Gregory Cahill, Ph.D. - US grants

DESCRIPTION (Adapted from the applicant's abstract): The long-term goal of this project is understand basic mechanisms of circadian rhythmicity in vertebrate retina. Many aspects of retinal physiology and metabolism are regulated by circadian clocks. These clocks are set by daily cycles of environmental and physiological stimuli, resulting in appropriately timed daily rhythms of visual system function. Rhythmic retinal activities include the expression of photoreceptor genes, the turnover of photoreceptive membrane, changes in synaptic structure and function, rod-cone dominance, and the synthesis and release of retinal neuromodulators. The ubiquity of circadian rhythmicity in the retina indicates a critical role for the circadian system in maintenance of retinal health and optimal visual function. Previous studies demonstrated that a fully functional circadian clock is located within the retinal photoreceptor layer, and suggested that individual photoreceptor cells are capable of circadian rhythm generation. This clock regulates melatonin synthesis within the photoreceptor cells. The photoreceptor clock can be entrained by cycles of either light or a neuromodulator, dopamine. The circadian clock, the melatonin synthetic and regulatory mechanisms, and the transduction pathways for entrainment of the clock by light and dopamine are all preserved in a cultured photoreceptor layer preparation from the retina of Xenopus laevis. This preparation makes it possible to test hypotheses about photoreceptor clock mechanisms without the complications of interactions with other cell types. The melatonin release rhythm of photoreceptor layers will be used in the proposed experiments as a measure of circadian clock responses to experimental manipulations. The specific goal of this project is to define transduction mechanisms by which light and dopamine entrain the photoreceptor circadian clock. Previous studies showed that light and dopamine have nearly identical ultimate effects on the timing of the clock. However, recent data indicate that the transduction pathways for these signal are distinct at the second messenger level, and suggest specific hypotheses about how components of these pathways interact to entrain retinal rhythms. The experiments proposed here will determine: (1) the characteristics and identities of the dopamine receptors and photopigments that mediate entrainment; (2) the roles of membrane potential and Ca2+ fluxes in the entrainment pathways; and (3) the role of a novel protein kinase mechanism in the entrainment pathways. The results of these studies will define basic mechanisms of retinal circadian rhythmicity, and will provide important information about how therapeutic interventions may affect the retinal circadian clock.

This proposal is submitted for funding the purchase of a tabletop ultracentrifuge. This type of centrifuge is particularly useful for the isolation of cellular fractions and purification of nucleic acid samples, where small volumes, high speed and short spin times are required. The instrument will be used in three major projects: (1) Membrane preparation: isolation and reconstitution of bacterial membrane fractions will be performed in the context of the electrophysiological study of Escherichia coli porins and other bacterial ion channels. (2) Isolation of RNA by cesium gradient centrifugation will be performed from embryonic explants in the context of the study of gene regulation during neural induction of Xenopus embryos. (3) RNA will be isolated by cesium gradient centrifugation from zebrafish pineal glands in studies of gene regulation by light and the circadian clock The productivity and success of our research efforts will be greatly enhanced by the acquisition of the proposed instrument, which will allow us to perform procedures that are not feasible with the present state of our instrumentation.

LAY ABSTRACT Principal Investigator: Cahill, Gregory Proposal Number: IBN 9728697 In all organisms, internal biological clocks drive daily (or circadian) rhythms in physiological states, resulting in sleep-wake cycles and other behavioral rhythms. Genetic studies of insects and non-animal organisms have identified genes that code for many of these molecular gears, but very few clock genes have been identified in vertebrate animals. The goal of this project is to understand how the components of the circadian system interact to produce daily rhythms, and to determine how the clock is assembled during embryonic development. Recent work in this laboratory has demonstrated that clock cells are located in the pineal gland and retina, and that these cells release a hormone, melatonin, in a circadian rhythm. Behavioral activity levels are also controlled by a biological clock. The first major objective of this project is to determine the causal relationships among these phenomena. Is behavioral rhythmicity driven by melatonin rhythms, and is behavior regulated by clocks in the pineal gland, the retinas or some other clock7 The second objective is to determine when and how rhythms of melatonin and behavior are initiated during development from a single cell embryo to a fully formed vertebrate animal. Fish embryos are fertilized and develop external to the mother. This allows manipulation of physiological and environmental conditions to test hypotheses about how the biological clocks that control melatonin and behavior are initiated and set. This initial characterization of the zebrafish circadian system will provide the baseline information necessary to fully exploit emerging zebrafish genetic information and technology in future studies of vertebrate circadian clock mechanisms.

The long term goal of this project is to define basic molecular mechanisms that underlie circadian rhythms of behavior in vertebrates. A wide variety of biochemical and physiological processes are regulated by circadian clocks, resulting in coordinated daily rhythms of gene expression, metabolism, neurochemical and hormonal functions, and behavior. Disruption of the human circadian system due to disease, aging or voluntary disruption of sleep-wake cycles can lead to diminished sensory and motor performance, sleep disorders or affective disorders. An understanding of the biological basis of circadian rhythmicity will be important for undertanding and treatment of these disorders. The specific goal of this project is to identify and characterize vertebrate genes that are involved in circadian rhythmicity through mutational analysis of the zebrafish circadian system. Recent advances in zebrafish genetics and genomics, together with recently-developed methods for efficient measurement of zebrafish circadian rhythms, make this a useful and economical model system for genetic studies of vertebrate circadian clocks. The specific aims of this project are to: 1) Screen for mutations that affect behavioral circadian rhythms in matugenized zebrafish, recover mutant lines, and characterize the phenotypes of these mutations at the system and cellular levels. 2) Map these mutations on the zebrafish genetic linkage map. 3) Clone and map zebrafish homologs of known circadian clock-related genes and determine whether any of these are disrupted by the newly identified mutations. 4) Use candidate and positional cloning techniques to clone mutated clock genes. This project is expected to result in the identification of novel vertebrate circadian clock genes, and in new information about the functional roles and mechanisms of action of previously- identified clock-related genes.

