditis elegans 3 1.3 Sensory deprivation in C. elegans 6 1.4 General methods 11 1.1.1 Non-localized mechanical stimulation 13 1.1.2 Localized mechanical stimulation 13 1.1.3 Conditioned plates 13 1.1.4 Shared air 14 1.1.5 Strains 14 2 Results 15 2.1 Isolate-reared worms were dwarfed and had an altered egg deposition schedule 15 2.2 Modulation of body size was not density dependent 16 2.3 The dauer pheromone is not the relevant conspecific cue 17 2.4 Determining the nature of the relevant cue through environmental manipulations 19 2.4.1 Non-localized mechanical stimulation did not alter body size 19 2.4.2 Localized mechanical stimulation did not alter body size 20 2.4.3 Neither stable soluble chemical cues nor colony volatiles were sufficient to alter body size 23 2.4.4 The presence of paralyzed worms reversed the effects of isolation on body size 24 2.5 Testing sensory perception mutants for the effect of isolation on body size 28 3 Discussion 37 References 44 111 LIST OF TABLES Table2.1 .36 iv LIST OF FIGURES Figure 1.1 12 Figure 2.1 18 Figure 2.2 22 Figure 2.3 27 Figure 2.4 34 Figure 2.5 35 V ACKNOWLEDGEMENT I offer my enduring gratitude to all members of the Rankin Lab, especially Andrew Giles, Conny Lin, and Tiffany Timbers, who have been here through it all. Thank you to my supervisor, Dr. Catharine Rankin, for guiding me through grad studies and introducing me to the wonderful world of C. elegans. Special thanks to my parents, who have supported me throughout my years of education, both morally and financially. vi 1 INTRODUCTION Li Role of conspecifics in development One of the most impressive features of our development is its sensitivity to the environment in which it occurs. A common method used to assess the effects of experience on development is to study organisms that have been deprived of the experience of interest. Environmental cues that influence development can come from many sources in nature, but of particular importance to many organisms is sensory input derived from conspecifics \u00E2\u0080\u0094 organisms of the same species existing in the same space and time. In mice, sexual pheromones from adult males accelerate the onset of puberty in immature females (McLellan et aL, 1998). In rats, deprivation of conspecific interactions through isolation rearing leads to thinner visual cortices (Volkmar & Greenough, 1972) and fewer synapses per neuron (Turner & Greenough, 1985). Furthermore, rat pups separated from their mothers at birth display suppression of growth hormone release and protein synthesis (Schanberg & Field, 1987) and decreased prepulse inhibition and performance in attention tasks (Lovic & Fleming, 2004). However, the effects of this maternal deprivation can be at least partially alleviated by tactile stimulation in the form of stroking with a paintbrush, which presumably mimics licking and grooming (Schanberg & Field, 1987; Lovic & Fleming, 2004). Upon delivery, preterm babies are often isolated in specialized incubators, depriving them of much of the mechanical stimulation they would otherwise experience through interactions with their mother. Schanberg & Field (1987) demonstrated that for small, premature neonates, 15 minutes of tactile-kinesthetic stimulation three times a day led to increased weight gain and activity, as well as more mature behaviours. Conspecific mediated developmental plasticity is not limited to vertebrates: Drosophila melanogaster reared in low-density cultures had fewer Kenyon cell fibers in their mushroom 1 bodies (Heisenberg et a!., 1995). Among insects, it is not uncommon for larval development to be dependent on population density. In some cases the relevant sensory input and receptor mechanism are known. For example, in the Central American tenebrionid beetle, Zophobas rugipes. fully grown larvae do not pupate under crowded conditions, although they do continue to go through larval-larval molts. Tschinkel and Wilson (1971) demonstrated that inhibition of pupation was mediated primarily by the mechanical stimulation resulting from physical contact with other larvae. Although the relevant sensory input may vary, inhibition of pupation by crowding appears to be widespread among tenebrionids. The proposed ecological explanation for this phenomenon is intraspecific predation - the relatively immobile and defenseless prepupae are often fed upon by the large active larvae. One of the most well-known effects of population density on animal biology is the density-dependent phase polymorphisms of the locust, Schistocerca gregaria. The phases are continuous with several forms existing between the two extremes \u00E2\u0080\u0094 the shy, cryptically coloured solitarious phase and the swarm-forming, conspicuously coloured gregarious phase. Each form can differ in a number of traits (morphology, colour, physiology and behaviour) and each trait is controlled by different suites of cues operating on different time courses. For example, the behavioural response to crowding occurs in a matter of hours and is mediated by non-species specific mechanical stimulation of the hind-legs (Simpson et al., 2001), gregarious black patterning is induced by the sight and/or smell of other locusts, and full yellow patterning requires direct contact with other locusts (Lester et aL, 2005). The transition from solitary to gregarious is complex and requires stimuli exerted by volatile pheromones, contact pheromones, visual cues, and mechanical touching. 2 1.2 Caenorhabditis elegans For this thesis I studied a simple and genetically tractable model system to identify relevant conspecific cues, the neurons through which these cues are registered, and the genes required to process them. Some 40-years ago Sydney Brenner chose the nematode Caenorhabditis elegans as the ideal model system for the study of developmental biology. The worm\u00E2\u0080\u0099s small size (approximately 1-mm), short life cycle (<4-days), and ability to survive freezing make it highly amenable to laboratory studies and its mode of reproduction is ideal for genetics; self-fertilizing hermaphrodites make it easy to homozygose and maintain recessive mutations and males allow for genetic crossing. The relatively small 97 Mb genome is mapped and sequenced and thousands of mutants are currently available to researchers. Morphologically C. elegans is very simple and its development is highly deterministic. As a result, the complete neural wiring diagram and cell lineage have been worked out. The adult hermaphrodite has only 959 cells, 302 of which are neurons forming about 5000 chemical synapses, 600 gap junctions, and 2000 neuromuscular junctions (White et al., 1986). Neurite morphology is predictable and the synaptic connections vary little from worm to worm, but C. elegans has proven to be exquisitely sensitive to its environment. Paradoxically, it is its \u00E2\u0080\u0098hardwired\u00E2\u0080\u0099 development that makes it such an excellent model for studies of plasticity - worms develop independently and predictably, leading to genetically identical colonies in which the influence of the environment can be assessed directly with little worry of confounding genetic factors. Despite the vast amount of information available on nearly every aspect of the worm\u00E2\u0080\u0099s biology, remarkably little is known about its ecology. its natural habitat is not known, but it can be found in farmland and garden soil, compost heaps, and on a variety of carrier invertebrates, such as snails, slugs, and isopods, millipedes, and other arthropods. It is not clear whether the 3 carrier organisms are for transportation and/or propagation. Although dense populations are rarely found in nature (Barri\u00C3\u00A8re and Felix, 2005), the reproductive mode is optimized for rapid population growth (Barnes & Hodgkins, 1991) and a boom and bust strategy of resource depletion (Riddle & Albert, 1997). Given this ecology, conspecifics are an obvious source of biologically relevant environmental cues. The dauer pheromone is one such cue. It is composed of several structurally related ascarosides (derivatives of the dideoxysugar ascarylose; Butcher et al., 2007) constitutively secreted by worms. Its concentration is used as an indirect measure of population density, which is an extremely important environmental factor in the life history of the worm, If reared under optimal conditions, worms develop through four larval stages (Li, L2, L3, and L4) to become fully reproducing adults 3 days after fertilization, However, high population densities, high temperatures, and a poor and limited food supply can lead to a developmental diversion into larval diapause, called \u00E2\u0080\u009Cthe dauer stage,\u00E2\u0080\u009D at the end of Li. Cell ablation studies have shown that under conditions favouring nondauer development, ADF, ASI, and ASG chemosensory neurons promote \u00E2\u0080\u0098normaF development, while ASJ chemosensory neurons promote dauer development (Bargmann & Horvitz, 1991). Mutant screens have identified more than 30 genes important for diversion to the dauer life cycle. Complex, overlapping pathways speak to the exquisite sensitivity of larvae to their environment. Appropriate response to these environmental cues is highly adaptive given the critical implications of life cycle choice on reproductive survival. Dauer formation highlights the vast impact that a conspecific cue can have on the biology of C. elegans. Specialized for long-term survival and dispersal, dauer larvae appear thin and dense and exhibit distinctly different behavioural patterns from developing larvae. Pharyngeal pumping is suppressed (Cassada & Russell, 1975) and movement is limited in order to conserve 4 energy. Dauer worms do respond to touch, but do not chemotax and unlike adult worms, dauer worms thermotax to novel ambient air temperatures (Hedgecock & Russell, 1975). Dauer