Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

In Caenorhabditis elegans, body muscles extend muscle arms in a chemotropic fashion to the nearest nerve cord and serves as a model for the investigation of guided cell migration. I found that the transmembrane receptor UNC-40/DCC is required, and functions cell-autonomously to regulate muscle arm extension. Surprisingly, both the canonical ligand of UNC-40 (UNC-6/Netrin) and the extracellular domains of UNC-40 are dispensable, suggesting that UNC-40 relies on a co-receptor or other polarizing pathways to direct muscle arm extension. Furthermore, through double mutant analyses and the use of a neomorphic phenotype induced by UNC-40 over-expression, I define a distinct UNC-40 pathway in which UNC-73/Trio, the WAVE actin polymerization complex, and components of the dense body likely act downstream of UNC-40 to regulate muscle arm extension. Distinct modes of UNC-40’s function in muscle arm extension compared to its role in neurons provide a more complete understanding of how this conserved guidance receptor functions.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

In Caenorhabditis elegans, body muscles extend muscle arms in a chemotropic fashion to the nearest nerve cord and serves as a model for the investigation of guided cell migration. I found that the transmembrane receptor UNC-40/DCC is required, and functions cell-autonomously to regulate muscle arm extension. Surprisingly, both the canonical ligand of UNC-40 (UNC-6/Netrin) and the extracellular domains of UNC-40 are dispensable, suggesting that UNC-40 relies on a co-receptor or other polarizing pathways to direct muscle arm extension. Furthermore, through double mutant analyses and the use of a neomorphic phenotype induced by UNC-40 over-expression, I define a distinct UNC-40 pathway in which UNC-73/Trio, the WAVE actin polymerization complex, and components of the dense body likely act downstream of UNC-40 to regulate muscle arm extension. Distinct modes of UNC-40’s function in muscle arm extension compared to its role in neurons provide a more complete understanding of how this conserved guidance receptor functions.

The field of neuroscience has recently seen optogenetics emerge as a highly utilized and powerful method of non-invasive neural activation and inhibition. This thesis seeks to enhance the optogenetic toolbox through the design, construction, and evaluation of a number of hardware and software modules for research in Caenorhabditis elegans neuroscience. In the first aim, we combine optogenetics, microfluidics, and automated image processing, to create a system capable of high-throughput analysis of synaptic function. In the second aim, we develop a multi-modal illumination system for the manipulation of optogenetic reagents. The system is capable of multi-spectral illumination in definable patterns, with the ability to dynamically alter the intensity, color, and shape of the illumination. The illumination system is controlled by a set of software programs introduced in aim three, and is demonstrated through a set of experiments in aim four where we selectively activate and inhibit specific neural nodes expressing optogenetic reagents in freely moving C. elegans. With the ability to target specific nodes in a freely moving animal, we can correlate specific neural states to behaviors allowing for the dissection of neural circuits. Taken together, the developed technologies for optogenetic researchers will allow for experimentation with previously unattainable speed, precision and flexibility.

The nematode Caenorhabditis elegans is a model organism for understanding sensory molecules of multicellular organisms. Ovulating hermaphrodites produce putative pheromone(s) that cause male attraction. Because pheromones are produced in such small quantities, adult conditioned-media from large-scale synchronous culture is necessary to analyze these pheromones. Current protocols for culture synchronization have volume constraints that limit large-scale synchronous cultures and current methodology for adult conditioned-media production is impractical. Modification of Tangential Flow Filtration (TFF) systems was investigated for use as a method to increase the volume limits of bleach egg harvest for C. elegans culture synchronization. Also, an adult retention device built within the culture vessel was investigated to optimize the environment for aseptic conditioned-media production from dense large-scale C. elegans cultures. During this investigation, we have shown that synchronous C. elegans cultures for adult conditioned-media production can be grown at scales larger than reported before, with potential for further scale up. Our growth methodologies have also yielded denser cultures than previously achieved at large scales. Since rapid bleach harvesting appears to be the bottleneck for large-scale production of synchronous C. elegans cultures, our approach of using modified TFF systems with mesh to retain C. elegans eggs increased the amount of eggs that could be bleach harvested at one time. Using this method we have been able to achieve up to 5x103 synchronous C. elegans per mL at a 50L scale. Since scale-up of TFF is straightforward, our results suggest that the technique reported here can easily be applied to larger scale systems for production of adult conditioned-media for C. elegans. Further, the adult retention device within the culture vessel can ensure that the whole process remains aseptic.

