Glutamatergic Synapse Formation in Developing Zebrafish Embryos
Fierro Jr., Javier
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Fierro Jr., Javier
In order for a human being to process complex thought, cells within the brain must communicate with each other in a very precise manner. The mechanisms which underlie the development of these connections, however, are poorly understood and thus require a thorough investigation. In this dissertation, we attempt to identify components involved in stabilizing synaptic contacts and the mechanisms by which synaptic proteins are trafficked to newly forming contact sites. Interestingly, we also identify a gene involved in the formation of the myotome. To identify proteins involved in stabilizing synaptic contacts, we characterized the function of 4.1B in developing zebrafish embryos. 4.1B is a scaffolding molecule involved in stabilizing protein complexes at sites of cell adhesion. We identified two 4.1B genes in the zebrafish genome, 4.1B-a and 4.1B-b, which are differentially expressed and have evolved divergent functions. 4.1B-a is expressed within the central nervous system, specifically within primary motor neurons. Knockdown studies show a reduction in the number of synapses and altered kinetics of touch evoked-responses, suggesting a role in synaptic stabilization. In contrast, 4.1B-b is primarily expressed in muscle cells. Knockdown of 4.1B-b results in severe muscle fiber disorganization as well as altered locomotor behaviors. Together, these data suggest the basic functions of 4.1B are evolutionarily conserved, with new roles described in the development of synapses and muscle fibers. To determine the mechanisms that underlie protein recruitment to newly forming synapses, we examined the recruitment of three distinct transport packets in the zebrafish spinal cord. During presynaptic assembly, we found synaptic vesicle protein transport vesicles preceded piccolo-containing active zone precursor transport vesicles, which in turn preceded synapsin transport vesicles. We identified the last transport packet as a unique and independent mechanism for the recruitment of synapsin, a protein involved in regulating the reserve pool of synaptic vesicles. Importantly, we found cyclin-dependent kinase 5 regulated the late recruitment of synapsin transport packets to synapses, thus identifying kinases as a key signaling molecule in the formation of synaptic contacts. Together, this work provides new insight into the mechanisms that underlie synaptogenesis. This dissertation includes both previously published and unpublished co-authored material.