The ability to express a gene of interest in a spatially restricted manner in a transgenic animal has greatly contributed to the successful use of Drosophila
in a wide variety of biological studies. This is especially true for the nervous system, where experiments often call for manipulating the activity of small, reproducible sets of neurons (reviewed in Venken et al., 2011
; Griffith, 2012
). Our objective was to generate and characterize a set of transgenic lines that can each drive expression of a reporter gene in a distinct, small subset of the approximately 150,000 neurons that comprise the adult central nervous system. The set of lines also needed to be large enough to ensure that all, or nearly all, neurons are represented in at least one line.
Transgenic animals in which the yeast transcriptional activator GAL4 is placed under the control of endogenous regulatory elements have proven to be powerful and versatile tools for manipulating gene expression (Fischer et al., 1988
; Brand and Perrimon, 1993
). Analysis of confocal imagery of whole mount preparations from animals in which a reporter gene is expressed under the control of GAL4 has been widely used for identifying and characterizing the morphology of neuronal populations, an approach pioneered and applied most extensively by Kei Ito’s laboratory (see for example, Ito et al., 2003
; Otsuna and Ito, 2006
; Tanaka et al., 2012
Prior studies have used collections of GAL4-expressing lines based on enhancer traps. In an enhancer trap, a transposon carrying a GAL4 gene is inserted at a large number of random genomic locations; at each insertion site, the pattern of GAL4 expression comes under the influence of local transcriptional regulatory elements (O’Kane and Gehring, 1987
). Enhancer traps have a number of properties that limit their use: the precise nature of the sequence elements driving expression is unknown, the varied genomic locations complicate genetic manipulations and, in general, GAL4 is expressed in more cells than optimal.
These limitations can often be overcome for anatomic studies. All that is required for such studies is the ability to recognize different neuronal populations under the microscope; stochastic labeling methods can be used to generate animals in which only a fraction of the cells in the parent GAL4 pattern express the reporter gene (reviewed in Venken et al., 2011
). But this is not the case when one intends to use the GAL4 driver to manipulate the activity of a specific population of neurons to study physiology or behavior. For such applications, the ability to direct expression of an exogenous protein to reproducible, small subsets of neurons in all animals in a population is critical.
The collection of GAL4 lines we generated for this project, based on the experimental design of Pfeiffer et al. (2008)
, begins to address those requirements. In these lines, a short fragment of genomic DNA controls GAL4 expression. As a result, GAL4 is, on average, expressed in fewer cells than in enhancer trap lines (Pfeiffer et al., 2008
). Since the transgenes are all inserted at a known location by site-specific integration, genomic position effects on expression are held constant. Moreover, because the DNA fragments driving expression in each line are completely defined, one can make constructs that reuse the same enhancer fragment to drive the expression of another protein. Importantly, the expression pattern of that protein will be predictable from the image data obtained with the original GAL4 line. Pfeiffer et al. (2010)
show examples of such enhancer reuse in making constructs expressing the transcriptional activator LexA and the repressor GAL80.
The ability to reuse the enhancer fragments greatly enhances the value of the image database we report here. One can use our database not only for neuroanatomy or to select a GAL4 line of interest for further studies, but also as a resource to select enhancer fragments to generate the constructs required for more sophisticated applications. For example, LexA and GAL4 expressing constructs can be combined in the same animal to separately control expression of two reporters in different cell populations. This is required for a variety of experiments used to determine neuronal connectivity in circuit mapping (reviewed in Griffin, 2012
Although the GAL4 lines we present here are expressed more sparsely than enhancer trap lines, in most cases even more restricted expression will be required for behavioral studies where the effects of altering the activity of a very small population of neurons is desired. For example, GAL4 expression can be restricted to cells in which expression of two enhancer fragments overlap by using the split-GAL4 method, developed by Luan et al. (2006)
and improved upon by Pfeiffer et al. (2010)
. This approach appears to be sufficient to reach single “cell-type” specificity. The modular nature of our constructs enables the facile construction of the required split-GAL4 transgenic lines.
With 6,650 lines successfully imaged, this is the largest dataset of GAL4-driven expression patterns in the adult brain and the only large dataset available of expression patterns in the adult ventral nerve cord (VNC). The expression patterns generated by these same lines in the embryonic nervous system and in third instar imaginal discs are described in accompanying papers (Manning et al., 2012
; Jory et al., 2012
). These combined data sets should be especially powerful for efforts to understand the logic underlying the cis
The GAL4 lines have been deposited in the Bloomington Drosophila Stock Center for distribution and the expression patterns of the lines in several tissues are documented on a web-accessible database. We also developed a number of software tools to support our production pipeline and to analyze the large image dataset we generated; we expect these tools and methods to be useful for other large-scale imaging projects.