<p>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 <em>Caenorhabditis elegans</em> 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 <em>C. elegans</em> and several <em>C. elegans</em> 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 <em>C. elegans</em>. We established <em>C. elegans</em> as a model for <em>Yersinia pestis</em> 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 <em>C. elegans</em> can identify bacterial pathogens using sensory neurons and activate an avoidance response that requires components of G-protein signaling pathways. We screened forty <em>C. elegans</em> strains containing mutations in chemosensory G-protein coupled receptors for altered survival on pathogen and identified <em>npr-1</em> to be required for full <em>C. elegans</em> 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 <em>C. elegans</em> is directly linked to the nervous system and establishes the nematode as a novel system to study neuroimmunology. From this work, we have identified <em>Y. pestis</em> virulence-related genes and <em>C. elegans</em> innate immune effector genes required for innate immunity to human bacterial pathogens.</p> / Dissertation

The nematode Caenorhabditis elegans, with its sequenced genome, compact nervous system and stereotyped behaviors is an ideal model organism in which to study the integration of sensory input with motor output. Male mating behavior is among the most complex of these behaviors and males utilize a number of sensory organs in its execution. Among these are the rays, which are nine pairs of sensory organs that are arranged laterally along the male tail. Each ray is composed of two ultra-structurally distinct neuron types, an A type and a B type, surrounded by a glia-like structural cell. Though compositionally identical, each pair of rays maintains a unique, genetically-hardwired identity based on wiring, morphology, and neurotransmitter fate. Three techniques were used to investigate the role of the rays in male mating behavior. First, cauterization of the tips of the rays removed their sensory endings, leading to ray neuron death. Second, a heterologous light-activated cation channel was utilized to activate specific ray neuron types. Finally, ray neuron types were genetically targeted to undergo apoptosis by expression of heterologous caspases. The results show that the rays play important roles in multiple steps of male mating behavior, including contact response, scanning, and turning. The rays as a whole mediate posture change and backing during contact response. The ability to respond to hermaphrodite contact is shared among the rays, as is initiation of backward locomotion, though all rays are required for efficient, prolonged backward scanning. Both A and B neuron types appear capable of initiating contact response. Direct activation of B neurons through ChR2 causes a contact response-like ventral tail flexure, and elimination of both A and B neurons reduces contact response. A neurons additionally have a unique role in turning.

The objective of this research is to develop automated and integrated microsystems for high-resolution imaging and high-throughput phenotyping / laser ablation of C. elegans. These microsystems take advantage of microfluidic technology for precisely handling animals and computer-aid automation for high-throughput processing. We demonstrated automated and high-throughput imaging / sorting and laser ablation of C. elegans. This thesis work is divided into four parts: development of a microsystem for imaging and sorting, development of a microsystem for laser cell ablation, development of a novel temperature measurement method, and development of pressure measurement method in microchannels. First, a microsystem was developed for high-throughput microscopy at high resolution and sorting. The microfluidic chip integrates novel microfluidic components to trap, position, immobilize, and sort/release animals. To characterize device operation and aid design of the device numerical models were developed. The experimental results demonstrate that the device operates robustly in a completely automatable manner. Additionally, a sophisticated control algorithm developed by Matthew Crane (Dr. Hang Lu¡¯s lab) automates the entire process of image acquisition, analysis, and sorting, which allows the system to operate without human intervention. This microsystem sorted worms based on their fluorescent expression pattern with over 95% accuracy per round at a rate of several hundred worms per hour. Secondly, the technologies developed for the imaging/sorting system were adapted and further improved to develop a microsystem for high-throughput cell laser ablation of C. elegans. The multiplex ablation module combined with the embryo trap module enables robust manipulation of embryos/L1-stage C. elegans. In addition, software for image processing and automation was developed to allow high-throughput cell ablations. This system performed ablation of a large number of animals and demonstrated accurate ablation by showing behavioral defects of the ablated worms in a chemotaxis avoidance assay. Thirdly, to aid future development of the microdevices, a novel in situ method for three-dimensionally resolved temperature measurement in microchannels was developed. This method uses video-microscopy in combination with image analysis software (developed by Jaekyu Cho in Dr. Victor Breedveld¡¯s group) to measure Brownian diffusion of nanoparticles that is correlated to temperature. This method offers superior reproducibility and reduced systematic errors. In addition, we demonstrated that this method can be used to measure spatial temperature variations in three dimensions in situ. Lastly, a method for pressure measurement in microdevices was also developed through collaboration with Hyewon Lee (Dr. Hang Lu¡¯s lab) to aid further device optimization. These micro pressure-sensors are composed of two flow layers with a polydimethylsiloxane (PDMS) membrane in between. The membrane deforms as a function of pressure and its deformation is quantified by a simple image-based method. These sensors offer high-precision pressure measurement in broad sensing ranges. In addition, a pressure transduction scheme combined with imaging-based method enables multiplex pressure measurement for simultaneously detecting pressures in multiple locations in a microsystem. Overall, the technologies developed in this thesis will establish a solid basis for continuous improvement of the microsystems for multi-cellular model organisms. This high-throughput technology will facilitate a broad range of biological and medical research.