Neuroanatomy of cells expressing clock genes inDrosophila: Transgenic manipulation of theperiod andtimeless genes to mark the perikarya of circadian pacemaker neurons and their projections

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Abstract
Subsets of brain neurons expressing the clock genes period (per) and timeless (tim) are involved in the generation of circadian behavioral rhythms. However, current knowledge of projection patterns of these neurons is limited to those immunoreactive to an antibody against a crustacean neuropeptide. The GAL4-expression system was utilized to visualize neuronal processes from all per and tim-expressing neurons in the central nervous system. Each of two types of GAL4-driver fusion genes, per-gal4 or tim-gal4, was combined in transgenic flies with marker genes-lacZ, and sequences encoding green fluorescent protein or TAU protein-under the control of the GAL4-responsive element UAS. This allowed visualization of the cytoplasm of GAL4-expressing cells. Thus, neurites of clock neurons in the adult brain as well as those of larvae and pupae were revealed. Among the anatomical patterns revealed by per-gal4- or tim-gal4-driven marker expression were a previously unknown, dorsally located neuronal cluster, along with the projections of these cells and of other dorsal neurons characterized in earlier studies only by the location of their perikarya. The similarity of projections from PER- or TIM-containing neurons during development to those in the adult implies that these features of mature clock neurons are established by the larval stages. Neurons that have never been identified as PER- or TIM-immunoreactive were also visualized in this assay system, indicating promoter activity of the clock genes in these cells and suggesting that their products cannot accumulate to detectable levels in certain neurons.