larvae may also climb objects and wave their body in the air (Croli & Matthews 1977), a behaviour that probably leads to insect-mediated dispersal in the natural environment. Dauer development leads to considerable reorganization of the nervous system, as several neurons adopt dauer-specific morphologies and positions. Changes in these neurons alter the worm\u00E2\u0080\u0099s perception of the environment and are likely responsible for the changes in chemotactic behaviour and recovery from the dauer state. Modification of the sensory endings may also serve to protect the neurons from the harsh environments often encountered by dauer worms. As would be expected, metabolism in the developmentally arrested worms also changes to meet the demands of long- term survival in the absence of food, Once favourable conditions return, dauer larvae recover to become \u00E2\u0080\u0098normal\u00E2\u0080\u0099 fertile adults. Recovery is mediated by the same three environmental cues (Golden & Riddle, 1984) and is more easily initiated in older dauer larvae (Riddle & Albert, 1997). The standard laboratory strain, N2, was the strain and the derivative of the strains used in the following experiments. Upon encountering a bacterial food source, N2 hermaphrodites reduce locomotion and disperse across the bacterial lawn, but N2 males actively seek out hermaphrodites, attracted to a diffusible chemical cue released by sexually mature animals (Simon and Sternberg, 2002; Chasnov et al., 2007). This chemical cue is composed of the same small signaling molecules as the dauer pheromone and at high concentrations it ceases to attract males as it signals a high population density and therefore limited resources (Butcher et al., 2007; Srinivasan et al., 2008). 1.3 Sensory deprivation in C. elegans One useful approach to studying the importance of a particular sensory input is to remove the input and see what happens. This can be done by manipulating the rearing environment or by impairing the function of sensory neurons with genetic or laser abiations. Peckol et al. (1999) demonstrated that normal axon morphology in several sensory neurons is dependent upon normal neural activity. They established an experimental paradigm for sensory deprivation using sensory mutants with defective sensory transduction. These mutations resulted in the formation of ectopic processes in the ASE, ASJ, ASI, and AWB chemosensory neurons. Sensory deprivation affected axon growth, but not axon guidance, as the ectopic processes did not appear until long after the wild-type axon reaches the nerve ring in late embryogenesis. Peckol et al. (1999) hypothesized that axon morphology of these neurons has two distinct phases of development, the activity independent initial axon outgrowth and the activity dependent maintenance of sensory neuron morphology. Apart from the neuromorphological changes described above, blocking the function of sensory neurons also results in an extended lifespan. Using sensory perception mutants, Apfeld and Kenyon (1999) demonstrated that environmental cues regulate the lifespan of C. elegans. They found that mutants with defective sensory signal transduction or unexposed or irregular sensory cilia lived longer than wild-type worms. Based on their mutant analysis, they proposed that the increased lifespan resulted from a decrease in the activity of the DAF-2 insulin/IGF- 1 pathway. In a follow-up study, Alcedo and Kenyon (2004) used laser ablation to identify specific sensory neurons with a role in longevity \u00E2\u0080\u0094 some inhibited, while others promoted it. They also showed that lifespan could be extended by RNAi knock-down of a putative chemosensory G protein-coupled receptor, str-2; providing further evidence that lifespan is influenced by the 6 perception of some unknown environmental chemical cue. Although the relevant environmental cue is likely not the dauer pheromone, as its addition did not affect longevity (Alcedo and Kenyon, 2004), there is some evidence that the cue may be derived in part from conspecifics. Kawano et al. (2005) showed that a crude extract of liquid culture medium was capable of extending lifespan via insulin signaling. This suggests that chemical cues from other worms may play a role in ageing. By manipulating the rearing environment, Rose et al. (2005) were able to directly assess the role of sensory input from conspecifics on C elegans development. They found that worms reared in isolation showed decreased adult responsiveness to mechanical stimuli, altered protein expression of both a pre- and a post-synaptic marker in the mechanosensory neural circuit, a delayed onset of egg-laying, and a smaller body size at 96-h. Forward swimming worms briefly reverse direction in response to a mechanical tap delivered to the side of the plastic Petri plate in which they reside. Rose et al. (2005) measured adult responsiveness to mechanical stimuli by tapping the Petri plate and measuring the distance the worm swam backwards. They found that adult worms reared in isolation responded less to the tap than worms reared in groups of 30 to 40 (colony worms). The neural circuit that mediates the response to a mechanical tap is composed of six sensory neurons (PVD (i) ALM (2), AVM (1), and PLM (2); Wicks and Rankin, 1995). These neurons synapse onto four pairs of command interneurons (AVD, AVB, AVA, and PVC), which synapse onto a large cluster of motor neurons. The mechanosensory neurons release glutamate and the command intemeurons express GLR- 1, a C. elegans homologue of a mammalian AMPA/Kainate-type glutamatergic receptor subunit, gir-] mutants do respond to tap, but fail to show the effect of isolation on the tap-withdrawal response (Rose et al,, 2005), thereby 7 implicating glutamate neurotransrnission in the decreased response seen in isolate-reared worms. Using confocal microscopy and transgenic worms expressing GLR-l fused to a green fluorescent protein (GFP), Rose et al. (2005) quantified GLR-1 ::GFP expression along the posterior ventral nerve cord of C. elegans and found a significant reduction in GLR-l :GFP in isolate-reared worms. This reduction came in the form of smaller, but not fewer, GLR-1 ::GFP expressing clusters. Because the GLR-l ::GFP construct did not discriminate membrane-bound and intracellular GLR- 1, the observed reduction in GLR- 1 likely resulted from degradation or removal from the cell process, as opposed to the cytosolic internalization and re-packaging seen in mammalian neurons. A down- regulation of gir-] also correlated with the decrease in tap responsiveness seen in worms that underwent long-term habituation training (Rose et al., 2003; Ebrahimi and Rankin, 2007). Changes in glutamate receptor expression have been found to affect synaptic strength and may be a mechanism for the formation of memories in mammalian systems (L\u00C3\u00BCscher & Frerking, 2001; Malinow & Malenka, 2002). Rearing condition also altered the presynaptic terminals of the mechanosensory neurons. snb-] encodes synaptobrevin, the synaptic vesicle protein that regulates vesicle fusion to the presynaptic terminal, SNB- 1 levels in the neuron can be used as an indirect measure of the number of synaptic vesicles in the pre-synaptic terminal. Rose et al. (2005) were able to quantify SNB-l expression in the mechanosensory neurons of the tap withdrawal circuit using a SNB I :GFP transgene under the control of the mec- 7 promoter. They found that isolate-reared worms had significantly smaller areas of fluorescence. Isolated worms are reared alone on a smooth agar plate and are therefore deprived of much of the mechanosensory stimulation they would otherwise experience. This suggests that the number of synaptic vesicles and glutamate receptors in the mechanosensory circuit is determined by the amount of mechanosensory input received 8 during development deprivation through isolation-rearing results in the development of weaker synaptic connections. Worms reared in isolation are not only deprived of mechanosensory stimuli, they are also deprived of the chemosensory cues of other worms. To address this issue, isolated worms were reared on plates which had once held a colony of wonns. Isolated worms reared on these conditioned plates still showed a decreased response to tap, suggesting that chemosensory input from stable chemical cues had little to no influence on the strength of the mechanosensory synapse. A role for chemosensory input could not be ruled out entirely because the conditioned plates lacked the volatile cues constitutively released by worms. However, colony worms with a mutation rendering them insensitive to chemosensory cues exhibited a greater response to tap than isolate-reared worms with the same mutation. Since these chemosensory mutants showed the isolate/colony effect on the response to tap, the requirement of chemosensory stimulation for normal development of the tap withdrawal response was ruled out. A number of studies using rodents have reversed the effects of environmental deprivation by applying mechanosensory stimulation, such as the \u00E2\u0080\u0098stroking\u00E2\u0080\u0099 of deprived rat pups. Stroking with a fine paintbrush mimics the touch stimulation normally provided to pups by the mother when she licks and grooms them (Schanberg & Field, 1987; Lovic & Fleming, 2004). If it was only the lack of mechanosensory stimulation during development that led to the decreased response to tap and the down regulation of the associated pre- and post-synaptic markers, snb-1 and gir-], respectively, then we would predict that receiving mechanical stimuli during development could rescue the effects of isolation on the tap-withdrawal response. This was indeed the case. In Rose et al (2005) mechanical stimulation was produced by putting all of the Petri plates with isolated worms into a box and repeatedly dropping