Generation of LexA enhancer-trap lines in Drosophila by an international scholastic network

Abstract

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic, and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II, and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and seventeen insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development, and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

Keywords: patched (ptc); 1360; Copia; L3 brain; LexA; Stan-X; enhancer trap; high school biology class; insulin producing cells; natural transposons.

Fig. 1.

wt P-element and lexA enhancer traps. a) wt P-element described in O’Hare and Rubin (1983). b) SE1 lexA enhancer trap used in Kockel et al. (2019). c) SX4 lexA enhancer trap used in this study. The SX4 enhancer trap encodes a lexA DNA-binding domain fused to the hinge and transactivation domain of Gal4, driven by the hsp70 promoter. The enhancer trap is marked by the white selectable eye color marker. See Supplementary Data File 1 for annotated sequence of SX4. d) Sequence logo (see Methods) derived from 281 independent 8 bp direct repeat sequences caused by SX4 insertion.

Fig. 2.

Map of novel Stan-X lexA enhancer trap insertions across chromosomal arms of X, II, and III. Chromosome arms are drawn to scale, and the enhancer trap positions are designated by their molecular coordinates. Scale below is in megabases (Mb). P-element insertions indicated below the chromosomal scaffold are oriented 3′ to 5′, and insertions above are oriented 5′ to 3′ relative to the reference sequence release 6 in FlyBase. Multiple insertions of identical orientation near identical genes are unified as single entry, and are separately listed in Supplementary Table 1. Insertions into natural transposable elements (natural TEs) are indicated in blue.

Fig. 3.

Expression of SX4 enhancer trap insertion into patched, ptc-lexA^SX4S8–9. Genotype: ptc-lexA^SX4S8–9/+; lexAop-CD8::GFP. a) Third instar larval brain, expression in VNC and CNS. b) Third instar larval gut, expression in enteric neurons located at proventriculus, caeca, and midgut. c) Third instar larval testis, expression in putative hub cells and spermatocytes. d) Third larval instar wing disc. Expression along the putative anterior–posterior boundary. Anterior to the right, except d) ventral to the right. Blue: DAPI. Green: anti-GFP. Scale bar 200 μm, except c) 100 μm.

Fig. 4.

Expression pattern of 6 representative SX4 enhancer traps crossed to lexAop-CD8::GFP in wandering third instar larval brains by IHC. Green: anti-GFP, blue: DAPI. a) SX4Aq845; lexAop-CD4::GFP. b) SX4Hb22-1B; lexAop-CD4::GFP. c) SX4La892; lexAop-CD4::GFP. d) SX4Lw221A; lexAop-CD4::GFP. e) SX4Pr4; lexAop-CD4::GFP. f) SX4S11-6; lexAop-CD4::GFP. All images were recorded with a 20× lens, scale bar = 200 μm.

Fig. 5.

Immunohistochemistry analysis of lex-A activity in insulin expressing cells (IPCs) of selected SX4 enhancer-trap lines. IPCs are marked by dilp2-Gal4, UAS-CD4::tdt (a–d), or anti-dilp2 co-stain (e and f), shown in red. Main images (a–f) were recorded with a 20× lens, scale bar = 200 μm. Inserts were recorded with 40× lens (A’, A”–F’, F”), scale bar = 100 μm. Green: anti-GFP. Red: anti-RFP (a–d) or anti-ilp2 (e and f). Blue: DAPI. A, A’, A”) dilp2-Gal4, UAS-CD4:tdt; SX4Et7; lexAop-CD8::GFP. B, B’, B”) dilp2-Gal4, UAS-CD4:tdt; SX4Et21; lexAop-CD8::GFP. C, C’, C”) dilp2-Gal4, UAS-CD4:tdt; SX4Et23; lexAop-CD8::GFP. D, D’, D”) dilp2-Gal4, UAS-CD4:tdt; SX4Et38; lexAop-CD8::GFP. E, E’, E”) SX4Pr5; lexAop-CD8::GFP. F, F’, F”) SX4Lw22e; lexAop-CD8::GFP.

Fig. 6.

Location, tagging, breakpoint cloning, and L3 brain expression pattern of natural TEs not present in FlyBase R6. a–d) Schematic representation of natural TE locations not represented in R6 and associated data. a) 1360 located at 2L:12,004,481 tagged by SX4Ch7, integrated 501 bp off the 3′ end of 1360. b) 1360 located at 2L:16,727,570 tagged by SX4Aq839, integrated 50 bp 3′ off the 3′ end of 1360, c) Copia located at 2R:9,237,980 tagged by SX4Et51, integrated 174 bp into 5′ of Copia, d) HMS Beagle located at 2R:15,951,007 tagged by SX4Et8 integrated 133 bp off the 5′ end of HMS Beagle. Blue line: genomic scaffold. Orange line: sequence originated by iPCR, spanning the breakpoint of the SX4 enhancer trap and the natural TE, and the breakpoint of the natural TE with the genomic scaffold. iPCR representation not to scale. Green lines: sequenced amplicons from w¹¹¹⁸, SX4; iso#32^II; iso#32^III genomic libraries, found by TE mapper (Methods). Sequence of amplicons is listed in Supplementary Table 2. Amplicon representations not to scale. Red box: natural TE. Arrow: direction of natural TE 5′–3′. A’–D’) Third instar larval brains of respective LexA enhancer traps crossed to w; lexAop-CD8::GFP. Genotypes: A’) w; SX4Ch7/+; lexApo-CD8GFP/+. B’) w; SX4Aq839/+; lexApo-CD8GFP/+. C’) w; SX4Et51/+; lexApo-CD8GFP/+. D’) w; SX4Et8/+; lexApo-CD8GFP/+. Blue: DAPI, Green: anti-GFP. Scale bar: 200 μm.

Fig. 7.

The Stan-X program. a) Timeline of school recruitment into the Stan-X program. Recruitment takes place during the school year, followed by training of the teachers in the Discover Now Teacher Academy in the summer (Supplementary Fig. 1). See text for details.