Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters

Abstract

Background: The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.

Results: Here we report the establishment of the GAL4/UAS binary expression system in Tribolium castaneum. Both GAL4 Delta and GAL4VP16 driven by the endogenous heat shock inducible promoter of the Tribolium hsp68 gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in Tribolium - 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on Tribolium, Drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.

Conclusions: With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in Tribolium. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in Tribolium and probably also other model systems.

Figure 1

Schematic representation of driver and responder constructs. Driver constructs: (a) Dm-hs-GAL4; (b) 3 × P3-GAL4-VP16; (c) 3 × P3-GAL4Δ; (d) Tc-hsp-GAL4-VP16; (e) Tc-hsp-GAL4Δ. Responder constructs: (f) UAS-Dm-hsp-LacZ; (g) UAS-Dm-hsp-Tc-giant; (h) UAS-SCP1-DsRed; this construct contains GAGA sites to prevent position effects. (i) UAS-Dm-hsp-Tc-bh-EYFP; (k) UAS-Tc-bhsp-tGFP.

Figure 2

The GAL4/UAS system is active in several Tribolium stages and tissues. Animals positive for both, the GAL4Δ driver and UAS responder construct, show strong tGFP fluorescence after heat shock. Shown are pupae (a-d) and adults (e-h). (a, e) No fluorescence is visible in the negative control (heat-shocked animals that carry only the responder line UAS-Tc-bhsp-tGFP#7, i.e. eyes with red fluorescence only). (b, f) Without heat shock, animals carrying driver (Tc-hsp-GAL4Δ#1) and responder (UAS-Tc-bhsp-tGFP#7) - eyes marked red and blue - do not show body fluorescence. (c, g) Immediately after heat shock, animals carrying driver and responder construct exhibit no fluorescence of tGFP. (d, h) The same animals 24 h after heat shock exhibit strong tGFP fluorescence. Note expression in the adult wings (h). (i-u). Fluorescence is detected in some internal organs upon activation of the system (m,q,u) but not before heat shock (k, o, s; note low level of autofluorescence at different wave length). Tested were male reproductive organs (i-m), female reproductive organs (n-q) and gut (r-u). (i, l, n, p, r, t) are the respective bright field pictures.

Figure 3

Endogenous versus exogenous promoters in transactivator and responder constructs. Only when Tribolium core promoters are used in the responder and transactivator constructs, activation of the reporter gene is observed via in-situ hybridization. Exogenous promoters are not capable of driving reporter gene expression. (a-h) Transactivator line Tc-hsp-GAL4Δ#1 was crossed to different responder lines and in-situ hybridization was performed with the respective antisense RNA probe. (a, b) In the positive controls (UAS-Tc-bhsp-tGFP) ubiquitous expression of tGFP is detected. In contrast, no reporter activity is detected in constructs utilizing a Drosophila core promoter (UAS-Dm-hsp-LacZ) (c, d) or an artificial "super core promoter" (UAS-SCP-DsRed) optimized for vertebrate cells (e, f). (g, h) A construct containing both the Drosophila basal heat shock promoter and the Tribolium hairy promoter (UAS-Dm-hsp-Tc-bh-EYFP) shows activation predominantly in the nervous system of advanced embryos. (i-k) The Drosophila heat shock promoter driving Gal4 shows no activity in one insertion line (i) while in the other some isolated cells show expression of the reporter (k).

Figure 4

Temperature dependence of the GAL4/UAS system in Tribolium. The transactivator line Tc-hsp-GAL4Δ#1 was crossed to the responder line UAS-Tc-bhsp-tGFP#7. Eggs from a 1h collection were heat shocked and checked for tGFP fluorescence directly after heat shock, at five one-hour intervals, and again after 24h. Each row shows the same embryos at a given time after heat shock kept at a certain temperature. As both lines are heterozygous, only about 25% of embryos were expected to exhibit tGFP fluorescence. In the bottom row, embryos of the heat shocked responder line at 30°C are shown as negative control. At 26°C, fluorescence is first seen 5 hours after heat shock (Af); at 28°C: 4-5 hours (Be; Bf); at 30°C: 3-4 hours (Cd; Ce); at32°C: 3 hours (Dd).

Figure 5

Fast onset of GAL4 driven expression relative to embryonic developmental time. Transheterozygous eggs were collected for one hour and aged for 18 hours before heat shock. They were then incubated at 25°C/77°F (a-m) or 32°C/90°F (n-y) respectively. At given time points, tGFP expression was detected by in-situ hybridization. (a, b) At 25°C, there is no expression of the reporter gene two hours after heat shock. (c) After 4 h weak expression is detected. (d) After 6 h strong expression of the reporter gene is seen. (n) At 32°C there is no expression directly after heat shock. The onset of expression is seen after 2 h (o) and remains high during the subsequent two hours (p, q). (e-m) and (r-y) show higher magnifications of the respective embryos above. Judged from head morphology and appendage outgrowth, embryonic development has progressed little between heat shock and first appearence of reporter gene expression (compare e, i to g, l and r, v to s, w).