Browsing by Subject "C. elegans"

Basement membranes (BMs) are conserved, cell-associated, sheet-like networks of extracellular matrices that provide structural support to tissues. BMs also function as a signaling scaffold, directing cellular processes including self-renewal, adhesion, and proliferation. However, the role of BMs in mediating interactions between tissues is an active area of inquiry. Using C. elegans, which are highly amenable to genetic and visual manipulation, I investigated the role of BMs in mediating inter-tissue interactions within the reproductive system: in tissue separation (gonad and body wall muscle), in tissue linkage (uterine utse and epidermal seam), and in stem cell-niche interactions (germ stem cells and niche). To probe BM function, I used live-cell imaging of endogenous localization of BM components, conditional knockdown, RNAi screening, and genetic mutant alleles. In Chapter 1, I discuss what is known about BMs in the separation and linkages of reproductive tissues, BM function in the germ stem cell niche, and introduce the advantages of using C. elegans as a model. In Chapter 2, I explore the role of BMs in mediating niche enwrapment. In Chapter 3, I show that failed BM separation between the gonad and body wall muscle leaks germ cells into the body cavity to become enwrapped by abnormally protrusive muscle cells. In Chapter 4, I find that discoidin domain receptor-2 directs adhesion of uterine utse BM and epidermal seam BM to connect reproductive tissues. In Chapter 5, I discuss the impact of these findings.

Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode C. elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

The C. elegans insulin-like signaling network supports homeostasis and developmental plasticity. The genome encodes 40 insulin-like peptides and one known receptor. Feedback regulation has been reported, but the extent of feedback and its effect on signaling dynamics in response to changes in nutrient availability has not been determined. We measured mRNA expression for each insulin-like peptide, the receptor daf-2, components of the PI3K pathway, and its transcriptional effectors daf-16/FOXO and skn-1/Nrf at high temporal resolution during transition from a starved, quiescent state to a fed, growing state in wild type and mutants affecting daf-2/InsR and daf-16/FOXO. We also analyzed the effect of temperature on insulin-like gene expression. We found that most PI3K pathway components and insulin-like peptides are affected by signaling activity, revealing pervasive positive and negative feedback regulation at intra- and inter-cellular levels. Reporter gene analysis demonstrated that the daf-2/InsR agonist daf-28 positively regulates its own transcription and that the putative agonist ins-6 cross-regulates DAF-28 protein expression through feedback. Our results show that positive and negative feedback regulation of insulin-like signaling is widespread, giving rise to an organismal FOXO-to-FOXO signaling network that supports homeostasis during fluctuations in nutrient availability.

L1 arrest (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR, and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. We show that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

For all organisms, the ability to sense and react to noxious environments is fundamental to their survival. For multi-celled organisms this process generally involves a nervous system and an extensive network of signal transduction pathways. TRPV ion channels have been shown to participate in signal transduction in response to noxious stimuli. At the cellular level these channels function in sensing of mechanical, thermal, and osmotic stimuli, and at the organismal level they function in homeostasis and nociception. TRPV ion channels participate in nociceptive signal transduction via cation influx, but exactly how these channels function at a mechanistic level and lead to activation of the cell or induction of a specific behavior is elusive. Previous research has shown that the pore-forming unit of an ion channel is critical for channel regulation, gating, ion selectivity, and ion permeation. Various regulatory domains have been identified to date in the pore-forming unit of TRP channels and a clearer picture of channel gating is beginning to emerge, but less is known about ion permeation.

To better understand the specific domains that are critical to ion capture, selectivity, and permeation in TRPV channels, we investigated the function of these regions using the C. elegans TRPV channel OSM-9 in vivo, and the mammalian TRPV channel TRPV4 in heterologous cell culture. OSM-9 is the functional ortholog of mammalian TRPV4 and it is likely that critical domains identified in OSM-9 are functionally conserved in TRPV4 and play a similar role in other TRPV channels. OSM-9 is expressed in the ASH neurons and is responsible for all of the behaviors initiated by that cell. The stereotypical avoidance behavior mediated by ASH, in response to noxious stimuli, serves as a model for nociception in vertebrates. As OSM-9 is necessary for all of these behavioral responses, activation of ASH acts as a read-out for OSM-9 function.

Through targeted mutagenesis of the OSM-9 loop domains and transgenic expression directed to the ASH head sensory neurons in an osm-9 null background, we discovered a critical role for the amino acids both N- and C- terminal to the pore helix in osmotic avoidance behavior. We confirmed the existence of a selectivity filter C-terminal to the pore helix and revealed that the turret is critical for channel function, possibly as a component of the inactivation gate.

