Analysis of twenty‐four Gal4 lines in Drosophila melanogaster

The Gal4/UAS system is a powerful technique that allows the expression of a particular target gene in a tissue-specific manner (Brand and Perrimon, 1993). We isolated twenty-four Gal4 insertions with interesting expression patterns and characterized their chromosome location and whether the insertions were associated with zygotic lethality (Table 1). The Gal4 enhancer trap insertion that we used is described in Brand and Perrimon (1993). We characterized a number of lines originally generated by G. Technau and by Manseau et al. (1997), which are labeled T and C insertions, respectively. We used two stocks: w; Sco/CyO and w; Ly/TM3, Sb, to establish balanced stocks and determine whether the chromosome that carries the Gal4 insertion was homozygous viable. We characterized the expression patterns in ovaries, testis, embryos, larvae, and imaginal tissues. To determine the expression pattern of Gal4, we chose to express a membrane tethered green fluorescent protein (GFP) fusion molecule that allows rapid in vivo imaging as well as the analysis of fixed tissue. We crossed each of the Gal4 lines to UAS-mCD8-GFP (Lee and Luo, 1999) and analyzed the GFP expression in the progeny using either a Leica TCS-NT confocal microscope or a Zeiss Axiophot 2 fluorescent microscope equipped with a Zeiss Axiocam CCD camera. Embryos were collected overnight, dechorionated in 50% bleach, and fixed with 10% formaldehyde in PBS for 20 min. A variety of specific expression patterns were found and are summarized in Table 2 (Fig. 1). All Gal4 lines show expression in salivary glands, presumably due to a salivary gland-specific enhancer in the original construct (Brand and Perrimon, 1993). To analyze the general larval expression pattern, whole late 3rd instar larvae were dissected in Ringer’s solution and visualized live using fluorescence microscopy (Fig. 2). For a more detailed analysis of specific larval tissue, we dissected the discs in Ringer’s solution and examined them live (Fig. 3). The Gal4 expression patterns in larval brains, larval wing discs, eye discs, leg discs, and whole larvae are summarized in Table 3. We also determined the Gal4 expression pattern in the male and female reproductive systems. Ovaries from 5-day-old, well-fed female progeny were dissected in PBS and fixed with 4% formaldehyde in PBS-0.1% Triton X-100 for 20 min. All stages of oogenesis were examined for particular Gal4 expression patterns (Fig. 4, Table 2). To analyze the male reproductive system, the reproductive tract was dissected in PBS and visualized live without prior fixation (Fig. 5). GFP expression was noted for any pattern in the male germ line cells and associated somatic lineages within the testis, for the muscle and pigment cells that ensheath the testis, and for the somatic cells of the seminal vesicles, ejaculatory duct, and accessory glands of the reproductive tract (Table 2). In