We are interested in providing a better understanding of the molecular mechanism underling memory formation. Fly biologists have uncovered the role of genes required for learning and memory formation and many of these affect the cAMP-PKA-Creb pathway, including dnc, rutabaga, amnesiac, DC0, and creb2. These genes are often expressed in mushroom bodies (MB) which is the seat of learning and memory formation. However, it is likely that other players are involved in learning and memory.
To identify new genes required for learning and memory in the adult fly we screened a collection of MiMIC,insertions (Venken et al., 2011; Nagarkar-Jaiswal et al., 2015) MiMICs that are inserted in coding introns were modified through recombination mediated cassette exchange (RMCE) to generate flies with an additional exon that encodes GFP. We stained adult brains of 500 MiMICs that were tagged with GFP to identify genes expressed in MB. Unfortunately, only about 15% of the stocks express detectable levels of GFP in adult brains whereas GFP expression was observed in 90% of the third insaar larval brains (Nagarkar-Jaiswal et al., 2015). We therefore also converted the MiMICs with the T2A-Gal4 “Trojan exon” casette (Diao et al., 2015). This leads to the production of the Gal4 protein in the proper spatial and temporal pattern and permits the detection of GFP (via UAS-mCD8-GFP) for nearly 100% of the Gal4 tagged genes. Using this approach, we characterized the expression pattern of 30 genes encoding protein kinases and observed that 12 were expressed in MB. To identify whether or not these protein kinases are required for learning and memory we performed the olfactory aversive conditioning assay. We removed the GFP tagged kinase in adult flies using the Degrad-FP system in MBs and restored the protein in the same flies. By this approach, we identified a novel kinase required for memory. Interestingly, the loss of function of this gene leads to a complete loss of anesthesia-sensitive and long-term memory but does not affect anesthesia-resistant memory and learning. Our preliminary data indicate that this novel kinase regulates some of the substrates that are phosphorylated by protein kinase A (PKA).
In summary, we show that the MiMIC system, coupled to the Trojan exon and DegradFP technologies, is a powerful platform to screen for genes that are required for memory formation and probably many other behavioral functions.