Thomas Kidd - US grants

Adhesion Molecule; Axon; Binding; Binding (Molecular Function); Blood Vessels; Brain; CRISP; Cell Adhesion Molecules; Cell Communication and Signaling; Cell Signaling; Cells; Chemicals; Chromosome 21; Chromosomes, Human, Pair 21; Class; Computer Retrieval of Information on Scientific Projects Database; Condition; Cues; Diabetes Mellitus; Down Syndrome; Down Syndrome Cell Adhesion Molecule; Down's Syndrome; Downs Syndrome; Embryo; Embryonic; Encephalon; Encephalons; Environment; Extracellular Protein; Fiber; Funding; Genes; Genetic Screening; Grant; Individual; Institution; Intracellular Communication and Signaling; Investigators; Knowledge; Langdon Down syndrome; Medulla Spinalis; Molecular Interaction; Mongolism; Myelopathy, Traumatic; NIH; NRVS-SYS; National Institutes of Health; National Institutes of Health (U.S.); Nerve; Nerve Cells; Nerve Unit; Nervous; Nervous System; Nervous System, Brain; Nervous system structure; Neural Cell; Neurocyte; Neurologic Body System; Neurologic Organ System; Neurons; Patients; Phenotype; Protein Family; Proteins; Receptor Protein; Receptor Signaling; Research; Research Personnel; Research Resources; Researchers; Resources; Signal Transduction; Signal Transduction Systems; Signaling; Source; Spinal Cord; Spinal Cord Trauma; Spinal Trauma; Spinal cord injured; Spinal cord injuries; Spinal cord injury; Surface; Symptoms; Thinking; Thinking, function; Trisomy 21; United States National Institutes of Health; Work; axon growth cone guidance; axon guidance; biological signal transduction; cell adhesion protein; cell type; chromosome 21 trisomy syndrome; congenital acromicria syndrome; congenital cardiac disorder; congenital heart disease; congenital heart disorder; diabetes; gene product; morbus Down; netrin receptor; netrin-1 receptors; neuronal; novel; pseudohypertrophic progressive muscular dystrophy; receptor; trisomy 21 syndrome; vascular

In the developing embryo, growing nerves have to navigate long distances to find their targets and to connect up the nervous system. The individual fibers of the nerves are known as axons, and understanding how axons navigate is of great interest as it explains how the initial complexity of the brain and nervous system arises. Growing axons respond to cues in their environment that can either be attractive or repulsive; the same cues can also stimulate overall growth. The Down Syndrome Cell Adhesion Molecule (Dscam) gene is present in most organisms with a nervous system. Dscam unexpectedly allows axons to detect well-known navigational cues and to respond to them. The working model that drives much of the work in this proposal is that Dscam interprets the cues as attractive signals and stimulates axon growth towards the cues. This model is being tested in the fruit fly Drosophila using a genetic approach. The fruit fly nervous system is simple enough to observe the growth of single axons and to analyze how axons are affected when Dscam activity is reduced or absent. The results from the experiments will allow evaluation of whether all or part of the model is correct. The work is supported by experiments on tissue culture cells examining how Dscam and the cues physically interact. The project provides state of the art genetic and molecular biology training for both graduate and undergraduate students. The results derived from this study are expected to advance our knowledge of how axons grow and navigate in the fruit fly, as well as in many other nervous systems including the vertebrate spinal cord.

DESCRIPTION (provided by applicant): About one in five thousand babies are born lacking neurons in the lowest part of the intestine. This condition is known as Hirschsprung Disease (HSCR) and requires corrective surgery. The progenitors of the affected neurons are born adjacent to the developing spinal cord and subsequently migrate to populate the entire length of the digestive tract, forming the enteric nervous system (ENS). The neural precursors use a receptor tyrosine kinase, RET, to detect and migrate towards a chemoattractive ligand, Glial Cell Line-Derived Neurotrophic Factor (GDNF). Genetic analysis in humans and mice supports a central role for RET signaling in HSCR, yet the actual role in vivo is under debate. A major challenge for the ENS field is to identify the molecular signals required for ENS formation in vivo. We believe that using Drosophila, with its powerful genetics, we can uncover these signals. Preliminary data indicates that Drosophila Ret is required for ENS formation, and we have identified an additional, parallel signaling pathway. Specific aim #1 will confirm and extend these findings. There is a remarkable conservation of molecular function between invertebrates and humans, so we believe our results will be relevant to HSCR in humans. Straightforward genetic analysis will determine whether Ret and a novel unrelated pathway are required for migration, proliferation, differentiation or axon guidance of ENS cells in vivo (aim #1). This information will be relevant to HSCR, and to adult ENS conditions, such as seen in diabetes. We have identified a candidate co-receptor and ligand and are testing these in tissue culture for physical association and signaling potential (specific aim #2). This could create an opportunity to analyze GDNF signaling in a simple organism, which would have relevance to non-ENS conditions such as Parkinson's disease. We will also develop new reagents for studying Drosophila ENS formation based on Ret promoter analysis (specific aim #3). Preliminary evidence suggests that the parallel signaling pathway we have uncovered will be relevant to vertebrate ENS formation. Our research may offer novel therapeutic approaches to HSCR, including factors necessary for successful enteric neuron transplantation. PUBLIC HEALTH RELEVANCE: The Ret gene plays a central role formation of the enteric (gut) nervous system, being frequently mutated in Hirschsprung's Disease (HSCR). Despite the contribution of animal models to our understanding of HSCR, no invertebrate model has yet been established. This proposal aims to develop a Drosophila model of HSCR with the aim of elucidating the basic mechanisms of action of Ret and interacting genes.