the box onto a flat surface 9 from a height of about 2 inches. Such stimulation rescued the effects of isolation on the worm\u00E2\u0080\u0099s mechanosensory circuit. Brief stimulation (30 drops) anytime during development reversed the effects of isolation on tap reversal and gir-] expression in adult worms (Rai & Rankin, 2007). Low levels of stimulation early in development also rescued snb-] expression in adults, but as the age at which stimulation was administered increased, so too did the amount of stimulation required to rescue snb-.I expression: 400 drops at L2 or L3 and 800 drops at L4 and YAD. This suggests that as the worm ages, snb-] expression becomes less susceptible to environmental manipulation. The differing ability to rescue GLR-1 and SNB-1 suggests that there is not a strong relationship between pre- and post-synaptic gene expression. This emphasizes the point that alleviating the effects of isolation on one aspect of development has in no way reversed the impact of isolation on the organism as a whole. Without rescuing both the pre- and post-synaptic markers, mechanosensory behaviour is still deficient. For example, isolate-reared worms that were stimulated enough to rescue GLR- 1, but not SNB- 1, habituated to tap more rapidly than group reared worms (Rose et al., 2005). It is worth noting that the ability to relate animal behaviour to both pre- and post-synaptic elements of identified neurons has not been possible in any other organism. Rose et al. (2005) also found that isolate-reared worms were shorter and narrower than colony worms at 4-days old and showed a later onset of egg-laying. Rai and Rankin (2007) were able to reverse the effect of isolation on body size by transferring isolated worms into colonies before the end of the third larval stage. However, if isolated worms were transferred into a colony at the start of L4 or later, they remained significantly shorter than colony worms. In a complementary study, worms reared in a colony condition were transferred to individual isolate plates. if the transfer occurred at the start of Li, L2, or L3, the worms remained significantly 10 shorter than colony worms. In contrast, transferring colony worms to individual isolate plates at the start of L4 or later did not affect body size. Fuj iwara et al. (2002) showed that worms with defective sensory neurons were smaller than wild-type worms, but had a normal brood size and were not delayed in development. This suggests that environmental factors can regulate neuroendocrine functions that control the body size of C. elegans. Although many groups are investigating the molecular mechanisms underlying body size determination, few are looking at the relevant environmental factors in body size determination. Studies on isolate-reared worms (Rose et al., 2005; Rai and Rankin, 2007) suggest that sensory input from conspecifics influences development. In this thesis I further characterize the effect of isolation on body size and explore the nature of the relevant conspecific cues, the neurons through which these cues are registered, and the genes required to process them. Teasing out which conspecific cues modulate growth will be accomplished by manipulating rearing conditions and utilizing a variety of readily available sensory perception mutants. Sensory perception mutants will also be used to determine which parts of the nervous system are required to sense and respond to conspecifics. L4 General methods Worms were reared in 60 x 15-mm Petri plates filled with 10-mL nematode growth medium agar and streaked with 1-drop (10-mm in diameter) of 0P50 E. coli. To get isolated worms, one adult hermaphrodite was left to lay eggs on a Petri plate with E. coil for 30-min, then the worm and all but one egg was removed from the plate. To get colony-reared worms, 3- adult worms were left to lay 10-20 eggs and then removed. Removal of the adult worms from the plate was time 0. Colony worms were derived from at least 2, but up to 5 different colony plates, 11 except for the longitudinal study and tax-4 rescue strain, where all colony worms originated from the same plate. Plates were stored upside down in a 20\u00C2\u00B0C incubator. Unless otherwise stated, body size was measured when worms were fully reproductive adults at 96-h old. Body size was defined as the total area of a worm in a 2-D image. Worms were recorded onto iMovie using a Leica IC A camera on a Leica Wild M3Z stereomicroscope at 40 or 60x magnification (Fig. 1.1 a). Images of worms were extracted from the video and body size was measured using NIH image software (Fig. 1.1 b). Many factors could potentially influence the size of a worm and I saw considerable variability between days. Therefore I did not make any between experiment comparisons. For all experiments, ci. was set top 0.05. Figure 1.1 a b Figure 1.1 a, Leica IC A camera on a Leica Wild M3Z stereomicroscope streaming into iMovie on a Mac. b, Captured video frames were opened in NIH image and the threshold was taken to measure the worm\u00E2\u0080\u0099s body area. 12 1.4.1 Non-localized mechanical stimulation An automated plate dropper was developed to give isolated worms continual non- localized mechanical stimulation. The automated plate dropper consisted of a wooden platform hinged to a wooden frame and a BBQ rotisserie (Hens Specialty Co., Ltd., Model #3 040) Petri plates housing isolated worms were placed on the platform and the BBQ rotisserie was situated such that its tines contacted and lifted the hinged platform approximately 2-inches before dropping it back onto the wooden frame. The rotisserie ran at 1 rpm and had 4 equally spaced tines, resulting in 1-stimulus every 15-sec. The automated plate dropper was stored in a temperature and humidity controlled room (20 \u00C2\u00B1 1\u00C2\u00B0C and 40 + 5% RH). 1.4.2 Localized mechanical stimulation In order to provide mechanical stimuli that were more localized and discrete than the plate drop, I added sephadex beads and fixed worm corpses to the plates of isolated worms. Sephadex beads (50-150-pPm in diameter, Sigma Chemical Co.) were added to liquid E. coil culture at a concentration of --500-beads/mL. Plates were seeded with a single drop of the E. coli and bead mixture (25 beads per plate). In another experiment, we added the fixed worm corpse of an L4, a young adult, and an adult worm to the plates of isolated worms at time 0. Worms were fixed overnight in 4% paraformaldehyde at 4\u00C2\u00B0C. 1.4.3 Conditioned plates In order to expose isolated worms to the soluble chemical cues of others, I reared them on plates which had been chemically conditioned with conspecifics. In one experiment, plates were conditioned by placing 4 young adult worms on plates for 4-h before the isolated egg was laid. In I\u00E2\u0080\u0094, 1.) a second experiment, conditioned plates housed 3-4 worms for 50-h as they developed from Li to young adults at 20\u00C2\u00B0C. 1.4.4 Shared air In the shared air experiment, I reared large colonies of worms on the Petri plate lids of isolated worms, so that the isolated worm would remain physically separated from the colony, but would still be exposed to the volatiles of the colony. This was done by placing a 10 x 10 x 10-mm chunk of agar seeded with E. coli on the lid, to which two 4-day old worms were added. These worms established large, mixed-age colonies over the course of the experiment. Their body size was not recorded. 1.4.5 Strains The following strains used in this study were obtained from the C. elegans Genetics Centre: N2 wild-type, DR476 daf-22 \u00E2\u0080\u0098m]3O,) , MT1O89 unc-58(h495,) , PR802 osm-3(\u00E2\u0080\u0099p802,), CX4 odr-7(\u00E2\u0080\u0099ky4,), CX1O osn2-9f\u00E2\u0080\u009Dky]O,), CB1 124 che-3(e1124), CB161 1 rnec-4(e161]), CX3937 urn 4(\u00E2\u0080\u0099ky403,), CX5922 ceh-36(\u00E2\u0080\u0099ky640,), FK1 03 tax-4 (ks28,) , PR678 tax-4(v678,), CX2205 odr 3(n2]50), and CX2065 odr-1 (n]936,), The strain PS5713 tax-4(p678); Ex(odr-4p::tax-4::GFP) was a gift from Paul W. Sternberg. Putative null alleles: che-3 (eI]24) , odr-7(Icy4), osm-9(kylO), osm-3(\u00E2\u0080\u0099p802), and tax-4(p678,) have early nonsense mutations. Loss of function alleles: ceh 36(ky640), odr-3 (n2150,), and lirn-4(ky4O3) have late nonsense mutations, tax-4(ks28,.) has a missense mutation, and odr-] (n1936,) has a splice donor mutation. Gain of function alleles: mec 4(e16]]) and unc-58(n495). 14 2 RESULTS 2.1 Isolate-reared worms were dwarfed and had an altered egg deposition schedule Rose et al. (2005) showed that at 96-h post-egg lay, isolate-reared worms were shorter and narrower than worms reared in colonies. Furthermore, they found that isolate-reared worms were delayed in the onset of egg-laying. Taken together, these results suggest that isolation retards development. However, Fujiwara et al. (2002) examined gonad maturation and found that although sensory perception mutants were smaller than age-matched wild-types, they were not delayed in development. Isolated worms then may not be developmentally delayed, but smaller with a lowered fecundity or defective egg-laying or perhaps delayed only in the onset of egg- laying, as opposed to all aspects of development. To gain further insight into the effects of isolation on development, we conducted a longitudinal study in which adult body size was monitored for 6-days and total progeny was determined. Following larval development in a colony, young adults (62-h old) were transfcred to their own Petri plate with E. coli. Isolate-reared worms were similarly transferred to a new Petri plate with E. coli. From this point on, worms were transferred to fresh plates daily and body size was measured into late adulthood (Fig. 2.1 a). These data suggest that isolated worms were in fact dwarfed compared to colony worms. The maximum body size of the isolated worms was 81% of the maximum body size of the colony worms. Replicating Rose et al. (2005), an unpaired t-test showed 4-day old colony worms (n = 35) were larger than isolate-reared worms (n = 24; t(57) = 2.4.