We first identified the boundaries of the selectivity filter to be M601-F609. We also determined what properties of those residues were critical to Ca2+ and Na+ selectivity. In vivo Ca2+ imaging strongly suggested that residues Y604, D605, and F609 are critical for Ca2+ entry into the cell. Patch-clamp electrophysiology of a chimeric ion channel consisting largely of rat TRPV4, but encompassing transmembranes 5 through 6 of OSM-9, revealed that OSM-9 conducts both Ca2+ and Na+. Mutation Y604G disrupted both Ca2+ and Na+ conductance, whereas mutations Y604F and Y606A increased or maintained Na+ conductance and severely reduced Ca2+ conductance, while maintaining avoidance behavior. Homology modeling of OSM-9, based on an alignment of OSM-9 to Kv1.2, suggests that Y604 and F609 serve structural roles in maintaining filter constraints. Thus, aromatic and negative residues in the OSM-9 selectivity filter are critical to ion permeation and selectivity.

Our studies involving the selectivity filter support previous research that the selectivity filter is critical for TRP channel function. We also provide evidence that the selectivity filter is critical for nocifensive animal behavior. Fewer studies, however, have investigated the TM5-pore helix linker, known as the turret. The turret is believed to function in the binding of ligands and toxins in K+ channels, and more recently was suggested to be critical for temperature sensing in TRPV1. We investigated the function of the turret residues in several sensory submodalities of the OSM-9 channel and found that all deletions tested result in channel defects, including gain- and loss-of-function phenotypes. Several charge reversal mutations in the OSM-9 turret also resulted in partial defects. The discovery of a gain-of-function mutation indicates that the turret functions in gating. When the turret is mutated in this way, the channel is unable to enter into the inactivated state, allowing continued ion influx after repeated stimulation. The loss-of-function phenotypes indicate that the secondary structure of the turret is critical to the function of the channel, and perhaps gating. These findings, combined with the observed charge-reversal defects, support the conclusion that the turret is necessary for transducing conformational changes in response to stimuli.

Our in vivo findings on the external pore forming structures increase the understanding of ion permeation in TRP channels and clarify mechanisms of activation in nociceptor neurons in vivo. Furthermore, these studies enhance our insights into evolution of mammalian nociception in view of the established functional orthology of OSM-9 and TRPV4.

Niches regulate stem cell fate during development and disease. It is thus critical to understand the cellular behaviors that underlie stem cell-niche interactions. Cellular enwrapment of stem cells by their niche is observed in the C. elegans germ stem cell niche, the D. rerio hematopoietic niche, and the D. melanogaster intestinal and lymph niches. This widespread niche behavior is nevertheless difficult to dissect. In the visually and genetically tractable C. elegans germ stem cell niche, the distal tip cell (DTC) functions as a single-cell niche that elaborates to form a plexus of cellular processes enwrapping germ stem cells. Using the DTC niche as a model and tools including live imaging, genetic screens, and tissue-specific knockdown, we elucidate the regulation and function of cellular enwrapping behavior in C. elegans stem cell-niche interactions. In Chapter 2, I identify a set of DTC-autonomous genes including lin-40/MTA1 that directly promote DTC plexus formation. I also identify a set of germline-autonomous genes that indirectly promote enwrapment by the DTC plexus by supporting germ progenitor cell fate, suggesting that germ progenitor cells produce a cue for enwrapment. DTC plexus formation promotes GLP-1/Notch signaling and stem cell fate in enwrapped germ cells, suggesting a positive feedback loop between the niche and stem cells that expands the stem cell pool. In Chapter 3, I show that the adhesion molecules hmr-1/cadherin and sax-7/L1CAM promote DTC plexus formation. Substantiating the idea that germ cells produce a cue for enwrapment, I show that outside the reciprocal interactions within the germ stem cell niche, escaped germ cells induce enwrapment by other somatic tissues. Outside of importance in normal niche establishment, these observations suggest that enwrapment could be coopted in disease states. Chapter 4 discusses the implications of this work.

