During embryonic development, transcription factors are essential for establishing a code that will determine the fate of neuronal precursors and neurons. However, most proteins that are responsible for a neuron's functional properties have a half-life ranging from minutes to several hours;they therefore must be tightly regulated long after neural development is over in order to maintain neuronal function. Surprisingly, very little is known about the role that transcription factors play in this process. We hypothesize that transcription factors that were initially characterized as early neuro-developmental genes, are required in fully developed motorneurons (MNs) to maintain neuronal function. We propose to drastically affect the levels of three transcription factors gsb, isl and eve within fully developed MNs and determine the neuronal functions they control. The long term goal of this research is to establish the molecular targets of these transcription factors. The Drosophila neuromuscular junction (NMJ) provides a model that is well-suited to the study of fully developed MNs. MNs have reached their muscle targets and are releasing neurotransmitter before the end of embryogenesis. We can test MN viability, synaptic structure and physiology 4 to 5 days after they are fully developed, near the end of the larval life, many times longer than the lifetime of the average protein. Moreover, the use of transgenic RNAi and conditional expression allows for knockout of gene expression in fully developed MNs, therefore bypassing the embryonic requirement for transcription factors. We have recently shown that gsb is ubiquitously expressed in fully developed MNs of the late larval CNS. Using immunohistochemistry, we will first show that eve and isl are expressed within a subset of fully developed MNs. In a second aim we will combine classical genetics and the use of the Gal4/UAS system in conjunction with Gal80TS to knock down or over-express transcription factors late, after development. We will then use immunohistochemistry and electron microscopy to determine whether the viability of the MN or the structure of the NMJ is affected by knocking down or over-expressing eve, isl or gsb within fully developed MNs. We will then perform intracellular electrophysiological recordings at the NMJ to determine whether eve, isl or gsb control synaptic release and homeostatic plasticity within fully developed MNs. This study will provide a conceptual template attributing a function to transcription factors within fully developed neurons;this in turn could lead to great insights into the molecular processes of neuronal aging and neuronal degeneration.
Very little is known about the biological mechanisms required to maintain neuronal function during life. This study proposes to show that transcription factors known to be embryonic key regulators are also required to maintain neuronal integrity and electrical properties. This will prove that transcriptional regulators have a broader role outside development and might be important in processes like aging and neurodegenerative diseases.
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