\u00E2\u0080\u009977,p = 0.016) and here we showed that isolate-reared worms never catch-up, as 8-day old isolate-reared worms (n = 24) were still smaller than 8-day old colony worms (n = 30; (52) = 2Z32,p = 0.024). I determined the total progeny produced by each isolate and colony worm by counting the number of adult worms that grew-up on each plate. No differences were found in the total 15 number of progeny produced by isolate and colony worms (colony-reared, 227.9 +1- 10, n = 31; isolate-reared, 229.6 +/-l4, n = 20; t(49) = O.ll,p = 0.92). Daily progeny production is shown in Figure 2.lc. Rose et al. (2005) found that colony worms laid significantly more eggs at the start of the egg deposition schedule and Figure 2.lc shows that isolate-reared worms produced more progeny at the end of the egg deposition schedule. Comparing the average number of eggs laid on day 8 and 9, an unpaired t-test showed that isolated worms were laying more eggs than colony worms (day 8: t(53) = 2.40,p = 0.010 and day 9: t(50) = 2.68,p = 0.0099). From this experiment, I concluded that isolated worms were smaller than colony worms and had an altered egg deposition schedule, but were no less fecund. 2.2 Modulation of body size was not density dependent Rose et al. (2005) grew worms in colonies comprising 30-40 individuals and Rai and Rankin (2007) used colonies of 20-30 individuals. Here I obtained the same results with colonies housing fewer worms (10-20). To determine if modulation of body size by conspecific stimulation was dose-dependent, I compared the body size of worms reared alone and in colonies of 2, 10, and 25 animals (Fig. 2.lb). An ANOVA showed overall differences in body size among the 4 rearing conditions (F(2,149) = 4.906,p = 0.0086). Fisher\u00E2\u0080\u0099s PLSD comparisons showed that isolated worms were smaller than worms reared in any of the 3 colony types: 2-worm colonies (p = 0.0025), 10-worm colonies (p = 0.0002), and 25-worm colonies (p = 0.0049). The number of individuals in the colony did not affect the size of its constituents, as worms in colonies of 2, 10, and 25 had body sizes that were statistically indistinguishable. Therefore, it only takes 1 worm to reverse the effect of isolation on body size. This suggests a binary response of body growth to the presence or absence of another worm. Isolated worms are alone on a smooth agar surface and 16 are therefore deprived of many of the sensory stimuli experienced by a worm reared with even one other conspecific. 2.3 The dauer pheromone is not the relevant conspecific cue The dauer pheromone is constitutively released by C. elegans and its concentration is used as an indirect measure of population density. If concentrations are too high, worms will make a developmental diversion into larval diapause, known as the dauer stage. The dauer pheromone is also used by males track down potential mating partners (Srinivasan et al., 2008). Based on this information, I hypothesized that the dauer pheromone may be the conspecific cue mediating the effect of isolation on body size. Although the gene has yet to be cloned, daf-22 mutants do not secrete the dauer pheromone (Golden and Riddle, 1985; Srinivasan et al., 2008). I measured the body size of daf-22 mutants to determine if the dauer pheromone was required for the effect of isolation on body size. I found that it was not (Fig. 2.4). Although the dauer pheromone was not present on this mutant\u00E2\u0080\u0099s colony plates, the colony worms were still larger than isolated mutants (t(55) = 2.96, p = 0.0046). Although the dauer pheromone may not be the cue inducing growth in colony worms, conspecifics are a rich source of sensory input. The next set of experiments explores what conspecific signals are sufficient for the modulation of body size. I systematically exposed isolated worms to 4 types of stimuli of which they are deprived - non-localized mechanical, localized mechanical, stable water-soluble colony chemicals, and colony volatiles. 17 Figure 2d a w 0. w .4- 0 0 0 E b Figure 2.1 a, Body size measured from the young adult stage into adulthood. Worms reared in isolation were dwarfed compared to worms reared in colonies of 10-20 worms, b, A dose-response curve for worms reared in different colony sizes. At 96-h, isolated worms are smaller than worms reared in colonies of 2, 10, or 25, but the number of worms in the colony did not influence the size of its constituents, suggesting a binary response to the presence of one other worm. c, Number of progeny produced per day by isolated and colony worms. Although total brood size was indistinguishable between isolated and colony worms, the egg deposition schedule differs, such that isolated worms laid more eggs at the end of the cycle. Error bars represent SEM, asterisks denote statistically significant differences, and numbers in bars represent N for each group. * 0.12 0.1 0.08 E 0 p4 >. 0 0 C \u00E2\u0080\u0094 colony isolate 0.12 * 0.11 _______ E 0.1.. 0p4 >. 0 0 .0 0.08 - 0.07 0.02 0 120 100 60 80 100 120 140 160 180 200 Age (h) 19 1 2 10 25 Number of worms per co\u00E2\u0080\u0099ony 60 40 20 \u00E2\u0080\u0094colony isolate * * 0 4 5 6 7 8 9 10 Age (day) 18 2.4 Determining the nature of the relevant cue through environmental manipulations 2.4.1 Non-localized mechanical stimulation did not alter body size Isolated worms exhibited a decreased behavioral response to mechanical stimulation and decreased expression of a pre- (snb-1) and post-synaptic marker (gir-]) in the neural circuit underlying this behavior (Rose et al., 2005). These effects of isolation could be reversed in isolated worms by applying small amounts of a non-localized mechanical stimulus, i.e. picking up and dropping the isolated worm\u00E2\u0080\u0099s Petri plate 30x (Rose et al., 2005; Rai and Rankin, 2007). Rai and Rankin (2007) stimulated isolated worms with up to 400 plate drops and found that they could not reverse the effect of isolation on body size. I hypothesized that greater amounts of non- localized stimulation were required to rescue the effect of isolation on body size and developed an automated plate-dropping system designed to give isolated worms continual non-localized mechanical stimulation throughout development. Worms received I plate drop every 15-sec for 96-h, for a total of 23,040 plate drops. I found that this level of stimulation had no effect on the size of a worm (Fig. 2.2a). An ANOVA comparing the body size of colony, isolated, and stimulated-isolated worms showed an overall effect of rearing environment (F(2,59) = 7.011, p = 0.00 19). Fisher\u00E2\u0080\u0099s PLSD comparisons showed that isolated worms were smaller than colony worms (p = 0.0039). Continual non-localized mechanical stimulation had no effect on body size, as stimulated isolated worms were indistinguishable from unstimulated isolated worms (p = 0.64) and smaller than colony worms (p = 0.00 14). Based on these data, I concluded that this form of mechanical stimulation is insufficient to reverse the effects of isolation on body size. One possible explanation is that the nature of the mechanical stimulus matters. The substrate vibrations induced by dropping the Petri plate were clearly different than the localized and discrete mechanosensory experience that would result from physically interacting with another 19 worm. Alternatively, the absence of mechanosensory stimulation may not be involved or may not be the only variable involved in explaining the smaller body size of isolate-reared worms. After all, isolate-reared worms are also deprived of chemosensory input from other worms. The following experiments attempt to parse out the roles of mechanosensory and chemosensory stimulation on the modulation of body size. 2.4.2 Localized mechanical stimulation did not alter body size To administer a more localized and discrete mechanical stimulus than that provided by the plate drops, I added sephadex beads (diameter: 50 \u00E2\u0080\u0094 1 50.im) to the plates of isolated worms. Sephadex were added as a source of tactile stimulation for the isolated worm, but I found that they had no effect on worm size (Fig. 2.2b). Although an overall ANOVA comparing the body size of colony, isolated, and stimulated-isolated worms was not statistically significant (F(2,87) = 2.72, p = 0.071), I was interested in specific planned comparisons decided on prior to testing and therefore still conducted the two post-hoc tests of interest. Fisher\u00E2\u0080\u0099s PLSD comparisons showed that isolated worms stimulated with sephadex beads were significantly smaller than colony worms (p = 0.045) and indistinguishable from unstimulated isolated worms (p = 0.87). This suggests that the addition of sephadex beads had no effect on the body size of isolated worms. However, a small spherical bead of 0.1-mm diameter is far from the shape of a 1-mm long roundworm. To administer a localized mechanical stimulus more similar to that experienced by worms in a colony, I used paraformaldehyde fixed hermaphrodites. In the absence of hermaphrodites, sexually mature male C. elegans will engage in exploratory mate searching behaviour, fixed hermaphrodites are capable of inhibiting this behaviour (Arantza Barrios, personal communication), suggesting fixed hermaphrodites provide a mechanosensory contact- 20 cue by which males recognize potential mates. I added fixed hermaphrodites to the plates of isolated worms to see if simply touching another worm would reverse the effect of isolation on body size. I found that worms reared in colonies of fixed worms were still dwarfed compared to colony worms (Fig. 2.2c). An overall ANOVA comparing body size of colony, isolated, and stimulated isolated worms showed an effect of rearing condition (F(2,100) = lO.96,p <0.0001). Fisher\u00E2\u0080\u0099s PLSD comparisons showed that colony worms were larger than both isolated worms (p > 0.000 1) and isolated worms stimulated with fixed worm corpses (p 0.002). The stimulated isolated worms were indistinguishable from unstimulated isolated worms (p = 0.58). Taken together, these results suggest that discrete, localized mechanical stimulation is insufficient to reverse the effect of isolation on body size. 21 Figure 2.2 96-h body size of colony worms, isolated worms, and isolated worms receiving various types of mechanical stimulation. a, Stimulated isolated worms received 23 040 plate drops over the course of development. This non-localized mechanical stimulation had no effect on the body size of the isolated worm. b, Approximately 25 sephadex beads (diameter: 50-150- were added to the plate of isolated worms as a source of localized stimulation. They had no effect on the body size of isolated worms c, The addition of 3 paraformaldehyde fixed worms to the plates of isolated worms failed to reverse the effect of isolation on body size. Error bars represent SEM, numbers in bars represent N for each group, and asterisks denote statistically significant differences. \u00E2\u0080\u00A2 colony D isolate stimuated-isolate \u00E2\u0080\u00A2 colony D isolate isolate + sephadex beads b 0.105 0.1 0.095 - ZN E E 0.09 N >. 