Comparative Analysis of mRNA Asymmetrically Localized in D. melanogaster Neuromuscular Junctions & Class IV Dendritic Arborization Neurons

Abstract

Messenger RNA (mRNA) localization and local protein synthesis are important for cellular development, maintenance, and function. Studying these processes is especially crucial in morphologically and functionally polarized cells, like neurons, but the traditional methods for visualizing localized mRNA are not optimally suited for use in such cell types. A new method employing both the GAL4/UAS and ms2-MCP systems has recently been used to screen for asymmetrically localized mRNAs in both D. melanogaster tracheal cells and class IV dendritic arborization (da) sensory neurons. The goal of my research was to adapt the screening protocol for use in the neuromuscular junction (NMJ), to compare the NMJ and class IV da screening results of ~100 EP-ms2 transgenic fly stocks, and conduct follow-up analysis of positive candidates to determine how their mRNA localization affects gene function. Due to stock availability and differences in technique, I was unable to develop appropriate reagents for NMJ screening. Yet, comparative analysis proved fruitful. I was able to identify escargot (esg) as a transcript asymmetrically localized to both the class IV da dendrites and the NMJ presynaptic boutons. RNAi knockdown and EP over-expression assays were conducted to investigate esg’s function in these two neuronal subtypes. My results suggest that a certain concentration of esg is required for proper NMJ morphogenesis, as structural defects were observed in both esgRNAi and EP{esgEU143} NMJs. Yet, these morphological defects do not necessarily translate into functional defects. In fact, while over-expressing esg in the NMJ resulted in decreased bouton numbers, it increased larval locomotive activity. On the other hand, over-expressing esg in class IV da neurons did not result in any significant morphological or functional defects. esgRNAi class IV da neurons, however, exhibited decreased terminal branch numbers and the larvae were significantly less sensitive to noxious stimuli. Taken together, these results indicate that esg may play a distinct role in class IV da morphogenesis (which then influences its function), while it may alternatively play a number of roles in the NMJ. These preliminary results highlight how a screen of this nature could uncover distinct mechanisms involved in mRNA localization and, potentially, neurodegenerative diseases known to afflict human beings.