The brain is a remarkably complex organ comprised of hundreds of unique cell types that are organized to form sophisticated neural circuits. Although we have made progress toward understanding brain function and development, it is clear there is still much to be learned. Currently, all genome-wide methods that could be brought to bear on functional studies of the brain are destructive, meaning that as a genomic analysis is performed on a population of cells, the cells are destroyed. This fact limits our ability to connec early molecular events in the cells of the brain with later behavioral or cellular changes. For example, it is currently impossible to connect transcriptional changes in a neuron with knowledge of whether or not the cell was successfully incorporated into a memory trace. Similarly, it is not feasible to connect the early molecular events that occur in a neuronal progenitor cell with the final cell fate decision made by the cell. We have set out to develop a transformative technology that can record molecular events at the time that they occur and can then be read out later after any defined period of time. We have a novel technology called transposon `Calling Cards' that, in culture, provides cells with a molecular memory of protein-DNA interactions that occur at a particular moment in time. Here, we propose to adapt this technology for use in vivo enabling a retrospective genomic analysis of molecular events. We will demonstrate the utility of this technology by completing four test-case experiments that cannot be done with existing methods. Specifically, we will test the method by: 1) retrospectively identifying candidate transcription factors that control the specification of cell types in the CNS 2) identifying features that distinguish neurons resistant to neurodegeneration in vivo, 3) identifying the neurons that become active during mouse vocalization behavior while simultaneously mapping the genome-wide binding of activity-dependent transcription factors in these neurons, and 4) identifying the molecular features that distinguish neurons that were incorporated into a fear memory trace from those that were not incorporated.
The brain is the most complex organ in vertebrates; many questions remain about its function and how it is formed. Which genes control the cell fate decisions that give rise to the myriad of neuronal cell types? What molecular events are required for a neuron to be incorporated into a memory trace? Why do some neurons die in neurodegenerative diseases like ALS while their neighbors do not? These are pivotal questions in neurobiology. We have developed a technology, Transposon 'Calling Cards,' to attack these questions in a novel way. This technology is unique because it provides cells with a molecular memory to record transient molecular events that occur during brain development or during normal brain functions. This record can be read out at a later time so that these early molecular events can be correlated with the later cellular phenotype or animal behavior. The tools that we propose to develop would contribute to the field of neurobiology by providing a unique and broadly useful technology for understanding brain function and development. The data that we propose to collect will deepen our understanding of brain development, memory formation and the progression of neurodegenerative disease. Ultimately, the information that we, and other users of this toolkit, will collect will be used to develop better ways to treat diseases of the central nervous system such as Huntington's disease, Alzheimer's disease, or Parkinson's disease.
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