Dan E. Wells Proposal # 9987937



Abstract

The Department of Biology and Biochemistry at the University of Houston proposes to establish a Research Experience for Undergraduate (REU) site in the field of Molecular and Cellular Biology. Along with a substantial cost-sharing from various administrative units at the University of Houston, NSF funds are requested which together will support twelve undergraduate students who will have the opportunity to conduct research and participate in other science-related activities during the summers of 2000-2002. The objectives of the Program are (1) to provide incentives for students to pursue a career in science by giving them the opportunity for a realistic and rewarding experience in a research environment, and (2) to make this opportunity accessible to a great diversity of students, in particular those from minority groups and those from Institutions with limited research resources. Through this multi-faceted experience, students will be provided with a rigorous, but stimulating training that will convey not only excitement about science, but also expose them to the realistic aspect of conducting basic research. Students will work closely with their mentors, who have designed realistic projects that will teach the practical and conceptual aspects of molecular and cellular Biology. In addition, during their 10-week training, the students will also become familiar with searching and assessing scientific literature, exchange of scientific ideas, and techniques for verbal and written presentation of scientific data. Workshops that expose students to issues such as ethics, safety, and career choices will also be organized. The Program will end with a poster session similar to those featured at scientific meetings, to be attended by the entire faculty and students. The participants who present the two best posters will be awarded with travel funds to attend a national meeting. The success of the Program will be evaluated through student and faculty questionnaires, and by long-term tracking of student participants.

DESCRIPTION (provided by applicant): The long-term goal of this project is to define basic molecular mechanisms that underlie circadian rhythmicity in vertebrates. Circadian clocks regulate a multitude of biochemical and physiological processes, resulting in coordinated daily rhythms of gene expression, metabolism and behavior. Disruption of the human circadian system due to disease, aging or voluntary disruption of sleep-wake cycles can contribute to diminished sensory and motor performance, sleep disorders or affective disorders. An understanding of basic circadian clock mechanisms may help to define the etiology of these disorders, and may help in the design of treatments. To approach the long-term goal, vertebrate circadian clock genes will be identified and studied by mutagenesis and antisense knockdown analyses in zebrafish. Accelerating advances in zebrafish genetics and genomics and a new method for high-throughput measurement of zebrafish circadian rhythms make this an efficient model system for these approaches. The specific aims are to: 1) Screen 1500-3000 mutagenized genomes per year for mutations that affect circadian rhythmicity, map each mutation to a chromosome arm, and make all mutants available to other researchers. 2) For selected mutants, clone the mutated gene, characterize phenotypes at the molecular, cellular and behavioral levels, and define the genes' roles in circadian rhythmicity. 3) Screen candidate clock genes for circadian function by knocking down their expression levels with antisense morpholino oligonucleotides. These mutant and morphant screens will exploit new transgenic zebrafish lines in which firefly luciferase expression is driven by the clock. Live larval fish of these lines glow rhythmically when incubated in luciferin, the substrate of luciferase. With available technology, bioluminescence rhythms can be measured simultaneously from 2,000 larvae, making a high-throughput screen for vertebrate clock genes feasible. "Early pressure" parthenogenesis will be used to reveal recessive mutations in a single generation and to produce mapping panels for centromere linkage analysis. This project is expected to identify novel vertebrate circadian clock genes, and to generate new hypotheses about the functional roles and mechanisms of action of both novel and previously identified clock gene products.

DESCRIPTION (provided by applicant): The long-term goal of this project is to define basic molecular mechanisms that underlie circadian rhythmicity in vertebrates. Circadian clocks regulate a multitude of biochemical and physiological processes, resulting in coordinated daily rhythms of gene expression, metabolism and behavior. Disruption of the human circadian system due to disease, aging or voluntary disruption of sleep-wake cycles can contribute to diminished sensory and motor performance, sleep disorders or affective disorders. An understanding of basic circadian clock mechanisms may help to define the etiology of these disorders, and may help in the design of treatments. To approach the long-term goal, vertebrate circadian clock genes will be identified and studied by mutagenesis and antisense knockdown analyses in zebrafish. Accelerating advances in zebrafish genetics and genomics and a new method for high-throughput measurement of zebrafish circadian rhythms make this an efficient model system for these approaches. The specific aims are to: 1) Screen 1500-3000 mutagenized genomes per year for mutations that affect circadian rhythmicity, map each mutation to a chromosome arm, and make all mutants available to other researchers. 2) For selected mutants, clone the mutated gene, characterize phenotypes at the molecular, cellular and behavioral levels, and define the genes' roles in circadian rhythmicity. 3) Screen candidate clock genes for circadian function by knocking down their expression levels with antisense morpholino oligonucleotides. These mutant and morphant screens will exploit new transgenic zebrafish lines in which firefly luciferase expression is driven by the clock. Live larval fish of these lines glow rhythmically when incubated in luciferin, the substrate of luciferase. With available technology, bioluminescence rhythms can be measured simultaneously from 2,000 larvae, making a high-throughput screen for vertebrate clock genes feasible. "Early pressure" parthenogenesis will be used to reveal recessive mutations in a single generation and to produce mapping panels for centromere linkage analysis. This project is expected to identify novel vertebrate circadian clock genes, and to generate new hypotheses about the functional roles and mechanisms of action of both novel and previously identified clock gene products.