A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth

Abstract

Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression.

Figure 1. Select VDRC KK RNAi lines enhance Yorkie-induced eye overgrowth.

(a) Wildtype eyes (F₁ flies of VDRC genetic background crossed to GMR-Gal4, UAS-EYFP). (b,c) Adult eye phenotypes of F₁ flies carrying GMR-Gal4, UAS-yki^S168A–YFP transgenes crossed to VDRC genetic background (b) or VDRC line 105838 (targeting Caf1) (c). (d–f) Posterior area of pupal eyes of wildtype (d) and F₁ flies carrying GMR-Gal4, UAS-yki^S168A–YFP transgenes crossed to VDRC genetic background (e) or a recombinant harbouring Gal4-responsive UAS repeats but no functional shRNA-coding sequence at 40D (‘40D^UAS') (f). Pupal eyes were fixed and stained with Discs large antibody to highlight individual cells. Yellow arrowheads indicate interommatidial cells. Scale bar, 20 μm.

Figure 2. Enhancement of Yorkie-induced eye overgrowth by VDRC KK RNAi lines co-segregates with insertion at 40D and is independent of knockdown of intended target genes.

(a–j) Adult eye phenotypes of F₁ flies carrying GMR-Gal4, UAS-yki^S168A–YFP transgenes crossed to VDRC genetic background (a), VDRC line 107151 (MBD-like) with double insertion (b), single insertion at 30B (c) and 40D (d), VDRC line 105838 (Caf1) with double insertion (e), single insertion at 30B (f) and 40D (g), VDRC line 110512 (CG3630) with double insertion (h), single insertion at 30B (i) and 40D. The transgene insertion at 40D in line 110512 does not carry a functional hairpin-coding sequence, hence it is called ‘40D^UAS' throughout this manuscript. (j). (k,l) Eye phenotypes of flies harbouring sav³ clones expressing Gal4 in the eye combined with the VDRC genetic background (k) and 40D^UAS (l). Sav is an upstream negative regulator of Yki, therefore the sav³ mutation causes Yki hyperactivation. Clones were generated using the Mosaic analysis with a repressible cell marker (MARCM) system under the control of the eyeless (ey) promoter.

Figure 3. Overexpression of tiptop enhances Yorkie-induced eye overgrowth and induces bantam expression.

(a,b) Adult eye phenotypes of F₁ flies carrying GMR-Gal4, UAS-yki^S168A–YFP transgenes crossed to UAS control (a) and UAS-tio (b). (c) Tio mRNA expression in eye imaginal discs of F₁ flies carrying GMR-Gal4 crossed to VDRC genetic background, 40D^UAS and UAS-tio (positive control). QPCR was performed using two different primer sets probing tio mRNA expression, and normalized against Act5c and Rp49 mRNA-loading controls, yielding identical results (only tio primer set 1 and Act5c results shown). Average results from three independent experiments are shown. Error bars indicate s.e.m. *P<0.05, ****P<0.0001, unpaired two-tailed t-test. (d,e) Actin-Flp-out clones (yellow arrowheads) expressing Gal4 in combination with UAS-tio (d) or 40D^UAS (e) in larval eye imaginal discs expressing the ban-lacZ reporter. Scale bars, 50 μm.

Figure 4. Separation of insertion sites in the Warts KK RNAi line reveals potential for false-negative screening results and shows the power of KK RNAi lines if the 40D integration is removed.

(a–c) Wings of control flies (a) and F₁ flies carrying en-Gal4 crossed to VDRC line 106174 (wts) with double insertion (b) or single insertion at 30B (c). Flies were reared at 18 °C. (d–h) Adult eye phenotypes of F₁ flies carrying GMR-Gal4 crossed to VDRC genetic background (d), and wts RNAi lines Bloomington TRiP 27662 (e), NIG 12072R-1 (f), VDRC GD line 9928 (g) and VDRC KK line 106174, insertion at 30B only (h).