Studies of the Drosophila Brain Using P[GAL4] Enhancer Trap Lines

This thesis is concerned with the application of enhancer trap technology to illustrate the structures of Drosophila brain and identify the genes relevant to the central complex function in the brain. Over 1400 novel enhancer trap lines bearing P[GAL4] insertions were generated by genetic crosses and they were then screened for GAL4-directed beta-gal expression in cryostat sections of the Drosophila head. More than 300 lines display interesting patterns in the brain from an anatomical perspective. Of these, as many as 100 are more or less restricted to specific regions or neuronal sub-populations of brain. Particularly exciting are lines that express GAL4 in the mushroom bodies and the central complex, structures that have been implicated in associative learning and memory. For most of lines, the chromosomal locations of P[GAL4] insertions were identified by in situ hybridisation to polytene chromosomes. P[GAL4] expression patterns have suggested multiple roles for certain Drosophila brain structures in integration of signals. In the mushroom bodies, the blue staining patterns have revealed axonal processes corresponding to Kenyon cells and showed that the Drosophila mushroom bodies are compound neuropils in which parallel subcomponents exhibit discrete patterns of gene expression. A strong prediction that different sub-sets of Kenyon cells perform different functional roles is made. In the ellipsoid body of the central complex, four different ring structures are revealed by P[GAL4] expression patterns. Developmental analysis indicates that the lacZ expression in the central complex lines begins at early pupal stages to the adults. The central complex of the Drosophila brain has been shown to act as a higher centre for locomotor activity and other behaviours. To identify the genes relevant to central complex function, seven P[GAL4] enhancer trap lines with staining patterns specific to the central complex were selected. Genomic DNAs flanking each insertion site were cloned by plasmid rescue. Rescued genomic DNAs from some of lines were used as probes for screening a cDNA head library. Corresponding cDNA clones were isolated. In the case of line c507, P[GAL4] staining is restricted to the ellipsoid body of the central complex of brain and to the Malpighian tubules in the Drosophila. Three genes, two located downstream and one upstream of the P[GAL4] element, are identified. Sequencing of a 1.8 kb cDNA clone from pMY51 which is located downstream of the P[GAL4] reveals a protein with significant homology to the alkaline phosphatase gene family in other organisms. The other cDNA clone, closely linked with pMY51, represented by pMY8 is sequenced and a full length predicted amino acid sequence identified. It has head-elevated expression as judged by Northern blot analysis. The gene which is located upstream of the P[GAL4] element is identified as calcineurin Al, the Ca2+/calmodulin-stimulated protein phosphatase. in situ hybridisation to tissue sections using cDNA probe generated from a whole insert of pMY51 reveals that the gene has expression patterns in the cell bodies of the ellipsoid body and the Malpighian tubules consistent with the X-Gal staining. Results indicate that the Drosophila "alkaline phosphatase" enhancer is trapped and the corresponding gene has been cloned by enhancer trap approach