Genetic Analysis of the Olfactory Circuit Organization in Drosophila

Abstract

The Drosophila olfactory system provides an excellent model for studying how complex neuronal circuits are assembled. In Drosophila melanogaster, each olfactory receptor neuron (ORN) class typically expresses a unique olfactory receptor (OR) gene, and synapses with their target projection neurons (PNs) within each class-specific and uniquely positioned glomerulus in the antennal lobe. Understanding how ORN axon terminals and PN dendrites are organized into these defined structural compartments is fundamentally important. It can bring us insights into common principles on how highly diverse neuronal populations are genetically controlled to form hardwired circuits, which might also be shared over evolutionary history. Over the past decades, many critical molecular players have been uncovered to control distinct steps of ORN and PN wiring. Among these, multi-member gene family encoding cell surface proteins are of interest due to their features of forming heterophilic interaction networks and cell-type-specific expression as cell surface codes, with the potential to mediate wiring specificity. Compared to the well-studied DIP-Dpr families in diverse neuronal contexts, the expression patterns and functions of another multi-protein interactome, Beat-Side, remain elusive in assembling the Drosophila olfactory circuits. Here, I thoroughly analyzed the expression pattern of the beat/side gene family in ORNs and PNs by leveraging the publicly available single-cell RNA-seq datasets and generating gene trap transgenic driver lines to faithfully probe the spatial expression pattern in vivo. My results reveal that each ORN and PN class expresses a specific combination of beat/side genes, which appears to be regulated by lineage-intrinsic mechanisms. Though ORNs or PNs from closer lineage tend to possess more similar beat/side profiles, I did find many examples of divergence among closely related ORNs and closely related PNs. This raises an interesting possibility that Beat/Side combinatorial expression might introduce variability on the cell surface that assists in the distinct glomerular targeting of these ORN/PN classes. To explore whether the class-specific combination of beats/sides defines ORN-PN matching specificity, I perturbed presynaptically expressing beat-IIa and postsynaptically expressing side-IV in two ORN-PN partners. However, disruption of Beat-IIa-Side-IV interaction did not produce any significant mistargeting in these two examined glomeruli, pointing to the redundancy and complexity of this multi-member cell surface interactome. Nonetheless, this comprehensive analysis of expression patterns lays a foundation for in-depth functional investigations into how Beats/Sides contribute to ORN-PN circuit formation. Additionally, this transgenic driver line collection offers a valuable resource for examining the beat/side expression and roles outside the olfactory system. This study is presented in Chapter 2. When I studied the functions of beats/sides by a targeted genetic screen using an RNAi collection, I also found an unexpected recurrent phenotype, which turned out to be RNAi-independent but linked to the transgenic docking site where UAS-RNAi transgenes are inserted. This landing site is attP40, which is widely used for the bacteriophage integrase-directed insertion of various transgenic constructs into this specific genomic locus by the Drosophila community. The attP40 landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. However, I found that the homozygous attP40 chromosome disrupts the normal glomerular organization of Or47b ORN class in Drosophila. This effect is not likely to be caused by the loss of function of Msp300, where the attP40 docking site is inserted. Moreover, the attP40 background seems to genetically interact with the second chromosome Or47b-GAL4 driver, and Or47b-GAL4/attP40 transheterozygotes yield a similar glomerular defect. I also found a short chromosome region near the attP40 site, covering only five genes, which likely contains the causal genetic lesion accounting for the attP40-associated glomerular phenotypes. Though the exact causal genes remain undetermined, my findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out the attP40 landing site-associated background effects, particularly in neuronal contexts. Moreover, the identification of this short chromosomal region provides a promising shortlist of candidate genes for future functional investigation to establish the causal link, which may also bring new biology into how ORNs are organized into morphologically specified glomeruli. This story is presented in Chapter 3.