The ability of metazoans to sense and interpret the external chemical environment is conferred by the chemosensory nervous system governing the senses of smell and taste. Chemosensory neurons convey external stimuli in the form of electrical impulses, as well as by changes in gene expression. This thesis describes the genetic elucidation of a Caenorhabditis elegans molecular pathway that transduces the presence of pathogens to activate the transcription of a neuroendocrine ligand. Previously, our group has showed that secondary metabolites produced by the pathogen P. aeruginosa cause an expression pattern change of the gene coding for the C. elegans TGF-beta ligand DAF-7. Using forward and reverse genetic approaches, we identified several cGMP-related components that are essential for the pathway, including a subunit for cyclic nucleotide-gated channels, CNG-2, and a cGMP-dependent kinase, EGL-4. We show that while CNG-2 induces daf-7 expression in a calcium-dependent manner, EGL-4 likely works in a calcium-independent manner to regulate daf-7 expression. Our data suggest that EGL-4 acts by selectively promoting the transcription of neuronal genes in response to appropriate stimuli. In a separate set of experiments, we also showed that the expression of daf-7 is discretely regulated by different classes of intraflagellar transport proteins that function in cilia.

Discrimination among pathogenic and beneficial microbes is essential for host organism immunity and homeostasis. Increasingly, the nervous system of animals is being recognized as an important site of bacterial recognition, but the molecular mechanisms underlying this process remain unclear. Chapter One discusses how the nematode Caenorhabditis elegans can be used to dissect the genetic and neuronal mechanisms that coordinate behavioral responses to bacteria. In Chapter Two, we show that chemosensory detection of two secondary metabolites produced by Pseudomonas aeruginosa modulates a neuroendocrine signaling pathway that promotes C. elegans avoidance behavior. Specifically, secondary metabolites phenazine- I -carboxamide and pyochelin activate a G protein-signaling pathway in the ASJ chemosensory neuron pair that induces expression of the neuromodulator DAF-7/TGF-[beta]. DAF-7, in turn, activates a canonical TGF-P signaling pathway in adjacent interneurons to modulate aerotaxis behavior and promote avoidance of pathogenic P. aeruginosa. This chapter provides a chemical, genetic, and neuronal basis for how the behavior and physiology of a simple animal host can be modified by the microbial environment, and suggests that secondary metabolites produced by microbes may provide environmental cues that contribute to pathogen recognition and host survival. Genetic dissection of neuronal responses to bacteria in C. elegans can also lend insights into neurobiology more generally. In Chapter Three we show that loss of the lithium-sensitive phosphatase bisphosphate 3'-nucleotidase (BPNT-1) results in the selective dysfunction of the ASJ chemosensory neurons. As a result, BPNT- 1 mutants are defective in behaviors dependent on the ASJ neurons, such as pathogen avoidance and dauer exit. Acute treatment with lithium also causes reversible dysfunction of the ASJ neurons, and we show that this effect is mediated specifically through inhibition of BPNT-1. Finally, we show that lithium's selective effect on the nervous system is due in part to the limited expression of the cytosolic sulfotransferase SSU-1 in the ASJ neuron pair. Our data suggest that lithium, through inhibition of BPNT- 1 in the nervous system, can cause selective toxicity to specific neurons, resulting in corresponding effects on behavior of C. elegans. In Chapter Four I discuss the future directions for the genetic dissection of pathogen recognition in C. elegans.

Cyclic guanosine monophosphate (cGMP) functions across all known living organisms as a second messenger that amplifies extracellular signals to downstream proteins in a multiplicity of signal-transduction cascades. In humans, cGMP-dependent pathways mediate cellular responses to contribute to a large number of important biological processes within the cardiovascular, neurological, immune, and metabolic systems. In Caenorhabditis elegans, cGMP is part of a complex series of protein networks that contribute to many critical functions including neuronal patterning, plasticity, chemosensation, thermosensation, phototransduction, metabolism, and life span. Here I examine the contribution of several key cGMP-dependent proteins and pathways including the G-protein kinase EGL-4, two guanylyl cyclases DAF-11 and ODR-1, and four phosphodiesterases PDE-1, PDE-2, PDE-3 and PDE-5 to the adaptation response of the chemosensory neuron AWC. Jin Lee, Damien O'Halloran, I, and others showed that there is a temporal correlation between the cGMP-dependent translocation of EGL-4 into the nucleus of the AWC and the adaptive behavioral change in C. elegans after long-term exposure to an attractive volatile odor in the absence of food. We showed that the nuclear localization of EGL-4 in the AWC neuron is both necessary and sufficient to induce long-term adaptation. Jin Lee and Damien O'Halloran demonstrated that the loss of function of the ODR-1 or the DAF-11 guanylyl cyclase results in constitutively nuclear EGL-4 in the AWC. Damien O'Halloran, I, and others also showed that loss of function in the four phosphodiesterases PDE-1, PDE-2, PDE-3 and PDE-5 block the movement of EGL-4 into the nucleus after prolonged odor exposure. Damien O'Halloran was also able to disrupt the EGL-4 translocation event with the treatment of animals with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Through this line of evidence, we deduced that reducing cGMP levels in AWC is necessary for the translocation of EGL-4 into the nucleus thereby inducing an adaptive response. To obtain a direct readout of cGMP levels in a living animal I developed a genetically encoded single fluorescent protein cGMP biosensor called WincG that stably expresses in C. elegans. Chantal Brueggemann and I used the WincG biosensor in living C. elegans animals immobilized in microfluidic devices to measure the dynamic changes in cGMP levels in the ASER amphid sensory neuron when exposed to NaCl and in the PHB phasmid neurons when exposed to sodium dodecyl sulfate polyacrylamide and 8-bromo-cGMP. These experiments established that the WincG cGMP biosensor was fully functional and also uncovered a number of novel findings related to the timing of the changes in cGMP levels in the stimulated ASER and PHB neurons. It also brought to light patterns in the increase and decrease of cGMP levels that appear to correlate with memory acquisition and retrieval models.

Comprehensive Overview of Advances in OlfactionThe common belief is that human smell perception is much reduced compared with other mammals, so that whatever abilities are uncovered and investigated in animal research would have little significance for humans. However, new evidence from a variety of sources indicates this traditional view is likely

Escherichia coli, commonly referred to as E. coli, has been the organism of choice for molecular genetics for decades. Its machinery and mobile behavior is one of the most fascinating topics for cell scientists. Scientists and engineers, not trained in microbiology, and who would like to learn more about living machines, can see it as a unique example. This cross-disciplinary monograph covers more than thirty years of research and is accessible to graduate students and scientists alike.