Basement membrane (BM) is the thin, dense, highly cross-linked form of extracellular matrix that underlies all epithelia and endothelia, as well as surrounds muscle, nerve and fat. These sheet-like networks function as physiological barriers to maintain tissue homeostasis. During normal developmental processes and immune surveillance, cells invade through BM to establish tissues and fight infection. Similarly, metastatic cancer cells are thought to co-opt normal programs for BM transmigration as they spread from primary tumors and colonize distant tissues. The difficulty of visualizing cell-BM interactions during invasion in vivo has left the cellular and molecular mechanisms used to breach BM undefined. Specialized F-actin-rich matrix-degrading membrane protrusions, termed invadosomes, have been described in cultured invasive cell lines for more 30 years. Invadosomes are hypothesized to mediate BM penetration during cancer metastasis. Despite promising advances in intravital imaging technologies, however, invadosomes have yet to be observed in cells transmigrating BM in vivo, leaving their physiological relevance unclear. Anchor cell invasion in C. elegans is a simple in vivo model of cell invasion that allows for combined visual and genetic analysis of BM transmigration. In this dissertation I develop high-resolution time-lapse imaging approaches to understand the dynamic interactions that occur at the AC-BM interface during invasion. Through the course of this work we identify an integrin-based mechanism that polarizes the AC towards the BM. We further discover protrusive F-actin-based invadosome structures that mediate BM breach during anchor cell (AC) invasion. We find that in most cases only one or two invadosomes penetrate the BM and then transform into an invasive protrusion that guides the AC through a single BM gap. Using genetics and quantitative single-cell image analysis we characterize several molecular regulators of invadosome formation in vivo. Our findings establish an essential role for invadosomes during BM transmigration in vivo, and support the idea that these structures are a core, conserved element of a normal invasive cellular strategy activated during cancer metastasis.

Recently, the study of invertebrate innate immunity has garnered considerable attention after the discovery that mammalian homologues of the Drosophila melanogaster

Toll pathway play a role in mammalian innate immunity. One invertebrate model system that has begun to be intensely studied is the nematode Caenorhabditis elegans. Immunity in C. elegans has been shown to be inducible in that it responds uniquely to different pathogens. These changes in gene expression require transcription factors in order for certain genes to be transcribed. We utilized an RNA interference screen of potential transcription factors to identify the GATA transcription factor ELT-2 as a possible transcription factor involved in immunity. We then demonstrated that ELT-2 was required for resistance to a wide range of pathogens and was responsible for regulating expression of the C-type lectin clec-67, a marker of immunity.

We also studied another transcription factor known to play a role in C. elegans immune function, the FOXO transcription factor DAF-16. We specifically focused in on the role of DAF-16 in germline-deficient mutants, and we demonstrated that such mutants are resistant to many different pathogens. This led to further investigation of the germline-deficient mutant glp-4, which should also show broad range resistance to pathogens but fails to do so. Through whole genome sequencing, we identified mutations that may be responsible for the glp-4 phenotype. We also demonstrated that DAF-16 was active in glp-4 mutants, leading to us proposing a model where glp-4 plays a role in influencing C. elegans immunity besides its involvement in germline development.

Glia make up roughly half of all cells in the mammalian nervous system and play a major part in nervous system development, function and disease. Although research in the past few decades have shed light on their morphological and functional diversity, there is still much to be known about key aspects of their development such as the generation of glial diversity and the factors governing proper morphogenesis. In the work presented here I started with a forward genetic screen using amphid sheath (AMsh) glia in the model organism C. elegans and uncovered various factors that govern different aspects of glial development including glial fate specification, migration and growth. First, I identified the function of the proneural gene lin-32/Atoh1 in repressing an AMsh glial fate. Gliogenesis is a fundamental process during nervous system development, and generating the appropriate number of specific glial cell types is required for proper nervous system function. lin-32 loss of function mutants possess additional AMsh glia beyond the normal pair. Interestingly, the ectopic AMsh cells at least partially arise from cells originally fated to become CEPsh glia, suggesting that lin-32 may be involved in the specification of specific glial subtypes. I also found that lin-32 acts in parallel with two other proneural transcription factors cnd-1/NeuroD1 and ngn-1/Neurog1 in negatively regulating an AMsh glia fate. Furthermore, expression of murine Atoh1 fully rescues lin-32 mutant phenotypes, suggesting potential functional conservation during glial fate specification. Next, I found that AMsh glial migration is regulated by vitamin B12 through isoform-specific expression of PTP-3/LAR PTPR (Leukocyte-common antigen related receptor-type tyrosine-protein phosphatase). The uptake of diet-supplied vitamin B12 in the intestine was found to be critical for the expression of a long isoform of PTP-3 (PTP-3A) in neuronal and glial cells. The expression of PTP-3A then cell-autonomously regulates glial migration and synapse formation through interaction with an extracellular matrix protein NID-1/Nidogen 1. Together, my findings demonstrate that isoform-specific regulation of PTP-3/LAR PTPR expression mediates vitamin B12-dependent neuronal and glial development. These results may also help inform our understanding of neurodevelopmental and degenerative disorders linked to vitamin B12 deficiency. Finally I identified a pathway that regulates AMsh cell growth involving the conserved cis Golgi membrane protein eas-1/GOLT1B. Coordination of cell growth is essential for the development of the brain, but the molecular mechanisms underlying the regulation of glial size are poorly understood. My research shows that that eas-1 inhibits a conserved E3 ubiquitin ligase rnf-145/RNF145, which in turn promotes nuclear activation of sbp-1/ SREBP, an important regulator of sterol and fatty acid synthesis, to restrict cell growth. At early developmental stages, rnf-145 in the cis Golgi network inhibits sbp-1 activation to promote the growth of glia, and when animals reach the adult stage this inhibition is released through an eas-1-dependent shuttling of rnf-145 from the cis Golgi to the trans Golgi network to stop glial growth. Furthermore, I identified long chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA), as downstream products of the eas-1-rnf-145-sbp-1 pathway that functions to prevent the overgrowth of glia. These findings reveal a novel and potentially conserved mechanism underlying glial size control. Taken together, my research reveals several pathways that regulate different stages of AMsh glial development. Since many of these pathways are conserved, study of C. elegans glial development may also help inform our understanding of glial biology in vertebrate systems.

