GAL4 Enhancer Trap Patterns During Drosophila Development Ellen J. Ward, 1 Iyarit Thaipisuttikul, 1 Mark Terayama, 1 Rachael L. French, 1 Stephen M. Jackson, 1 K. Amber Cosand, 1 Kyle J. Tobler, 1 Jennie B. Dorman, 2 and Celeste A. Berg 1,2 * 1 Department of Genome Sciences, University of Washington, Seattle, Washington 2 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington Received 3 June 2002; Accepted 10 July 2002 To identify genes expressed in interesting spatial and temporal patterns during development, we mobilized transposons carrying the yeast transcriptional activator, GAL4, to create new insertions throughout the Drosoph- ila genome. At these new sites, neighboring enhancers drive expression of GAL4 in a pattern similar to that of nearby genes (Brand and Perrimon, 1993). Since GAL4 binds to a UAS target sequence, flies containing a GAL4 transposon can activate transcription of a UAS-linked gene, thereby resulting in controlled expression of that secondary gene. Thus, the GAL4 P element is a useful tool both for identifying genes of interest and for ectopi- cally expressing genes in novel tissues or at specific developmental stages. We mobilized a GAL4 insertion on the second chro- mosome (P{GawB} CY2; Queenan et al., 1997) to gen- erate a collection of GAL4-expressing enhancer trap lines. F1 “jumpstarter” males (w 1118 /Y; GAL4/CyO; Sb 2-3/+) were mated in vials to attached-X (C(1)DX/Y) females. From 200 such crosses, red-eyed, curly-winged, long-bristled sons (bearing a new GAL4 insertion) were selected and mated individually to w 1118 /w 1118 females to establish 50 lines. Chromosome-mapping data, en- hancer trap pattern, and other phenotypic characters demonstrated that four insertions were duplicates, yield- ing a total of 46 independent lines (Table 1). All X and third chromosome lines are viable, although three lines exhibit reduced viability or fertility (lines 15, 45, and 50). We could not determine the viability of the CyO- linked lines since the balancer chromosome is homozy- gous lethal. The genotypes of the resulting stocks are: X-chromosome lines, w 1118 P{GawB}, second-chromo- some lines, y w; Pin/CyO, P{GawB}, and third chromo- some lines, y w; III P{GawB}. Line 15 also carries the FM6, y w Bar balancer while lines 45 and 50 contain TM3, Sb Ser. Finally, line number three exhibits a striking eye phenotype consisting of a variable loss of eye pig- ment in a gradient along the anterior/posterior axis. Surprisingly, this line lacks detectable GAL4 expression in larval eye discs. We characterized sequences flanking a subset of the insertions in our collection by the inverse PCR method (http:www.fruitfly.org/methods/). Table 1 lists neigh- boring predicted or known genes. To identify and visualize the morphology of cells ex- pressing GAL4, we crossed females from each line to males carrying UAS-taulacZ, which links the -galacto- sidase enzyme to the tubulin-binding protein Tau (Hildago et al., 1995). Some lines (1, 7, 10 (males only), 14, 22, 25, 26, 32, 35, 36, and 41) were lethal in pupal stages when crossed to this reporter; therefore, to assay ovary and testis expression, we crossed these strains to UAS-lacZ.NZ, which contains a nuclear-localization sig- nal (Y. Hiromi and S. West, unpubl. results). Tables 1 and 2 describe the expression patterns for each line and Figure 1 shows selected patterns that illustrate key points. More detailed descriptions of all the patterns are available upon request. All lines exhibit interesting staining in at least one developmental stage. As commonly noted, we observe patchy or variable GAL4 expression in many lines. For example, GAL4 drives embryonic PNS expression in 18 lines (Table 1). While all embryos within a given strain exhibit a general PNS pattern that is consistent, within an individual em- bryo, slight variations often occur between segmental repeats (e.g., brackets in Fig. 1a). Only a few lines express GAL4 in a single tissue at a specific developmental stage. For example, line 20 drives expression in the longitudinal visceral mesoderm from stage 12 onwards (Fig. 1c). When crossed to UAS- GFP, line 20 can be used to visualize this highly migra- tory tissue in vivo. As Tables 1 and 2 indicate, many lines express GAL4 in identical tissues. Nevertheless, impor- tant differences may exist in the distribution of expres- sion within each tissue. For example, Fig. 1f–i shows representative wing discs from four lines that express GAL4 in all imaginal discs, the brain, and the central nervous system (IGD, brain, and CNS). The distinct pat- terns shown in the wing discs presumably reveal specific regulatory elements that govern expression of nearby * Correspondence to: Celeste A. Berg, Department of Genome Sciences, Box 357730, University of Washington, Seattle, WA 98195-7730. E-mail: berg@gs.washington.edu Contract grant sponsors: the NIH (to EJW and SMJ), HHMI (to RLF and JBD), and NSF (to CAB). Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/gene.10138 © 2002 Wiley-Liss, Inc. genesis 34:46 –50 (2002)