Learning can be either non-associative or associative, though the molecular mechanisms underlying both remain enigmatic. The nematode Caenorhabditis elegans can adapt to both the AWC sensed odorants benzaldehyde (Bnz) and isoamyl alcohol (IsoA) and reciprocally cross-adapt. Surprisingly, however, these four adaptation permutations actually represent two distinct forms of learning: non-associative habituation and associative learning by pairing with starvation. Conditioning to the single odorant IsoA leads to both associative and non-associative memory traces, which can be preferentially accessed by choice of a Bnz or IsoA retrieval stimulus, respectively. Furthermore, we show that the molecular mechanisms underlying each form of memory can be genetically double dissociated, with insulin signalling and egl-4 being required for associative learning and osm-9 and arr-1 being essential for IsoA olfactory habituation. This represents the first demonstration where the form of learning displayed after conditioning to a single stimulus is a function of the retrieval stimulus employed.

Synapses are specialized sub-cellular junctions that transmit signals between neurons and their targets. In Caenorhabditis elegans (C. elegans) the F-box protein FSN-1 and the PHR family member RPM-1 form the SCFFSN-1 E3 ubiquitin ligase, which plays an important role in regulating synaptic growth factors. This SCF complex is evolutionarily conserved across species, and regulates many cellular processes including axon outgrowth, apoptosis and synaptogenesis. This thesis focuses on identifying targets of SCFFSN-1 that contribute to synaptogenesis. Forward genetics was employed to screens and isolate mutants that exhibit genetic interactions with fsn-1. I have identified an allele of the MAPK pmk-3(hp246) and three alleles of the MAPKKK dlk-1(hp180, hp192, hp195) that suppress fsn-1 defects. In addition, I have isolated five fsn-1 suppressing alleles and evidence suggests that these suppressors are likely novel fsn-1 suppressors.

Learning can be either non-associative or associative, though the molecular mechanisms underlying both remain enigmatic. The nematode Caenorhabditis elegans can adapt to both the AWC sensed odorants benzaldehyde (Bnz) and isoamyl alcohol (IsoA) and reciprocally cross-adapt. Surprisingly, however, these four adaptation permutations actually represent two distinct forms of learning: non-associative habituation and associative learning by pairing with starvation. Conditioning to the single odorant IsoA leads to both associative and non-associative memory traces, which can be preferentially accessed by choice of a Bnz or IsoA retrieval stimulus, respectively. Furthermore, we show that the molecular mechanisms underlying each form of memory can be genetically double dissociated, with insulin signalling and egl-4 being required for associative learning and osm-9 and arr-1 being essential for IsoA olfactory habituation. This represents the first demonstration where the form of learning displayed after conditioning to a single stimulus is a function of the retrieval stimulus employed.

The over-expression of Down syndrome cell adhesion molecules (DSCAMs) is partially responsible for the mental retardation associated with Down syndrome. Previous work in our lab showed that a DSCAM homolog in C. elegans, Y32G9A.8, is expressed at all developmental stages and appears to be crucial for survival. In an effort to map the expression pattern, I used the Genome Sciences Centre’s primer design program (http://elegans.bcgsc.bc.ca/gfp_primers/) to design a GFP promoter fusion product that was used to monitor gene expression. The results indicate that Y32G9A.8 is expressed in the animal’s gut, suggesting that it may function in the worm’s innate immune response. I also designed a primer set to amplify the Y32G9A.8 transcript. RT-PCR of the entire Y32G9A.8 coding region resulted in a single product; there appears to be no alternative splicing. Although this gene shows homology to other N-CAMS, results indicate that this gene may function in the innate immune system of C. elegans.

