A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species

Abstract

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

Keywords: Cis-regulatory elements; Drosophila melanogaster; Tribolium castaneum; Enhancers; Gene regulatory network; Reporter assay.

Figure 1

New suite of reporter vectors for diverse insects. (a) Expanding the functionality of the DSCP-based reporter construct by selecting different modes of transgenesis, reporter genes, and transgenic markers. A wing enhancer of Tribolium is shown as an example. A putative wing enhancer can be isolated from the Tribolium genome, cloned into one of our reporter vectors, and transformed into both Tribolium (i.e. the native species) via piggyBac transgenesis and Drosophila (i.e. a cross-species setting) via PhiC31 transgenesis, and evaluated for its enhancer activity using DSCP as a universal basic promoter. (b) Linear maps of the previously published vector piggyGUM (i) and the new constructs presented in this study (ii–x). An enhancer of interest can be cloned into the GATEWAY cassettes (attR1-attR2; i–ix) through an LR clonase reaction. Only the components relevant to reporter constructs are shown. ampR Ampicillin-resistance gene, chlR Chloramphenicol resistance gene, ori Replication origin.

Figure 2

Comparison of enhancer-reporter vectors in Drosophila with piggyGUM-TcNub1L. (a–h) Expression driven in the Drosophila imaginal discs by the previously published vector piggyGUM-TcNub1L (a) and the new suite of reporter vectors (b–h). The enhancer activity was visualized by mCherry (a), EGFP (b, e), UAS-Red Fluorescent Protein (RFP) (c–d), and UAS-tdTomato (f–h). All new constructs (b–h) recapitulated the expression pattern driven by the previously published piggyGUM-TcNub1L (a) in Drosophila imaginal discs. Scale bars: 50 $\mu$m.

Figure 3

Comparison of enhancer-reporter vectors in Tribolium . (a–i) Expression of the Tc-nub enhancer trap line pu11 (a–c), piggyGUM-TcNub1L (d–f), and piggyGUE-TcNub1L (g–i) in Tribolium. Wing expression is shown in the larval thorax (a, d, g), dissected elytral (b, e, h) and hindwing discs (c, f, i). The new piggyGUE construct (g–i) recapitulated the expression pattern of previously published piggyGUM-TcNub1L (d–f). Scale bars: 100 $\mu$m (a, d, g); 50 $\mu$m (b–c, e–f, h–i).

Figure 4

Expression of Gal4-UAS vectors in Tribolium. (a, a’) piggyGUG-TcNub1L/UAS-tGFP without heat-shock (at 30 °C, a), and with heat-shock (at 48 °C, a’). tGFP expression is seen in the wing stripe only after the heat-shock (arrowhead in a’). (b) piggyGUGd-TcNub1L/UAS-tGFP at 30 °C. tGFP expression is seen in the wing stripe (arrowheads), as well as in the cells scattered throughout the larval and pupal body (arrows). (c) piggyPhiGUGdTomI-TcNub1L at 30 °C. tdTomato expression is seen in the wing stripe (arrowhead), and also in internal tissues throughout the body (arrows). (d) piggyGUE-TcNub1L. The whole wing activity of TcNub1L in piggyGUE (d) differs significantly from its activity tested with the Gal4-UAS reporter constructs (a–c). Scale bars: 0.5 mm.

Figure 5

Workflow to identify position effect-free PhiC31 attP landing sites with piggyLANDR. (a) Enhancer traps can be detected on any of three promoters based on the expression of tdTomato, ECFP, or activation of a separate UAS-reporter by Gal4d. (b) Silencing position effects (including heterochromatin formation shown in the diagram) can be assessed via activation of UAS-tdTomato with a separate Gal4 driver (such as a wing-Gal4 in the diagram). (c) Once the ideal landing site unaffected by both enhancer trapping and silencing position effects is identified, the reporter function of the piggyLANDR construct will be removed via Cre-loxP recombination, leaving a 3xP3-ECFP marked PhiC31 attP landing site free of position effects.

Figure 6

Examples of enhancer traps in piggyLANDR lines. (a–f) Enhancer trap expressions detected in the third instar larvae of six piggyLANDR lines crossed with UAS-EGFP. In most lines, UAS-tdTomato expression was highly similar to UAS-EGFP expression (i.e. enhancer trapping on DSCP-Gal4d) (a–c’, f, f’). In some lines, only EGFP (arrowheads, d, d’) (i.e. enhancer trapping on DSCP-Gal4d, with silencing on UAS-tdTomato), or tdTomato (arrowheads, e, e’) (i.e. enhancer trapping on UAS-tdTomato) was detected. The minimal overall enhancer trap expression of piggyLANDR-1 (a) makes this insertion site a good candidate for an ideal PhiC31 landing site free of position effects. Scale bars: 0.5 mm.

Figure 7

piggyLANDR enhancer traps in larval imaginal discs. (a) tdTomato enhancer trap expression of piggyLANDR-3a and piggyLANDR-10 in the imaginal discs. Only two out of 11 piggyLANDR lines exhibited enhancer trap expression in the imaginal discs. (b) EGFP expression of piggyLANDR-3a and piggyLANDR-10 when crossed to UAS-EGFP. piggyLANDR-3a had overlapping expression of EGFP and tdTomato. In contrast, piggyLANDR-10 failed to activate UAS-EGFP in the imaginal discs, suggesting an enhancer trap on UAS-tdTomato but not on DSCP-Gal4d in the imaginal discs of this line. The absence of any observable enhancer trap expression in the imaginal discs of piggyLANDR-1 makes this insertion site a good candidate for an ideal PhiC31 landing site free of position effects. Scale bars: 50 $\mu$m.

Figure 8

The lack of detectable enhancer traps and silencing effects at potential landing sites. (a) The lack of active and lineage trace expression in three piggyLANDR lines, except for some neurons in the eye of piggyLANDR-8. (b) EGFP and tdTomato expression of the three piggyLANDR lines when crossed to piggyGUGd-TcNub1L; UAS-EGFP. Overlapping expression of tdTomato and EGFP suggests no silencing effect on these loci in the developing appendages. Scale bars: 50 $\mu$m.

Figure 9

Removing the DSCP-Gal4d UAS-tdTomato cassette from piggyLANDR. (a, a’) Ubiquitous epidermal expression of Gal4d and tdTomato in piggyLANDR-10. (b, b’) Loss of UAS-tdTomato expression upon crossing with hsp-Cre, Tb, demonstrating successful removal of DSCP-Gal4d and/or UAS-tdTomato from piggyLANDR in all but a few small groups of cells (arrowheads, b). 3xP3-ECFP expression in the anal pads (arrow, b’) confirms that piggyLANDR is not simply absent. (c, c’) Confirmation of the removal of the DSCP-Gal4d UAS-tdTomato cassette from piggyLANDR-10 by UAS-EGFP. Lack of UAS-tdTomato (c) and UAS-EGFP (c’) expression indicates successful removal of both DSCP-Gal4d and UAS-tdTomato, while 3xP3-ECFP expression in the anal pads confirms the presence of piggyLANDR (arrow, c’). Some background expression of UAS-EGFP (independent of Gal4) is still visible (asterisks, c’). Scale bars: 0.5 mm.