Generation of novel tools to study IroC gene function of IroC target.

Objective: The olfactory system depends on the proper gene expression of olfactory receptor (OR) genes in olfactory receptor neurons (ORN) and the proper transfer of olfactory signals to the brain. ORNs have to make two important choices. They have to choose to express one gene from the large repertoire of OR genes and send their axons to a special destination in the brain. Our studies focus on the elucidation of the role of the Iroquois complex (IroC) family, a family of transcription factor genes consisting of three conserved genes called araucan (ara), caupolican (caup) and mirror (mirr) in the differentiation of odorant receptor neurons. Methods: To elucidate the function of the IroC genes, we tried to generate mutants for all three genes using the CRISPR/Cas technology. In addition, to examine localization of each gene we aimed to add fluorescence proteins to each gene locus using the CRISPR/Cas. The obtained mutants were examined using RNASequencing to identify differentially expressed genes. The identified genes were confirmed by qRT-PCR. Verification by in situ hybridization is underway. Furthermore, the effects of iroC genes on the differentiation of cells are followed by the lineage- tracing experiments. Results: In this study we showed that the IroC genes are expressed in ORNs. While the maxillary palp iroC is expressed in the pb2 subset of the ORNs, expression in the adult antenna appears to be more dispersed. In order to identify the iroC target genes, we first used RNASeq analysis using triple mutant fly antennae and maxillary palps and identified the differentially expressed genes. We show that 11 OR genes and 23 transcription factors are differentially expressed. qRT-PCR experiments confirmed that expression of the OR genes is decreased. Conclusion: Our results confirm that iroC genes play a role in OR gene regulation. However, further experiments will show if this regulation is direct or indirect. [ABSTRACT FROM AUTHOR]