All complex organisms possess a nervous system which they use to monitor environmental and internal stimuli. In higher vertebrates, the nervous system is comprised of billions of cells which form highly plastic neural networks from their synapses. These large neural circuits modulate complex behaviors. The nematode roundworm Caenorhabditis elegans uses a small but highly-interconnected nervous system to carry out complex behaviors. The nervous system of C. elegans is a tractable model to determine the effects of changes on a nervous system at the systemic, cellular, genetic, and molecular levels. The C. elegans male’s nervous system detects environmental conditions, mating cues, attractants, repellents, and the location and composition of possible food sources and integrates these inputs to compute the decision of whether or not to mate. Mating behavior in the C. elegans male is regulated at a number of steps by cholinergic signaling from various sensory and sensory-motor neurons, but a comprehensive model of how cholinergic signaling controls this circuit has not yet been elucidated. Previous studies have thoroughly dissected the cellular structure, neural connectivity, and signaling pathways of the male’s peripheral circuits located in the genital regions of the animal’s tail. However, no studies have been conducted to determine what role the cephalic cholinergic neurons have in regulating mating behavior. I hypothesized that cephalic cholinergic neurons exert regulatory control over the male-specific mating circuit. I inserted the transmembrane light-activated ion pore Channelrhodopsin-2 fused to YFP and expressed from the Punc-17small promoter into these neurons and selectively stimulated them using high-intensity blue light. Stimulation induced mating-like behaviors in the male tail consistent with behaviors seen during copulation with a hermaphrodite. Using behavioral assays, I demonstrated that these behaviors were male-specific and only occurred after direct stimulation in the absence of a hermaphrodite. Incidence of mating-like behaviors increased significantly as the worm aged, and the mating circuit retained a memory of the stimulus, indicated by the latency between stimulation and onset of mating-like behaviors. Brief food deprivation, which normally downregulates excitability of the mating circuit via UNC-103 ERG-like K+ channels, caused an unexpected increase in the number of blue light-stimulated behaviors displayed. Pharmacological assays using acetylcholine (ACh) agonists showed that stimulation of the cephalic cholinergic neurons increased propensity for spicule protraction in the presence of an ACh agonist, and partially restored the decline in spicule protraction associated with temporary food deprivation. I sought to identify the cephalic cholinergic neuron or neurons responsible for regulating mating-like behavior in the tail circuits. I looked for a reduction in mating-like behaviors after stimulation after removal of a cephalic cholinergic neuron pair via laser micro-ablation. Two cholinergic and chemosensory neuron pairs in the inner labial sensilla (IL2L/R and IL2VL/R) appear to generate and/or relay the signal that induces mating-like behaviors in the tail. I hypothesize that these neurons sense environmental cues before the male contacts a mate, and modulate lasting motivational changes within the male mating circuit.

In this thesis I used Caenorhabitis elegans as a model of Fetal Alcohol Spectrum Disorder (FASD) to study the physical and behavioral effects of ethanol exposure during embryonic development. Davis et al. (2008) found that ethanol exposure during larval development in C. elegans produced physical/developmental and behavioral effects; however, whether exposure during embryonic development might produce similar outcomes remained to be elucidated. Because the type and degree of effects caused by developmental ethanol exposure was dependent on the pattern of ethanol treatment, in the first part of the thesis I investigated the physical/developmental effects of embryonic exposure to various ethanol doses, exposure durations, onsets and frequencies. I found that exposure to >30% ethanol for an hour during embryonic development was necessary to lower hatch rate, delay reproductive onset, and reduce body size in C. elegans. Furthermore, exposure during early embryonic development caused a larger effect than exposure during later stages, and multiple exposures produced a worse outcome than a single exposure for a comparable duration. In the second part of the thesis, I investigated locomotory activities and habituation of adult C. elegans exposed to various patterns of embryonic ethanol treatment. I found that the rate of locomotion was altered differently by chronic and acute embryonic ethanol exposure, but I did not find any effect in short- or long-term habituation. In summary, I have characterized the pattern of embryonic ethanol exposure necessary to produce physical/developmental effects in C. elegans, and identified the types of exposure conditions that would cause worse outcomes than others; in addition, I have found that embryonic ethanol exposure affects the rate of locomotion in C. elegans. In this thesis, I have established a foundation for the future investigation into the physical and motor defects caused by embryonic ethanol exposure in C. elegans.