0.085 0 0 0.08 0.075 - 0.07 Figure 2.2 a 0.105 - 0.1 0.095 ZN E .. 0.09 N >. 0.085 0 0.08 0.075 - 0.07 C 0.105 0.1 0.095 P4 0.09 N >. 0.085 . 0 0.08 0.075 0.07 \u00E2\u0080\u00A2 colony O isolate isolate + fixed worms * * 22 2.4.3 Neither stable soluble chemical cues nor colony volatiles were sufficient to alter body size Although fixed worms are the right shape, they obviously differ from living colony worms in that they cannot interact with the isolated worm upon contact and they do not secrete any chemical cues. To address this issue, isolated worms were reared on plates which had once held either 4 young adult worms for 4-h or 3-4 worms for 50-h, as they developed from the first larval stage into young adults. Rose et al. (2005) found that chemically conditioned plates had no effect on the body size of worms and I replicated that finding here (Fig. 2.3a,b). An overall ANOVA comparing the body size of colony worms, isolated worms reared on unconditioned plates, and isolated worms reared on the plates with 4-h of conditioning showed a significant effect of rearing condition (F(2,88) = 8.32,p = 0.0005). Fisher\u00E2\u0080\u0099s PLSD comparisons showed that isolated worms were smaller than colony worms (p = 0.0004). Chemical conditioning of the plates had no effect on the body size of isolated worms, as Fisher\u00E2\u0080\u0099s PLSD comparisons showed that isolated worms on conditioned plates were still smaller than colony worms (p = 0.0 15) and indistinguishable from isolated worms reared on unconditioned plates. Similar results were obtained with plates conditioned for 50-h, i.e. an overall ANOVA showed a significant effect of rearing condition (F(2,55) = 4.49, p = 0.0 16) and Fisher\u00E2\u0080\u0099s PLSD showed that isolated worms reared on chemically conditioned plates were smaller than colony worms (p = 0.0083) and indistinguishable from isolated worms reared on unconditioned plates (p = 0.46). This suggests that chemosensory input from stable conspecific chemical cues is not sufficient to reverse the effects of isolation on body size. Because chemically conditioned plates lack volatile colony cues, the next experiment tested isolated worms which shared air with a worm colony. 23 In another study, I measured the body size of isolated worms which were physically separated from worm colonies, but exposed to the volatiles they released (Fig. 2.3b). An overall ANOVA comparing the size of these worms, isolated worms, and colony worms showed an effect of rearing condition (F = 3.44, p = 0.035) and Fisher\u00E2\u0080\u0099s PLSD comparisons showed that colony worms were larger than both isolated worms (p = 0.044) and isolated worms stimulated with colony air (p = 0.03 7). Stimulated and unstimulated isolated worms were indistinguishable in terms of body size (p = 0.75). This suggests that colony volatiles alone are insufficient to modulate body size. 2.4.4 The presence of paralyzed worms reversed the effects of isolation on body size Based on the experiments in this section, I concluded that an isolated worm exposed to the stable, soluble chemical cues of a worm colony developed as if it was deprived of all sensory input from conspecifics, as did isolated worms exposed to the volatile chemical cues of a colony. Perhaps this is not surprising, as the isolated worm is already exposed to its own volatile and soluble compounds and since we\u00E2\u0080\u0099re dealing with an age-matched isogenic population, I would not expect worms to be capable of distinguishing their chemical profile from that of a genetic clone of the same age +1- 30-mm. It does not seem to be a dose-dependent phenomenon either, as the addition of one worm results in the larger body. However, simple physical contact with fixed worms was not enough to induce growth in an isolated worm. I developed three hypotheses to explain these results: 1) it is through extended two-way physical interactions (i.e. swimming together) that worms recognize each other and since the fixed worms could not move, they were not registered, 2) colony worms integrate mechanosensory and chemosensory input to \u00E2\u0080\u0098sense\u00E2\u0080\u0099 others and modulate body size accordingly, and 3) the relevant conspecific cue is a contact 24 pheromone, which would not have been deposited on the chemically conditioned plates nor released as a volatile. To test hypothesis 1, I needed to determine if extended two-way tactile interactions with a conspecific were required for the larger body size of colony worms. To test this I reared single wild-type worms in colonies of 10-20 paralyzed mutants of mixed developmental stage. If extended two-way tactile interactions with other worms induce body growth in colony worms, a wild-type worm reared in a colony of paralyzed mutants would be essentially isolated and therefore have a dwarfed body size. If on the other hand, extended two- way tactile interactions are not involved in modulating body size, the wild-type worm should be indistinguishable from colony-reared wild-type worms because paralyzed mutants are still physically present and releasing chemical cues. unc-58 encodes a TWK potassium channel subunit; gain-of-function mutations in this gene, like in the mutant I used, result in paralysis because of hypercontraction of the body wall muscle, Although unc-58 mutants are alive and physically present on the plate, they cannot respond to contact from the wild-type worm. Despite this, the presence of unc-58 worms was sufficient to induce growth in otherwise isolated worms (Fig. 2.3d). An overall ANOVA comparing wild-type worms reared in colonies of other wild- types, wild-type worms reared in unc-58 colonies, and isolate-reared wild-type worms showed an effect of rearing condition on body size (F(2, 75) =4.84,p = 0.01). Fisher\u00E2\u0080\u0099s PLSD comparisons showed that isolate-reared wild-type worms were smaller than wild-type worms reared in colonies of wild-types (p = 0.0027) and wild-type worms reared in colonies of unc-58 mutants (p = 0.047). It didn\u00E2\u0080\u0099t matter if the worms in the colony were wild-type or paralyzed, as the body size of colony-reared wild-types was indistinguishable from the body size of wild-types in unc 58 colonies (p = 0.24). Paralyzed worms are a source of simple physical contact-cues, but cannot engage in extended two-way interactions because they cannot move. Despite this, paralyzed 25 worms provide the necessary sensory input. This allowed me to refute hypothesis 1: extended two-way interactions with conspecifics result in the larger body size of colony worms. In summary, the presence of sephadex beads or fixed corpses on the plates of isolated worms had no effect on body size, but the presence of paralyzed worms resulted in a larger worm. While all three manipulations were a source of mechanosensory input, only the paralyzed worms created the chemical milieu of a colony. This suggests that chemical cues from conspecifics are involved in modulating body size. However, stable, soluble chemical cues and volatiles alone are insufficient to reverse the effect of isolation on body size (Fig. 2.3a,b,c). I had two hypotheses to explain this data: 1) colony worms integrate mechanosensory and chemosensory input to \u00E2\u0080\u0098sense\u00E2\u0080\u0099 others and modulate body size accordingly or 2) the relevant conspecific cue is a contact pheromone. To distinguish between these two hypotheses, I used genetic ablations to disrupt the function of different subsets of sensory neurons and determine which were required for colony worms to grow larger than isolated worms. 