1 Our ultimate goal is to discover how cells targeted for destruction during degenerative disease 2 can be regenerated from adult stem cells. The specific goal of this proposal is to create 3 transgenic zebrafish that can be used to model the regeneration of motor neuron (MN) cells, the 4 cell type lost in all motor neuron diseases (MND). Zebrafish have a remarkable capacity for 5 cellular regeneration that extends even to the nervous system, including MN cells in adult 6 zebrafish. Zebrafish are also an established model system for large-scale forward genetic 7 screens, whereby the genome is randomly mutated to identify genes which are required for a 8 specific biological process. To combine these attributes, we have developed simple screening 9 methods around an inducible cellular ablation platform that can be used to identify regeneration- 10 deficient mutants, in this case, genes required for MN regeneration. Specifically, transgenic 11 methods will be used to target the expression of a pro-drug converting enzyme, nitroreductase 12 (NTR), to MN subpopulations. NTR functions to convert water soluble pro-drugs into cellular 13 toxins, thereby ablating the MN specifically expressing the enzyme. A fusion between NTR and 14 a fluorescent reporter (NTR-FP) allows the presence or absence of targeted cells to be easily 15 monitored over time in living zebrafish. In this system FP loss would indicate MN degeneration 16 while subsequent gains in FP signal provide evidence of MN regeneration. By using high- 17 throughput plate readers for quantitative detection of fluorescent reporters in living zebrafish, a 18 large-scale genetic screen could be performed (Phase II) to identify multiple genes required for 19 MN regeneration. Thus, the identification of molecular factors which promote MN regeneration 20 in a vertebrate model system such as zebrafish should provide a means to explore the 21 possibility of regenerative therapies for human MND.
Public Health Relevance: Motor neurons are the cell type lost in a number of debilitating degenerative diseases, including Lou Gehrig's disease. The goal of this proposal is to discover genes required for motor neuron regeneration by identifying mutations that disrupt motor neuron regeneration in a small model organism, the zebrafish - a species with a regenerative capacity that extends even to the nervous system. The motor neuron disease models produced and genetic insights gained during these studies will facilitate efforts to discover drugs that promote motor neuron regeneration, thus suggesting possible avenues of regenerative therapies for human motor neuron diseases.
Thesaurus Terms: 21+ Years Old; Als; Ablation; Acute; Adult; Afferent Neurons; Amyotrophic Lateral Sclerosis; Animal Model; Animal Models And Related Studies; Assay; Bioassay; Biologic Assays; Biological Assay; Biological Function; Biological Models; Biological Process; Brachydanio Rerio; Cell Communication And Signaling; Cell Signaling; Cells; Chemicals; Chronic Disease; Chronic Illness; Danio Rerio; Degenerative Disorder; Detection; Development; Disease Progression; Disease Model; Drug Precursors; Drugs; Enhancer Elements; Enhancer Elements (Genetics); Enhancers; Enzymes; Fishes; Fluorescence; Gehrig's Disease; Generations; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Screening; Genetic Defect; Genetics-Mutagenesis; Genome; Goals; Hepatic Cells; Hepatic Parenchymal Cell; Hepatocyte; High Throughput Assay; Human; Human, Adult; Human, General; Humulin R; Hydrogen Oxide; Insulin; Insulin (Ox), 8a-L-Threonine-10a-L-Isoleucine-30b-L-Threonine-; Insulin, Regular; Intracellular Communication And Signaling; Lead; Life; Liver Cells; Lou Gehrig Disease; Mammalia; Mammals; Mammals, General; Man (Taxonomy); Man, Modern; Medication; Methods; Methods And Techniques; Methods, Other; Model System; Modeling; Models, Biologic; Molecular; Molecular Biology, Mutagenesis; Monitor; Mother Cells; Motor Cell; Motor Neuron Disease; Motor Neuron Disease, Amyotrophic Lateral Sclerosis; Motor Neurons; Muscle, Cardiac; Muscle, Heart; Mutagenesis; Mutate; Mutation; Myocardium; Nrvs-Sys; Natural Regeneration; Nerve Cells; Nerve Unit; Nervous System; Nervous System Structure; Neural Cell; Neural Stem Cell; Neurocyte; Neurologic Body System; Neurologic Organ System; Neurons; Neurons, Afferent; Neurons, Sensory; Nitroreductases; Noise; Novolin R; Pathway Interactions; Patients; Pb Element; Persons; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Pro-Drugs; Prodrugs; Progenitor Cells; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; Reader; Reagent; Regeneration; Regulatory Element; Regulatoryelement; Relative; Relative (Related Person); Reporter; Research; Role; Screening Procedure; Sensory Cell Afferent Neuron; Signal Transduction; Signal Transduction Systems; Signaling; Specific Qualifier Value; Specified; Stem Cells; System; System, Loinc Axis 4; Techniques; Time; Toxin; Transgenic Organisms; Variant; Variation; Water; Zebra Danio; Zebra Fish; Zebrafish; Adult Human (21+); Adult Stem Cell; Biological Signal Transduction; Cardiac Muscle; Cell Killing; Cell Type; Chemical Genetics; Chronic Disease/Disorder; Chronic Disorder; Degenerative Condition; Degenerative Disease; Disorder Model; Drug/Agent; Functional Restoration; Gene Discovery; Gene Product; Genetic Enhancer Element; Genome Mutation; Heart Muscle; Heavy Metal Pb; Heavy Metal Lead; High Throughput Screening; In Vivo; Insight; Model Organism; Motoneuron; Motor Neuron Degeneration; Mutant; Nerve Stem Cell; Neural Progenitor Cells; Neuron Cell Death; Neuron Loss; Neuronal; Neuronal Cell Death; Neuronal Loss; Neuronal Progenitor; Neuronal Progenitor Cells; Pathway; Public Health Relevance; Ratiometric; Regenerate; Regenerative; Regenerative Therapy; Restore Function; Restore Functionality; Restore Lost Function; Screening; Screenings; Social Role; Therapeutic Target; Transgenic
