High-resolution confocal and multiphoton imaging has recently provided unique insights into how neural circuits form by facilitating near real-time resolution of neuronal development in living model organisms. As a model system, zebrafish facilitate such studies due to that fact that they develop externally and are highly transparent during embryonic and larval stages. A number of transgenic zebrafish lines expressing fluorescent reporter proteins in specific neural subsets have been established and have proven useful for detailing how neuronal morphologies develop. However, in terms of neural circuit formation, methods must be established which promote simultaneous imaging of both pre- and postsynaptic partner populations. The use of complimentary """"""""colors"""""""" of fluorescent reporters facilitates imaging of both dendritic and axonal elements and has been successfully applied to reveal mechanisms underlying the formation of stratified subcircuits in the retina. However, practical issues hamper widespread implementation of this approach: 1) Fluorescent reporter expression levels in available transgenic lines are often suboptimal for detailed and/or long-term imaging studies. 2) Identifying regulatory DNA sequences competent for directing transgene expression to distinct neuronal subpopulations, let alone subsets of neuronal partners, is a time consuming process. Proposed here is the implementation of new transgenic approaches designed to circumvent current practical limitations and create zebrafish lines expressing cellular reporters and/or neuronal signaling molecules in unique neural subsets, including neuronal partner subpopulations. These lines will be instrumental for investigating molecular and cellular mechanisms underlying proper and aberrant circuit formation from a variety of subcircuit-specific perspectives. A series of stable transgenic zebrafish lines will be derived comprised of one of two modular transgene expression systems;a LexA-based system will be developed to complement the existing Gal4/UAS system. Having two binary expression systems available will promote maximum versatility regarding the expression of transgenes in specific subsets of neurons. For instance, two different neuronal subpopulations can be differentially labeled with fluorescent reporters. In cases where the two subpopulations interact, neuronal circuit formation can be visualized. Moreover, in addition to promoting cell labeling, this system can be used to express any gene(s) of interest within specified neurons. Transgenes designed to alter neuronal activity, label synapses, trace neural circuitry, etc., facilitate an array of assays for investigating mechanisms underlying circuit formation and function. Because the proposed lines represent universally adaptable toolsets these resources will remain relevant for many years to come.
Tools that promote fluorescent labeling of individual neuronal subtypes have been instrumental in revealing how single components of the brain develop. Proposed here are new toolsets that allow separate subpopulations of neurons to be differentially labeled with blue, green, yellow, and red fluorescent proteins. Moreover, these toolsets have been designed to facilitate direct visualization and molecular manipulation of neuronal circuit formation as it occurs in a living animal model system, providing a unique window into how brain cells get """"""""wired up"""""""".
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