26 Figure 2.3 Figure 2.3 96-h body size of colony worms, isolated worms, and isolated worms receiving different sources of chernosensory input from conspecifics. Isolated worms were reared on chemically conditioned plates that had held either 4 young adult worms for 4-h, a, or 3 \u00E2\u0080\u00944 worms for 50-h, as they developed from Li through to young adult at 20\u00C2\u00B0C, b. Neither plate treatment had any effect on the body size of the isolated worm. c, Isolated worms were physically separated, but reared in the same Petri plate as a mixed-stage colony of worms. These isolated worms were exposed to colony volatiles, but could not touch the other worms. Sharing the same air space as a worm colony had no effect on the body size of isolated worms. d, Single N2 wild-type worms were reared in colonies of paralyzed unc-58 mutants. Although these mutants could not interact with the isolated wild-type worm, they were a source of physical contact and chemical cues. Wild-type worms reared in unc-58 colonies grew as though they were reared in wild-type worm colonies. Error bars represent SEM, numbers in bars represent N for each group, and asterisks denote statistically significant differences. I colony U isolate isolate on 4-h conditioned plate I colony b 0.105 0.1 0.095 Eg 0.09 0.085 0 0.08 * U isolate isolate on 50-h * conditioned plate 0.075 a 0.105 0.1 0.095 0.09 a) 0.085 a 0.08 0.075 0.07 C 0.105 0.1 0.095 E E 0.09 5) N U, > 0.085 0 a 0.08 0.075 0.07 I colony U isolate isolate with shared air * 0.07 d 0.105 0.1 0.095 0.09 0.085 0.08 I colony U isolate isolate + unc-58 worms 0.075 0.07 27 2.5 Testing sensory perception mutants for the effect of isolation on body size Hypothesis 1 states that colony worms integrate physical and chemical cues to \u00E2\u0080\u0098sense\u00E2\u0080\u0099 other worms and modulate body size accordingly. If this hypothesis is correct I should be able to identify specific mechanosensory and chemosensory neurons required for the effect of isolation on body size. C. elegans has thirteen putative mechanoreceptor neurons. ALM(L/R), PLM(L/R), AVM, PVM, PVD(L/R), ADE(L/R), and PDE(L/R) are situated in the body wall, while ASH(L/R), FLP(L/R), OLQ(DL/VL/DRIVR), OLL(L/R), CEP(DL/VL/DRJVR), and IL1(L/RIDL/VL/DR/VR) are in the nose-tip. ALM, PLM, AVM, and PVM are nonciliated neurons whose processes are tightly coupled to the cuticle and contain unusually large 15- protofilament microtubules unique to the touch receptor neurons (Chalfie & Suiston, 1981). They respond to gentle mechanical stimulation along the body wall and vibrations in the substrate (Chaifie et al, 1985; Wicks and Rankin, 1995). These touch cells might be activated by tactile interactions with conspecifics. Mechanotransduction in the touch receptor neurons is mediated by an ion channel complex comprised of at least 4 proteins: MEC-4, MEC-lO, MEC-2, and MEC-6. MEC-4 and MEC-lO are members of the DEG/ENaC (degenerin/epithelial Na channel) superfamily and are thought to form the pore of the channel. Gain-of-function mutations in these genes resu1t in degeneration of the touch cells (Chalfie and Sulston, 1981). We tested an allele of one of these genes: mec-4 (e161 1) (Fig. 2.4). Despite being insensitive to mechanical stimulation along the length of the body, colony worms were larger than isolate-reared worms (t(63) = 5.36, p < 0.000 1). Therefore, the 6 touch receptor neurons are not required for a colony worm to sense the presence of another. There are five specialized sense organs in the head of C. elegans: 6 outer labial sensilla, 6 inner labial sensilla, 4 cephalic sensilla, 2 deirid sensilla, and 2 amphid sensilla. The amphid is 28 the principal sensory organ of C. elegans containing chemoreceptors for both taste and smell, thermoreceptors and mechanoreceptors. It is a prominent bilaterally symmetrical structure located at the side of the head, It is composed of two non-neuronal support cells (the sheath and socket cells) and the ciliated endings of 12 neurons, eight of which make direct contact with the environment through a pore in the cuticle through. A similar chemosensory organ, the phasmid, is found in the tail. All of these sensilla contain the ciliated nerve endings of at least one sensory cell. In total C. elegans has 60 ciliated neurons that are critical for chemosensation, thermosensation, and mechanosensation. Mutants with defective cilia have impaired sensory perception, increased lifespan, and a smaller body size (Perkins et al., 1986; Apfeld and Kenyon, 1999; Fujiwara et al., 2002). To confirm the hypothesis that isolated worms are smaller than colony worms due to the sensory deprivation resulting from isolation, I tested a mutant with broad defects in sensory perception. che-3 encodes a dynein heavy chain isoform required for establishing and maintaining sensory cilia in all ciliated cells (Wicks et al., 2000). In C. elegans, all ciliated neurons serve a sensory modality. Worms with a null mutation in che-3 have impaired chemotaxis to odorants and water-soluble compounds (Bargmann et al., 1993; Wicks et al., 2000). If the large body size of colony worms is mediated by a neuroendocrine response to sensing another worm, a che\u00E2\u0080\u00943 mutant should develop as though it was reared in isolation, even when reared in colony conditions. My results supported this hypothesis. The body sizes of the isolated and colony worms were statistically indistinguishable and trending in the opposite direction of wild-types (t(79) = l.8O,p = 0.075; Fig. 2.4). This mutant failed to show the effect of isolation on body size presumably because colony worms could not sense the relevant conspecific cues. From this mutant, I concluded that ciliated sensory neurons were required to 29 modulate body size in response to conspecifics. Because ciliated neurons play a role in both mechano- and chemosensation the next step was to determine whether either or both modalities played a role in the effect of isolation on body size. To do this, I tested mutants which disrupted subsets of ciliated cells with identified functions. Of the 302 neurons in C. elegans, eight are doparninergic (Suiston, 1975). They are the cephalic (CEP), deirid (ADE), and postderid (PDE) ciliated mechanosensory neurons which would have been disrupted in the che-3 mutant. These neurons function redundantly to sense the texture of the bacterial food source (Sawin et al., 2000). Upon activation they release dopamine, which results in slowed locomotion. These neurons may also be activated by physical contact with a conspecific, cat-2 encodes the dopamine biosynthetic enzyme tyrosine hydroxylase. The dopaminedeficient cat-2 mutants failed to show the effect of isolation on body size (Fig. 2.4). An unpaired t-test showed that isolated and colony worm body size was statistically indistinguishable (t(93) = 0.22, p = 0.823). This suggests that signaling from the dopaminergic mechanosensory neurons was required for the effect of isolation on body size. Hypothesis 1 predicts that colony worms integrate mechanosensory and chemosensory input to \u00E2\u0080\u0098sense\u00E2\u0080\u0099 others and modulate body size accordingly. I propose that the dopaminergic neurons detect the mechanical input. Hypothesis I also predicts that mutations disrupting one or more chemosensory neurons should also disrupt the effect of isolation on body size. Of the 60 ciliated cells disrupted by che-3 mutations, 26 are exposed to the external environment through the amphid, phasmid and inner labial sensilia (Bargmann and Horvitz, 1991). These exposed neurons sense soluble chemical cues (taste). Formation of the distal cilia of these neurons requires osm-3, which encodes a homolog of the heavy chain subunit of heteromeric anterograde motor kinesins (Tabish et al., 1995). Mutations in osm-3 result in .3 impaired chemotaxis to water-soluble compounds because the cilia do not extend to the environment. I measured the body size of osm-3 mutants and found that despite being defective in sensing the soluble cues released by conspecifics, colony worms were still larger than isolate- reared worms (Fig. 2.4; t(55) = 2.50,p = 0.0 16). This suggests that soluble chemical cues of conspecifics do not modulate body size. To further investigate which chemosensory neurons were required for the effect of isolation on body size, I tested worms with mutations in genes required for signal transduction. Two signal transduction systems are prominent in chemosensation: one that uses a cGMP-gated channel (TAX-2/TAX-4) and one that relies upon a transient receptor potential cannel (OSM 9/OCR-2). tax-2 and tax-4, encode the cyclic nucleotide gated cation channel required for many sensory processes. tax-2 and tax-4 are coexpressed in 11 chemosensory neurons: AWC, ASE, ASG, ASJ, ASI, AWB, ASK, BAG, AQR, PQR, and URX, as well as the thermosensory neuron, AFD (Coburn and Bargmann, 1996). Worms with mutations in these genes exhibit defective thermotaxis and water-soluble and volatile chemotaxis (Komatsu et a!., 1996; Coburn and Bargmann, 1996; Hallem and Sternberg, 2008). I tested two tax-4 alleles, tax-4(ks28) and tar 4(p678), in the isolate/colony paradigm and found that neither exhibited the effect of isolation on body size (Fig. 2.4). An unpaired t-test comparing the size of tax-4(ks28) colony and isolated worms showed that they were statistically indistinguishable (t(73) = 0.75, p = 0.46), as were colony and isolated tax-4(p678) worms (t(73) = 0.28, p = 0.78). This suggests that sensing the conspecific cues that modulate body size requires tax-4. odr-] encodes a guanylyl cyclase and is believed to be a source of cGMP for TAX 2/TAX-4 signaling in AWB, AWC, ASI, ASJ, and ASK. Worms with mutations in this gene are defective in both AWC and AWB olfactory responses (L\u00E2\u0080\u0099Etoile and Bargmann, 2000). odr-1 31 mutant colony worms were larger than the isolated mutants (Fig. 2.4; t(76) 3.478,p= 0.0008), suggesting this gene was not required for the modulation of size in response to conspecifics. Apart from TAX-2/TAX-4 dependent signaling, the other major signal transduction pathway in chemosensory neurons requires the TRP channel OSM-9/OCR-2. Mutations in ocr-2 and osm-9 are thought to impair all amphid sensory functions spared in tax-4 null mutants (Colbert et al., 1997; Tobin et al., 2002). To test if the OSM-9/OCR-l pathway was required for colony worms to respond to the presence of others, I measured the body size of an osm-9 mutant (Fig. 2.4). I found that mutants reared in colonies were larger than those reared in isolation (t(63) = 3.73,p = 0.0004). This suggests that the OSM..9/OCR-2 signal transduction pathway was not required for the