One of the great successes of biological research over the past three decades has been the use of genetic analysis to discover the pathways that control animal development. The success of this approach was largely dependent on three factors: (1) the genes themselves directly encode proteins, the primary functional units of these pathways; (2) nearly all the genes had been enumerated and described through analysis of genome sequences; and (3) powerful methods had been developed both to screen all the genes for their contribution to a given process and to inactivate, in a controlled way, the function of individual genes.
It is clear that the building blocks of the nervous system and individual neuronal circuits are not genes, but cells. And so the genetic methods that were so powerful in elucidating embryonic development will be of limited use in probing the function of the nervous system. Instead, we will need to be able to assay and manipulate the function of individual cells, if not the individual connections between cells, with the same facility as we can now manipulate genes.
A variety of genetically encoded probes have been developed to allow experimenters to monitor and alter the activity of individual cells, and this remains an active and important area of research in many laboratories. The utility of these probes depends on the precision with which their expression can be directed to small subsets of cells in reproducible, controllable, and convenient ways. Providing the tools required to accomplish this task in the larval and adult nervous system of Drosophila is the primary objective of our current research.
Drosophila researchers have known for more than 20 years how to determine and, to some extent, manipulate the DNA sequences—called promoters and enhancers—that control the temporal and spatial expression of individual genes. We are using our knowledge of high-throughput molecular and genetic methods to define the sequences controlling the neuronal expression patterns of about 1,100 genes. We selected these genes because their predicted function, as well as existing data on their expression, suggests that they are expressed in some, but not all, cells in the nervous system. We will directly analyze the expression patterns produced by fragments of the control sequences of these genes by generating and analyzing more than 7,000 lines of transgenic animals over the next two years. Prior work has established that separate modules, each of which produces a subset of a gene's total expression repertoire, are used to produce the complex expression patterns of many genes. Thus we expect to be able to define DNA fragments that generate patterns much more limited than those of the genes from which they were derived. Our analyses of the first 2,000 lines confirm this expectation.
We are implementing additional methods to allow us to further refine expression patterns through intersectional strategies. For example, we can limit labeling to cells contained in the overlap of the patterns driven by two different fragments. Alternatively we can limit labeling to cells contained in one pattern, but not in the other. We are confident that application of these methods will give us the specificity we require.
In parallel to generating these tools, we will explore, in collaboration with other laboratories, ways of applying these tools to study nervous system anatomy and function.