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Genetic Dissection Of Axon Guidance Mechanisms In the Drosophila Nerve Cord
Biochemistry and Molecular Biology
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Axon guidance is the process by which the neurons in a developing organism extend their axons and make the connections that will carry vital information and commands throughout the brain and body. The interactions of the various cues and receptors that govern this process are very intricate and still understood incompletely. We know of only a handful of diseases that are a result of disruptions of axon guidance, but that should not be misconstrued as meaning that other diseases are not impacted by errors of axon guidance. Since the flow of signals along the axons is vital to a functioning organism, any number of vital processes (and diseases that affect them) could potentially be affected by an error in axon guidance. Thus, arriving at a better understanding of axon guidance is a vital scientific goal that could have far-ranging implications for human health, as well as other potential applications. Only a very small number of axon guidance cues and receptors have been discovered to date, and this seems like an insufficient number to account for all the variability in phenotypes that have been discovered. The two most obvious solutions to this issue would be that either there are more cues and receptors waiting to be discovered, or there are additional relationships between existing cues and receptors that we have not yet elucidated (or, of course, some combination of the two options). While the search for additional molecules is ongoing, existing results have included a number of strange experimental oddities that could point the way towards discovery of additional interactions. This dissertation will explore the nature of the relationship between the known axon guidance cue slit and receptor Dscam. Prior to this work, while there was evidence that indicated a potential relationship between the two molecules, the nature of this interaction had yet to be elucidated and its effects noted. Here, I report my findings that despite Dscam having over 150,000 different isoforms, expression of only one pan-neuronally via the UAS-GAL4 system leads to a rescue of the <italic>Dscam fra</italic> nerve cord phenotype. Further, Slit, the major midline repellant, appears to transduce an attractive guidance signal via Dscam. By overexpressing <italic>slit</italic> at the midline in the absence of its repulsive receptor Robo, I was able to observe an attractive effect, which was subsequently suppressed by loss of <italic>Dscam</italic>. Given that the Slit receptor Robo2 appears to act variably in either an attractive or repulsive manner, I tested whether it would similarly suppress the attractive phenotype, which it did not do. Additional inquiries provided more evidence of Robo2's contradictory behavior, although they support the idea that Robo2 is acting not in an attractive manner, but an anti-repulsive one. This dissertation will then present my efforts to map the binding site of Slit to Dscam. While these molecules have been observed to bind in culture, the location of the binding is unknown, and could shed interesting light on the relationships between Dscam's homophilic binding and the signals generated by its binding to ligands. While I was unable to map the binding site, I have generated a number of techniques and reagents that can be used in the future to continue exploring this question. Additionally, this dissertation shows my contribution to an ongoing lab effort to characterize the role of a gene we refer to as hog, found within an intron of the <italic>NetrinB</italic> gene in <italic>D. melanogaster</italic>. As NetrinB is one of the canonical midline attractants, we wish to understand the contribution of <italic>hog</italic> to the mutant phenotypes resulting from <italic>NetrinB</italic> deletion. While hog does not seem to be directly involved in axon guidance, it does seem to play a role in gastrulation, and its lack can lead to developmental missteps that lead to subsequent physical disruption of <italic>D. melanogaster</italic>'s nerve cord. Thus, it can have an indirect effect on axon guidance.