Behavioural and Molecular Studies of the Drosophila Brain Using the P[GAL4] Enhancer-Trap

[摘要] The P[GAL4]UASG enahancer-trap system of Drosophila has been used to determine the cell-type specific expression patterns of genes flanking the transposon, and also to target expression of any specific desired gene products to the marked cells. In this thesis, P[GAL4] enhancer-trap lines with specific patterns of expression in the adult brains have been used to address the relationship between neural structure and function of sexual orientation and to identify enhancer-trapped genes according to their patterns of expression. Gynandromorphs of specific sub-domains within the male brains of 24 P[GAL4] lines were generated by GAL4-mediated expression of female-specific transformer (tra) transcripts, and the subsequent courtship behaviour towards male and female targets were tested. Feminisation of the mushroom bodies, which are thought to be involved in the olfactory pathway, appears to lead to non-discriminatory behaviour. The gene (in heat-shock construct pHSBJCaSpeR) has been reported to lead to male-male courtship (Zhang and Odenwald, 1995). As the gene is used as a marker in both P[GAL4] and UASG-tra constructs, male-male courtship was tested by mutagenesis of the mini-white+ gene in both the UASG-tra line and the P[GAL4] line 201Y which has specific expression in the mushroom body and shows a transformed bisexual behaviour when tra is expressed through the GAL4AJASG system (O'Dell, et al., 1995). Significant male-male courtship was only observed in 201Y mini-white+ homozygotes, but not 201Y mini-white heterozygotes. Mini-white+ has no such effect in the UASG-tra line. All the 201 YAJASG-tra flies, no matter whether they were homozygous or heterozygous for mini-white+, or lacked it entirely, displayed bisexual behaviour, which shows that the transformed behaviour of line 201Y is absolutely determined by tra expression, and not a consequence of mini-white+- Further, RT-PCR examination of the transformed dorsal brain tissue in male 201Y showed the female transcripts of double sex (dsx) and fruitless (fru) beside the male transcripts of these genes, providing evidence of the expression and functioning of female tra in the targeted cells. An analysis of the genomic DNA flanking 10 P[GAL4] insertions were carried out by plasmid rescue. These lines have brain specific expression patterns of the mushroom bodies, the central complex and the optic lobes. Detailed genomic restriction maps around the insertion sites were generated of mushroom body expression lines c739 and c772, and line c819 which shows expression in the ellipsoid body of the central complex. The cDNA of the AMP-activated protein kinase (AMPK)gamma subunit gene was isolated by cDNA library screening using the downstream region of the c819 insertion as a probe. The central complex of the Drosophila brain has been shown to act as a higher centre for locomotor activity and other behaviours. Anti-beta-gal antibody staining shows the expression in the central complex of line c819 begins at later pupal stages and continues to the adults. Developmental Northern and tissue in situ hybridization in the brain show that the AMPKgamma gene is expressed from the pupal stage and seems to have a specific expression pattern in the cell bodies of the ellipsoid body and the optic lobe. These results imply that the Drosophila AMPKgamma enhancer is likely to have been trapped by the c819 insertion and the corresponding gene has been cloned through the P[GAL4] enhancer-trap.

[发布机构] University:University of Glasgow

[关键词] Molecular biology, Neurosciences