L'élongation embryonnaire contrôle la transformation embryonnaire de C. elegans qui passe d'un embryon ovoïde à une larve vermiforme. C'est un modèle idéal pour la dissection de voies de signalisation qui contrôlent la morphogénèse des tissus et l'intégration de ces signaux dans les diverses couches cellulaires. L'élongation peut être divisée en deux parties : l'élongation précoce qui implique la contraction de l'hypoderme, alors que l'élongation tardive implique l'action synergique des muscles et de l'hypoderme. La contraction des filaments d'actines est régulée par le niveau de phosphorylation des chaînes légères de la myosine (mlc-4). Les GTPases Rho sont des protéines de signalisation régulées par l'action de 3 familles protéiques : les « GTPase-activating proteins » (GAPs), les « Guanine nucléotide exchange factors » (GEFs) et les « Guanine nucléotide dissociation inhibitors » (GDI). Les GTPases Rho contrôlent un large éventail de processus biologiques. Il y a trois GTPases Rho qui contrôlent l'élongation de C. elegans. Notre laboratoire a identifié une quatrième GTPase contrôlant l'élongation, CDC-42 et son régulateur, RGA-7. CDC-42 a été associée à la polymérisation de filaments d'actines dans les évènements de polarisation, de migration et de trafic membranaire (Harris KP. et Ulrich Tepass U., 2010). Nos résultats suggèrent que le gène rga-7 coderait pour trois formes protéiques résultant d'une initiation alternative de la transcription et que ces trois protéines seraient impliquées dans le contrôle de l'élongation. La délétion ok1498 induit un phénotype de létalité embryonnaire ayant une pénétrance variant entre 25 et 30%. Cette létalité est le résultat d'hypercontractions dorsales pendant l'élongation. L'activité catalytique du domaine GAP de rga-7 a révélé une affinité élevée pour la GTPases CDC-42 et faible pour les GTPases RHO-1 et MIG-2. Des analyses d'épistasies suggèrent que rga-7 contrôlerait l'activité de cdc-42 ainsi que de ses effecteurs wsp-1 et mrck-1 au cours de l'élongation. Nous émettons l'hypothèse que rga-7 pourrait contrôler la dynamique du recyclage des jonctions adhérentes (cadhérines) comme son orthologue humain probable PARG1, hypothèse que nous testerons lors d'études subséquentes. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : RGA-7, élongation, CDC-42, endocytose, GTPases, signalisation cellulaire, développement embryonnaire, filaments d'actine, jonctions adhérentes.

<p>Cadmium is a human carcinogen and a persistent environmental pollutant of increasing concern. Yet, the exact molecular targets of cadmium toxicity and the molecular mechanisms by which cadmium influences gene expression have not been fully elucidated. Therefore, the characterization of cadmium-inducible genes will provide a better understanding of the underlying mechanism involved in sensing cadmium-stress and the subsequent signaling pathways important for cellular defense against cadmium toxicity. To this end, we characterized two cadmium-responsive genes of no known biological function from the nematode Caenorhabditis elegans (C. elegans), numr-1 and numr-2.</p><p>Expression analysis of numr-1 and numr-2 revealed the same temporal and spatial expression patterns of both genes in the absence and presence of metal treatment. In the absence of metal, constitutive expression of numr-1/-2 was developmentally regulated. When adult animals were exposed to metal, numr-1/-2 expression dramatically increased. We show that worms overexpressing numr-1/-2 were more resistant to metal stress and longer lived than control animals; whereas reducing numr-1/-2 activity resulted in increased sensitivity to metal exposure. Furthermore, in the absence of metal, the two numr-1 mutant alleles, tm2775 and ok2239, exhibited decreased muscular functions. The molecular characterization of numr-1 and numr-2 also revealed that the expression of these two genes, at least in part, was regulated by changes in intracellular calcium concentrations ([Ca2+]i). This finding lead us to reevaluate the role of calcium mobilization in cadmium-induced transcription. </p><p>While several studies have indicated that exposure to cadmium resulted in increased [Ca2+]i, the mechanism by which cadmium can effect [Ca2+]i and concurrent effects on gene expression remain poorly understood. Therefore, we investigated the effects of low-level cadmium exposure, sufficient to induce transcription of cadmium-responsive genes, on the regulation of [Ca2+]i. In these studies, we utilized the protein-based calcium sensor YC 3.60 stably expressed in a HEK293 cell line. YC 3.60 is insensitive to cadmium ions, and thus is useful to monitor changes in [Ca2+]i following cadmium treatment. Exposing HEK293 cells to 1-30 µM cadmium was sufficient to induce transcription of cadmium-responsive genes such as metallothionein. Cadmium exposure from 1-10 µM had no effect on cell viability, [Ca2+]i mobilization, or increased transcriptional activity of calcium-responsive genes. In contrast, exposure to 30 µM cadmium significantly decreased cell viability, reduced intracellular calcium stores, and significantly altered the transcriptional activity of calcium-responsive genes. Taken together, these data indicate that low-level cadmium exposures (1-10 µM) can induce transcription of cadmium-responsive genes such as metallothionein independent of [Ca2+]i mobilization. </p><p>To gain further insight into the mechanistic relationship between cadmium and calcium we investigated the effects of cadmium exposure on the defecation cycle of C. elegans. Defecation is a highly rhythmic behavior that is regulated by calcium oscillations. We found that low-level cadmium exposures, sufficient to induce expression of cadmium-responsive genes such as numr-1/-2, significantly shortened the defecation cycle but did not alter the rhythm of the cycle or the magnitude of the intestinal calcium oscillations. Modulation of lipid metabolism in C. elegans results in a similar shortened defecation cycle, whereas modulation of [Ca2+]i results in lengthened and arrhythmic defection cycles, suggesting that the mechanism by which cadmium alters defecation is independent of [Ca2+]i mobilization.</p><p>In summary, the data in this work demonstrates that low-level cadmium exposure induces expression of cadmium-responsive genes independent of calcium mobilization. Thus, modulation of intracellular calcium is unlikely the primary mechanism by which cadmium regulates transcription at low-levels of exposure.</p> / Dissertation

