Immunocytochemical study of choline acetyltransferase in Drosophila melanogaster: an analysis of cis-regulatory regions controlling expression in the brain of cDNA-transformed flies

We have analyzed the cis-regulatory regions in the 5' flanking DNA of the Drosophila melanogaster choline acetyltransferase (ChAT; E.C. 2.3.1.6) gene by using germline transformants. These transformants are carrying wild-type ChAT cDNA fused to different lengths of 5' flanking sequence of the ChAT gene. Appropriate genetic crosses were used to introduce the transgene into animals with a presumptive null genetic background for endogenous ChAT. Expression of ChAT protein could thus be attributed exclusively to the transgene. Using a monoclonal antibody against Drosophila ChAT, we have investigated the spatial distribution of transgenic ChAT and compared it to the normal distribution of ChAT protein in wild-type animals. The brains of 7.4 kb cDNA transformants showed a ChAT expression pattern similar to that of wild-type animals in the first- and second-order sensory neuropil but reduced expression in other highly ordered neuropil. Several lines that were transformed with 1.2 kb or 0.8 kb of 5' flanking DNA demonstrated relatively normal expression in sensory neuropil. In addition, these lines also showed ectopic expression in higher order neuropil. In the optic lobe, the expression pattern directed by 7.4 kb of 5' flanking DNA was very similar to that of wild-type ChAT expression. In contrast, 1.2 kb or 0.8 kb transformants showed reduced levels of expression and a more limited pattern of distribution in the optic lobe. Our results suggest that the 5' flanking DNA of the ChAT gene can be divided into several separable positive and negative regulatory regions, which define various subsets of cholinergic neurons in the nervous system.