DESCRIPTION (provided by applicant): Nerve connections between the left and right hand sides of the nervous system are critically important for coordinated and independent movements. Disruption of these connections in the spinal cord can lead to curvature of the spine (scoliosis), a condition that can restrict physical activity, cause pain and lead to problems breathing. Many growing nerves in the central nervous system (CNS) make a decision whether or not to cross to the other side during embryonic development. They do this by sensing navigational cues in their environment, using specialized proteins on their cell surface known as receptors. Controlling which receptors make it to the cell surface allows neurons to control whether or not they can respond to different cues in their environment. Understanding how this process is controlled is poorly understood. In the fruit fly, "commissureless" or comm mutants have no nerve connections between their left and right sides at all. The Comm protein controls whether or not a class of receptors called Robos can make it to the cell surface. In humans, genetic variants of Robo receptors have been implicated in such diverse conditions as scoliosis and developmental dyslexia. To date, comm genes have not been found outside of insects. This proposal aims to test candidate genes from other species by using well-defined assays in fruit flies. Identification of comm genes from other species will open new avenues to studying control of receptor localization, and add to our understanding of how nerves accurately navigate long distances in the embryo. It may also help identify a novel locus for scoliosis and other disorders in which nerve wiring patterns are altered. PUBLIC HEALTH RELEVANCE: The commissureless gene plays a central role in nerve navigation at the CNS midline of fruit flies, yet vertebrate homologues have not been identified. This proposal aims to identify functional homologues, increasing our understanding of spinal cord development.

Information encoded in the DNA of an animal's genome is used to generate all the proteins found in their cells. This instruction set must be "read out" by each cell in a selective way, so that cells only generate the proteins they need according to what type of cell they are going to become, how early or late along the developmental timeline it is, and where in the embryo they are. These DNA instructions direct the formation of messenger RNA templates that are used to build the proteins. This project investigates how cells in the nervous system (neurons) interpret this genetic information in a more subtle way, to make unique, longer RNAs that are not found in other non-nervous system cells. A new gene-editing technology called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) will be used to eliminate the genetic information that causes these longer RNAs to be constructed in fruit fly neurons, to see how this affects the wiring of the developing nervous system. Experiments will discover the mechanisms by which molecules expressed in neurons control the generation of these longer RNAs. Such knowledge is vital for understanding an entire class of mechanisms underlying brain cell development that is currently very poorly understood. This project has a broader impact of training undergraduate and graduate students in neuroscience and molecular biology in Nevada, a state with a historically low percentage of adults with bachelors or advanced degrees. An outreach program called, "Editing the future", will be implemented in AP biology classes at a local high school. Students will learn about the huge potential impact of CRISPR gene-editing technology, and receive hands-on experience performing experiments on fruit flies, including using a technique called polymerase-chain reaction (PCR) to amplify and visualize DNA from CRISPR-mutated flies.

The 3´ Untranslated Region (3´ UTR) can regulate the stability, subcellular localization and translation of an mRNA. This post-transcriptional control is particularly important for neurons given their highly polarized morphology and capacity for localized translation in axons and dendrites. Most genes in higher organisms express at least two alternative 3´ UTR isoforms generated by alternative cleavage and polyadenylation (APA). In Drosophila, hundreds of genes express extended 3´ UTRs that are specific to neural tissues; however, the functional importance of this is unknown. Many genes involved in axon guidance generate extended 3´ UTR isoforms in a manner dependent on the neural-expressed RNA binding protein ELAV. This project tests the hypothesis that extended 3' UTR isoforms generated by ELAV are required for correct axon guidance. A novel approach using CRISPR genome editing to functionally remove 3´ UTR isoforms of axon guidance genes will be carried out. Characterization of neurodevelopment in these mutants will be performed. The impact of extended 3´ UTRs on RNA localization and translation will be determined. In parallel, the precise mechanisms of 3´ UTR extension of axon guidance genes by the RNA-binding protein ELAV will be elucidated. Extended 3´ UTRs are found for nine genes involved in axon guidance, and this project will focus on three - comm, Dscam1 and fasI. The project has the potential to transform our understanding of APA by establishing it as an essential component of nervous system development.

Axon guidance is the study of how developing nerves navigate in response to external signals. A remarkably small number of navigational signals have been identified, raising the question of how the complexity of the brain is generated. One solution is that a single ligand can signal in different ways either through the presence of different receptors or through processing that alters receptor binding. Slit is a large secreted protein that typically repels growing axons using Robo receptors. Slit is cleaved into two fragments, Slit-N and Slit-C, which display new biological activities in the nervous system and that in many other tissues. However, the role of the Slit fragments is controversial. The Slit-N fragment contains the Robo binding site and has been shown to be repel axons in in vitro culture systems and so is thought to be the active signaling molecule. Multiple lines of in vivo preliminary evidence in Drosophila suggest that only the full-length (Slit-FL) protein repels axons. This proposal takes an in vivo molecular genetic approach in flies and mice to separate the signaling of Slit-FL from that of the Slit fragments.These two systems will allow us to address the effects of Slit on axon growth, axon branching and regulated adhesion (fasciculation). This work will allow us to determine how a single signal can achieve different biological outcomes. The proposed work has implications for a diverse range of fields, including infectious disease, cancer and nerve regeneration.