<p>Basement membranes are dense, sheet-like forms of extracellular matrix that</p><p>surround the epithelial tissues of metazoan organisms. While these structures are</p><p>critical for epithelial support and tissue organization, basement membranes also pose</p><p>formidable barriers to most cells. However, certain specialized cells are able to breach</p><p>these barriers and move between tissues. Acquisition of cell invasive behavior by some</p><p>tumor cells is thought be an important step in cancer progression. Due to the clear basic</p><p>and clinical importance of understanding the mechanisms underlying cell invasion</p><p>through basement membranes, cell invasive behaviors has been an area of intense study.</p><p>In this work I examine a developmentally regulated model of cell invasive behavior in</p><p>the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels</p><p>basement membrane to connect two epithelial tissues, the uterus and vulva. Using this</p><p>model I identify a novel role for UNC-6/Netrin signaling during this process through basement membranes. I show that Netrin signaling is a third regulatory input for AC invasion that functions partially in parallel to fos-1a and the vulval signal. Further I link netrin signaling to the formation of invasive protrusions that penetrate basement membrane.</p> / Dissertation

The nervous system coordinates a sequence of muscle movements to give rise to animal behaviors. In complex invertebrates or lab-studied vertebrates, due to the large number of cells in their nervous systems and the complexities of their behaviors, it is difficult to address how circuits process information to direct each motor output of the behavior. In this dissertation, I used the Caenorhabditis elegans male copulation behavior as a model to address how a compact circuit coordinates different behavioral programs. Insertion of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial mating. However, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the C. elegans male cloaca is positioned over the hermaphrodite’s vulva as he attempts to insert his copulatory spicules repetitively. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, I show that contraction of the male oblique muscles is required to sustain genital contact between the sexes. These muscles are innervated by the postcloacal sensilla (p.c.s.) sensory/motor neurons, which secret ACh to activate the levamisole-sensitive AChR and the ACR-16-containing ionotropic AChR on the oblique muscles. For spicules to rhythmically thrust during genital contact, activity of the oblique muscles and the gubernacular muscles is transmitted to the spicule protractor muscles instantaneously via gap junctions between these muscles and causes shallow protractor contractions. The rhythmic protractor contractions eventually switch to sustained contraction, as the SPC sensory-motor neurons integrate information of spicule position at the vulva with inputs from the hook and cloacal sensilla. The ERG-like K+ channel, UNC-103, which decreases the spicule circuit excitability, is likely to set a threshold requirement for integration of these inputs, so that sustained spicule muscle contraction is not stimulated by fewer inputs. In addition, I demonstrate that a cholinergic signaling pathway mediated by a muscarinic acetylcholine receptor, GAR-3, is used to enhance the ionotropic AChRs-mediated fast synaptic transmission in the copulation circuit. GAR-3 is expressed in multiple cells of the copulation circuit, but mainly in the cholinergic p.c.s. neurons and SPC neurons. Activation of GAR-3 is coupled to Gαq to trigger downstream signal transduction events that modulate neurotransmitter release from these neurons. Males with a loss-of-function allele of the gar-3 gene are defective in inserting their spicules into the hermaphrodite’s vulva efficiently. Since the p.c.s. neurons regulate the male’s contact with the hermaphrodite’s vulva, and the SPC neurons are required for spicule insertion during mating, GAR-3 probably facilitates male mating behavior via enhancing synaptic transmission from these neurons to their postsynaptic partners.