effect of isolation on body size. I also tested a tax-4 mutant with TAX-4 function rescued in a subset of chemosensory neurons. This rescue strain allowed me to 1) show that it was specifically a loss of tax-4 function in chemosensory neurons that caused tax-4 mutant colony worms to grow as though they were isolated and 2) identify a subset of TAX-2/TAX-4 signaling neurons whose function was sufficient to mediate the effect of isolation on body size. In the rescue strain a tax-4::GFP transgene was driven by an odr-4 promoter in the tax-4(p678) mutant background. The odr-4 promoter drives gene expression in amphid chemosensory neurons AWA, AWC, AWB, ADF, ADL, ASG, ASH, ASI, ASJ, and ASK and in the two types of phasmid neurons, PHA and PHB (Dwyer et al., 1998). TAX-4 is endogenously expressed in 6 of these: AWC, AWB, ASG, ASJ, ASI, and ASK. Colony worms of the tax-4(p678); odr-4p::tax-4::GFP rescue strain were larger than isolated worms (Fig. 2.4; t(10) 3.l4,p = 0.0 10), suggesting that tax-4 acts in the AWC, AWB, ASO, ASJ, ASI, and/or ASK to modulate body size in response to conspecifics. These 6 amphid chemosensory neurons are classified as either gustatory or olfactory neurons based on 32 their morphology. ASG(L/R), ASJ(L/R), ASI(L/R), and ASK(L/R) are eight of the 26 exposed gustatory neurons which are defective in the osm-3 mutant described earlier in this section. AWC and AWB, along with AWA are unexposed olfactory neurons embedded in the amphid\u00E2\u0080\u0099s sheath cell. To determine whether these olfactory neurons were sensing and responding to an odourant released by conspecifics, I measure the body size of a mutant with an abnormal odourant response. The G-protein alpha subunit ODR-3 is expressed in AWC, AWB, AWA, ASH, and ADF, where it acts downstream of olfactory and nociceptive 0 protein-coupled receptors. Worms chemotax to a wide range of organic volatiles, including alcohols, ketones, aldehydes, esters, amines, sulfhydryls, organic acids, aromatic and heterocyclic compounds (Bargmann et al., 1993). Overexpression or loss of function of odr-3 results in severe olfactory defects (Roayaie et al., 1998). 1 measured odr-3 mutants to determine if this gene was required for the effect of isolation on body size. I found odr-3 colony worms developed as though they were reared in isolation (Fig. 2.4). The body sizes of colony worms were statistically indistinguishable from the body sizes of isolated mutants (t(73) = 0.28, p = 0.78), suggesting that the relevant chemical conspecific cues are sensed via an odr-3 pathway. Of the odr-3 expressing cells, AWC and AWB utilize TAX-2/TAX-4 signal transduction, while AWA, ASH, and ADF utilize OSM-9/OCR-2 signal transduction. Because tax-4 and not osm-9 was required for the effect of isolation on body size I propose that the chemical conspecific cue required for the modulation of body size is sensed by AWB and/or AWC olfactory neurons. Consistent with this conclusion, experiments with the osm-3 mutant suggest that the exposed gustatory neurons are not required for conspecifics to stimulate growth in colony worms, There appears to be functional redundancy in the circuit, as cell specification mutants, ceh-36, lim-4, and odr- 7 have impaired function and 33 morphology of AWC (Koga and Oshima, 2004; Lanjuin et al., 2003), AWB (Sagasti et al., 1999), and AWA (Sengupta et al., 1994) olfactory neurons, respectively, but each mutant still grew to a larger size in colonies than in isolation (Fig, 2.4; ceh-36: t(61) = 2.22,p = 0.03; lim-4: t(96) \u00E2\u0080\u0094 2.65,p = 0.0095; odr-7: t(58) = 3.84,p = 0.0003). By way of summary, Table 1 lists the mutants I tested, the function of the mutated gene, which neurons were affected, and whether or not the mutant showed the effect of isolation on body size. Figure 2.4 Figure 2.4 For several strains, the body size of colony and isolate-reared worms was measured at 96-h. Average colony worm size for each strain is set as 100% and the average size of isolated worms for each strain is plotted as the proportion of that strain\u00E2\u0080\u0099s colony worm size. che-3, tax-4, odr-3, and cat-2 isolated mutants do not show the reduction in body size seen in wild-type (N2) worms. Error bars represent SEM and asterisks denote statistically significant differences. Raw values are plotted in Figure 2.5. 110 100 N U, 0 0 0 U -o 4- 80 70 5\u00E2\u0080\u0099) 2 > 34 Figure 2.5 0.11 0.105 Icolony 0.1 * C isolate 0.095 * \u00E2\u0080\u0098i[i11iIiii[ti1i[ [I 4\u00E2\u0080\u0099 \ (\u00C2\u00B0 .. o o S\u00E2\u0080\u0099 ec\u00E2\u0080\u009D \u00E2\u0080\u00980 Figure 2.5 Raw values of data summarized in Figure 2.4. Comparing the 96-h body size of different strains reared in colonies and in isolation. che-\u00E2\u0080\u00A23, tax-4, odr-3, and cat-2 mutants fail to show the effect of isolation on body size. Error bars represent SEM and asterisks denote statistically significant differences. N2 colony n = 59, isolate n = 30; daf-22 colony n = 42, isolate n 15; che-3 colony n = 52, isolate n = 29; mec-4 colony n = 42, isolate n = 23; cat-2 colony n = 70, isolate n = 24; osm-3 colony n = 43, isolate n = 14; tax-4(ks28) colony n = 58, isolate n = 17; tax-4(p678) colony n = 48, isolate n = 27; tax-4(p678);odr-4::tax-4 colony n = 7, isolate n = 5; odr-1 colony n = 55, isolate n = 23; osm-9 colony n = 41, isolate n = 24; odr-3 colony n = 46, isolate n = 25; lim-4 colony n = 73, isolate n = 25; ceh-36 colony n 41, isolate n = 22; odr-7 colony n = 42, isolate n = 18. 35 Table 2.1 Isolation Gene Product Allele Defect Expression pattern effect daf-22 unknown m130 fails to secrete dauer unknown yes pheromone che-3 dynein heavy chain e1124 cilia structure all ciliated neurons no mec-4 degenerin/epithelial e1611 degeneration of ALM, PLM, AVM, yes Na channel (gf) touch receptor PVM neurons cat-2 tyrosine e1112 dopamine deficient CEP, ADE, PDE hydroxylase no osm-3 kinesin heavy chain p802 cilia structure of ADF, ADL, ASE, ASG, yes exposed neurons ASH, ASI, ASJ, ASK, L2, PHA, PHB tax-4 cGMP-gated cation ks28; signal transduction AWC, AWB, AFD, no channel p678 ASE,ASG,ASI,ASJ, ASK, BAG, AQR, PQR, URX odr-1 guanylate cyclase n1936 signal transduction AWC, AWB, ASI,ASJ, ASK yes osm-9 transient receptor kylO signal transduction AWA, OLQ, ADL, 1L2, yes potential channel AWC, ASE, ADF, ASG, ASH, ASI, ASJ, ASK, FLP, OLQ, PVD odr-3 Gprotein a subunit n2150 signal transduction AWC, AWB, AWA, ASH, ADF no Iim-4 LIM homeodomain ky403 cell specification AWB, RID, RIV, protein RMD,RME,SAA,SIA yes ceh-36 Homeobox ky640 cell specification AWC, ASE yes transcription factor odr-7 Nuclear receptor ky4 cell specification AWA yes 3 DISCUSSION A neuroendocrine pathway regulated by sensory neurons is thought to be responsible for controlling adult body size in C. elegans (Fujiwara et al., 2002). Although the underlying neural network is not yet defined, it has been shown to include amphid chemosensory neurons: Fujiwara et al. (2002) found that mutations resulting in a loss of function of all ciliated sensory neurons resulted in a small body size phenotype, but that this phenotype could be reversed by restoring function to overlapping subsets of amphid chemosensory neurons. Therefore, the nervous system has a mechanism through which it can adjust body size in response to environmental signals, but what are the relevant environmental signals? Body size determination is undoubtedly affected by countless variables, including nutritional status, humidity, and temperature. It has been shown in C. elegans that there is a positive relationship between body size and the amount of food ingested (M\u00C3\u00B6rck and Pilon, 2006), as well as simply the amount of food sensed in the environment (Tam et al., 2008). In replication of these findings, I have seen that larger bacterial lawns result in larger worms (data not shown). Despite considerable variability in average body size between experiments (Fig. 2.2 & 2.3), I consistently found that worms reared in isolation were small relative to colony worms. Based on the studies reported here, I propose that sensory input from conspecifics can modulate body size. Worms reared in isolation were deprived of all conspecific interactions and displayed a dwarfed body size. This appears to be due to the sensory deprivation resulting from isolation, as several sensory perception mutants (che-3, tax-4, and odr-3) failed to show the effect of isolation on body size. odr-3 encodes a Gprotein a subunit required for olfaction and nociception (Roayaie et al., 1998) and of the required genes, is expressed in the smallest number of neurons: AWC, AWB, AWA, ASH, and ADF. This suggests that the larger body size of colony worms .3 requires activation of some subset of these 5 chemosensory neurons. In AWC and AWB the TAX-2/TAX-4 cation channel is downstream of odr-3 and in AWA, ASH, and ADF, odr-3 signals through the transient receptor potential channel, OSM-9/OCR-2. I found that mutations in tax-4, but not osm-9 disrupted the effect of isolation on body size. This suggests that the conspecific cue that results in the larger body size of colony worms is detected by the olfactory neurons AWC and/or AWB. Genetically ablating either AWC or AWB alone using cell specification mutants, ceh-36 and lim-4, respectively, did not disrupt the effect of isolation on body size. This suggests that there is functional redundancy or developmental compensation in the circuit. However, residual function in the neurons of the cell specification mutants cannot be ruled out until I have laser ablated AWB and/or AWC. If laser ablation of either cell disrupts the effect of isolation on body size, I would conclude that there was residual functioning in my cell specification mutants. If the singly ablated animals still show the effect of isolation on body size, but the doubly ablated animals do not, I would