The innate immune system is the front line of host defense against microbial infections, but its rapid and uncontrolled activation elicits microbicidal mechanisms that have deleterious effects. Increasing evidence indicates that the metazoan nervous system, which responds to stimuli originating from both the internal and the external environment, functions as a modulatory apparatus that controls not only microbial killing pathways but also cellular homeostatic mechanisms. Exploiting simple organism Caenorhabditis elegans, we performed whole-animal screen and identified antibiotic colistin and dopamine antagonists as immune activators that target conserved immune pathways. The goal of this work is to investigate the role of dopamine signaling and underlying neuronal circuits in regulating the C. elegans innate immune response.

Through genetic and pharmacological studies, we identified a D1-like dopamine receptor, DOP-4, which suppresses the conserved PMK-1/p38 immune pathway in C. elegans. We also demonstrated that the manipulation of a dopaminergic neural circuit can alter the immune response upon pathogen infection. Previous studies showed that an octopamine receptor, OCTR-1, functions in chemosensory neurons to inhibit innate immunity. We found that OCTR-1-expressing neurons, ASH, and interneurons, AIA, are involved in controlling the resistance to pathogen infection. This work provides direct evidence that a neuronal network exists in C. elegans to orchestrate defenses against pathogen invasion.

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- β and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-β and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-β signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Basement membranes are dense, sheet-like forms of extracellular matrix that

surround the epithelial tissues of metazoan organisms. While these structures are

critical for epithelial support and tissue organization, basement membranes also pose

formidable barriers to most cells. However, certain specialized cells are able to breach

these barriers and move between tissues. Acquisition of cell invasive behavior by some

tumor cells is thought be an important step in cancer progression. Due to the clear basic

and clinical importance of understanding the mechanisms underlying cell invasion

through basement membranes, cell invasive behaviors has been an area of intense study.

In this work I examine a developmentally regulated model of cell invasive behavior in

the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels

basement membrane to connect two epithelial tissues, the uterus and vulva. Using this

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.

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.

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.

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.

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.

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.

An estimated twenty-five percent of the fifty-seven million annual deaths worldwide can be directly attributed to infectious disease. Mammals contain both adaptive and innate immune systems to deal with invading pathogens. The genetic model organism Caenorhabditis elegans lacks an adaptive immune system, which makes it a powerful model organism to study the innate immune system without the added complexity of an adaptive immune system. Multiple human pathogens can cause lethal infections in C. elegans and several C. elegans innate immune pathways have been identified that are conserved with mammals and protect the nematode from infection. The goal of this work was to identify novel bacterial virulence factors and innate immune defenses against pathogens by using the genetic model organism C. elegans. We established C. elegans as a model for Yersinia pestis infection and used this model to identify novel bacterial virulence factors that were also important for virulence in a mammalian model of infection. Previous studies demonstrated that C. elegans can identify bacterial pathogens using sensory neurons and activate an avoidance response that requires components of G-protein signaling pathways. We screened forty C. elegans strains containing mutations in chemosensory G-protein coupled receptors for altered survival on pathogen and identified npr-1 to be required for full C. elegans defense against pathogens. We found that activation of the NPR-1 nervous circuit enhances host susceptibility to microbial infection while inhibition of the circuit boosts innate immunity. This data provides the first evidence that innate immunity in C. elegans is directly linked to the nervous system and establishes the nematode as a novel system to study neuroimmunology. From this work, we have identified Y. pestis virulence-related genes and C. elegans innate immune effector genes required for innate immunity to human bacterial pathogens.