Files
Abstract
For a functional brain development neural circuits must be organized precisely. Any aberrations throughout the development of connectivity of the neural circuit can lead to neurodevelopmental defects. Therefore, understanding the early mechanisms underlying the organization of a functioning neural circuit is essential. Precision of synaptogenesis depends on target recognition and synaptic assembly mediated by cellular contacts between the partner cells. For studying how early recognition events contribute to functional neural circuit development, we use the embryonic Drosophila neuromuscular junction (NMJ) as a model system. The NMJ in Drosophila is a simple, stereotypically organized neural circuit made up of 36 motor neurons and 30 muscles. During NMJ synaptogenesis, both the presynaptic motor neurons and the postsynaptic muscles extend long and thin actin-containing membrane processes, called filopodia, that contribute to contact-based communication. While roles of filopodia on the presynaptic terminal are widely studied and relatively well understood, the functions of post-synaptic filopodia have been less studied. In this dissertation, first we report a retrograde lipophilic dye labeling method to labeling neurons using single or multi-color dyes allowing high resolution access to fine cellular processes like neurites, dendrites as well as filopodia. Second, we report a super-resolution microscopy method, stochastic optical reconstruction microscopy (STORM), to study molecular and morphological organization of in vivo and in vitro neurons. Third, we report on a set of orthogonal binary expression systems (Gal4/UAS and LexA/LexAop) that allow the study of single NMJs using live imaging and discuss other applications of these lines for studying muscle development and specification of targeting. Through live imaging studies we uncover that membrane morphologies between muscles are different and discuss their implications in synaptic specificity. Fourth, we explore the characteristics and roles of muscle-derived filopodia in synaptic targeting, axon guidance and synaptogenesis. Altogether, this work will provide improved resolution to study cellular structures like filopodia at synapses as well as provide insights into contributions of filopodia activity to neural circuit development in living animals.