Molecular and Genetic Studies of robo2 Transcriptional Regulation in the Central Nervous System of Drosophila melanogaster

Drosophila Robo2 axon guidance receptor is a member of the evolutionarily conserved Roundabout (Robo) protein family that is involved in directing axons that cross the midline to the other side of the animal body. Robo2 roles mainly depend on two factors: The functional domains of the Robo2 protein, which is extensively studied, and the dynamic transcription of robo2 in various subsets of cells throughout embryogenesis which is not fully understood. Thus, knowing robo2 enhancers that transcriptionally regulate robo2 during embryogenesis is significant. To investigate robo2 potential enhancers, we screened 17 transgenic lines of Drosophila that were generated by Janelia Research Center. These lines contain 17 fragments distributed within and around the robo2 gene. We identified six fragments that regulated robo2 expression by the GAL4-UAS-GFP system suggesting that they were promising enhancers. Using these identified regulatory fragments in addition to three fragments generated in our lab, we built the HA-Robo2 transgenic constructs. These constructs were introduced into Drosophila which allowed us to test robo2 expression and its dependent axon guidance phenotypes in the embryonic CNS. GMR28G05 and GMR28F02 fragments showed the strongest robo2 expression in the lateral pathway. To further study these fragments, we introduced them separately or together into robo2 null mutant background. We found that Robo2’s dynamic expression pattern is specified by multiple regulatory regions. We utilized these fragments to generate and characterize an equivalent set of robo2 transgenes expressing the axonal marker TauMyc instead of the HA-Robo2 and hsp70 promoter instead of robo2 promoter. The results show that GMR28F02 fragment drove strong expression of TauMyc in a subset of the lateral neurons, cell bodies, and commissural axon from which Robo2 protein is expressed. CRISPR/Cas9 system was used to further investigate the importance of our findings. Cas9 protein and specific gRNAs were used to target and delete robo2 potential enhancers (GMR28G05 and GMR28F02) separately or together. Applying bioinformatics tools and literature I predicted three transcription factors (Hb9, Nkx6.1, and Lhx2) that have a high probability to bind robo2 potential enhancers. In summary, robo2 has potential enhancers located in the first intron and upstream of the gene, and multiple enhancers more efficiently regulated robo2 expression in Drosophila