conclude that AWC and AWB function redundantly to sense the body-size modulating conspecific cue. This could also arise by developmental compensation. White et al. (2007) demonstrated developmental compensation in the neural circuit underlying male attraction to hermaphroditic pheromone. They found that genetic ablation or early laser ablation of AWC, AWA, and the male-specific sensory neuron, CEM, disrupted male chemotaxis to hermaphrodites, but ablation of any two of these neurons had no effect. This suggests functional redundancy between AWC, AWA, and CEM. However, when they performed laser ablation later in development (at the fourth larval stage), they found that a loss of any one of these neurons disrupted male chemotaxis, This shows that in unablated, wild-type animals each of these neurons function non-redundantly in the circuit, but any one of them can compensate for an early loss of either two. Functional redundancy or developmental 3 compensation may explain why I was unable to narrow the search to a single chemosensory neuron required for the effect of isolation on body size. Physical contact with other worms also plays a role in the determination of adult body size. The dopaminergic neurons of C. elegans are the cephalic (CEPV(L/R), CEPD (L/R)), dereid (ADE (L/R)), and postdereid (PDE(L/R)) mechanosensory neurons. The dopamine deficient cat-2 (tyrosine hydroxylase) mutants failed to show the effect of isolation on body size, suggesting a role for these mechanosensory neurons in the modulation of size in response to other worms. To verify that the effect of the cat-2 mutation is caused by a dopamine deficiency, future studies should isolate-rear these mutants in the presence of exogenous dopamine. In the presence of dopamine, cat-2 colony worms should be larger than isolated worms. Another interesting mutant to test would be trp-4, a gene which encodes the C. elegans homologue of the mechanosensitive TRPN channel and the gentle touch channel of the dopaminergic mechanosensory neurons. If the dopaminergic neurons are involved in sensing conspecifics, trp 4 mutants should not show the effect of isolation on body size. A major limitation to the use of sensory perception mutants in this paradigm is the potential for a floor effect to mask a reduced body size in isolated worms. These mutants are already dwarfed and may be unable to get any smaller. To address this issue, I tested sma ](ru]8) mutants. sma-1 encodes a f3H-spectrin required for normal morphogenesis. In sma-] mutants actin dissociates from the apical membrane skeleton during morphogenesis and the embryos fail to elongate (Praitis et al., 2005). As a result, sma-] mutants are less than 60% of the length of wild-type worms, a reduction far greater than that seen in sensory perception mutants, which are -8O% the length of wild-type worms (Fujiwara et al., 2002). Despite this massively dwarfed body size, I found that sma-] mutants are even smaller when reared in isolation (data not shown). Furthermore, some of the sensory perception mutants were smaller than wild-type worms and still showed the effect of isolation on body size (e.g. osm-9, Fig. 2.5). Although the possibility of a floor effect is an important caveat to keep in mind, it does not invalidate the mutant studies, which support and extend what I found by manipulating the rearing environment of N2 wild-type worms. In the environmental manipulations I found that neither physically touching worms, nor being exposed to their chemical cues was sufficient to reverse the effects of isolation on body size. There are however some important caveats to these manipulations. Firstly, the chemically conditioned plates would only contain stable chemical compounds. Secondly, in the shared air experiment, the volume of shared air is very large and the colony volatiles may have been too dilute to reach the isolated worm below. However, this is unlikely, as volatile odors can be sensed in the nanomolar range and are thought to be used for long-range chernotaxis. The mutant studies suggest that both olfactory and mechanosensory neurons are required for the effect of isolation on body size. This suggests that I should be able to restore a large body size in isolated worms that are exposed to both fixed worms and colony volatiles. The study using paralyzed worms as a source of stimulation has already demonstrated that a combination of volatile, soluble, and simple contact-cues was sufficient to reverse the effects of isolation on body size. Based on these experiments I concluded that an increase in body size in colony worms resulted from the integration mechanosensory and chemosensory input from other worms and that the chemical conspecific cues are detected by olfactory neurons AWB and/or AWC and the mechanical cues are detected by CEP and/or ADE and/or PDE. Using the neural wiring diagram described by White et al. (1986), I found that CEP, ADE and AWB are all synaptic partners with RMG, a motor and interneuron in the nerve ring. CEP and ADE synapse onto RMG and RIVIG is 40 electrically coupled to AWB. This neuron may be the site of integration for the mechanosensory and chemosensory circuits. How is information about the environment integrated by the nervous system and decoded at the genomic level to influence various aspects of development? Fujiwara et al. (2002) found that the effect of defective chemosensory organs on body size was mediated by egl-4, a gene encoding a cGMP-dependent protein kinase. egl-4 null mutants have an increased body length, altered sensory perception, and defective egg-laying (Daniels Ct al., 2000). EGL-4 is expressed in several head neurons, some muscle cells, and in the intestine (Hirose et al., 2003), but expression in sensory neurons is sufficient to rescue a wild-type body size (Fujiwara et al., 2002). This suggests tat EGL-4 plays a role in processing sensory information and regulating body size. It is thought that EGL-4 activity modulates body size by regulating expression of the TGF- 13 ligand dbl-]. Mutations in dbl-] (homologous to Drosophila\u00E2\u0080\u0099s Dpp and vertebrate\u00E2\u0080\u0099s BMP) and sma-6 (TGF- B type I receptor) result in worms that are only about half the size of wild-types. DBL-l is expressed in neurons and appears to control transcription of genes regulating body size by activating the SMA-6 receptor in the hypodermis (Suzuki et al., 1999; Yoshida et al., 2001). Rose et al., (2005) found that the effect of isolation on body size was dependent upon egl-4, suggesting a role for the TGF- B pathway in the modulation of body size in response to other worms. Animals can increase body size by increasing the number or size of their cells. Because of C. elegans\u00E2\u0080\u0099 invariant cell lineage, it is thought that an increase in cell size underlies adult growth. Lozano et al. (2006) provide compelling evidence that the TGF- B pathway regulates body size via endoreduplication of the nucleus in hypoderrnal cells. Future studies should 41 compare hypodermal cell ploidy of isolate and colony worms to test if colony worms increase size by increasing cell ploidy. But why evolve a mechanism to modulate body size in response to other worms? In nature C. elegans likely faces much intra- and interspecific competition for limited resources. Isolated worms have a smaller body size and delayed onset of egg-laying compared to worms reared in colonies. A hypothetical ecological explanation for more rapid development in colony worms is a selective pressure to produce progeny which can utilize an ephemeral food source. The absence of conspecifics may also be inhibiting growth and development in isolated worms. Perhaps the absence of conspecifics is a warning sign that this environment may not be hospitable. Alternatively, by staying small, an isolated hermaphrodite may be saving resources for its own progeny. An interesting set of experiments to shed some light on the ecological significance of my findings, will be to test how hermaphroditic C. elegans respond to colonies of males, other species of Caenorhabditis, and other species of nematode. It is important to note that although isolated and colony worms did not differ in total progeny produced, all experiments were conducted under ideal laboratory conditions. This may he masking some evolutionary tradeoff to growing big and laying eggs fast. C. elegans offers a unique opportunity to identify cells and genes involved in phenotypic plasticity - changes in life-history, size or shape in response to environmental cues. With its determinant cell lineage and predictable neural circuits, C. elegans development seems fairly predetermined and suggests it would not be a good model for studies of phenotypic plasticity. However, C. elegans turns out to be exquisitively sensitive to its environment. For example, the presence of one other worm induces growth and alters the temporal pattern of egg deposition (Fig. 2.1). C. elegans has 302 neurons. Using several well-characterized mutants I was able to 42 identify 5 sensory neurons necessary for the modulation of body size in response to conspecifics. In the future, I would like to restore function just to these neurons to test if they are sufficient for sensing and responding to conspecifics. Using the neural wiring diagram I propose RMG as an interneuron through which these inputs are integrated. Future studies can move inward and outward from here. Outward studies could identify the chemical compound or compounds and receptors modulating body size and inward studies could explore how sensory input from two modalities are integrated and registered by the genome to change something as fundamental as body size. 43 References Alcedo, J. & Kenyon, C. (2004) Regulation of C-elegans longevity by specific gustatory and olfactory neurons. 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