An RNAi Screen for Genes Required for Growth of Drosophila Wing Tissue

Abstract

Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development.

Keywords: Drosophila; endoreplication; polyploid; tissue growth; wing disc.

Figure 1

Screen strategy to identify genes required for wing growth. The dpp-Gal4 / TM3 Sb Ser strain females was crossed to different UAS-shRNA strain males from the TRiP collection. The UAS-shRNA / + ; dpp-GAL4 / + progeny have expression of the shRNA expression in a dpp-GAL4 expression domain along the anterior-posterior boundary of the larval wing disc (red), which in the wing pouch is fated to become the region of the adult wing between longitudinal wing veins 3 and 4 (L3 and L4) (red shading). The L3-L4 intervein region of these UAS-shRNA / + ; dpp-GAL4 / + progeny (Sb⁺ phenotype) was scored for total area and wing hair size, organization, and morphology relative to other wing regions, with UAS-shRNA / + ; TM3 Sb Ser / + (Sb^- phenotype) siblings serving as additional internal controls.

Figure 2

Adult wing phenotypes of shRNA strains that impaired growth. (A – S) Bright field images of adult wings from a wild type dpp-GAL4 /+ control (A) or after expression of a UAS-shRNA targeting the indicated gene (B-S). Insets are higher magnifications to show wing hair phenotypes. Shown are the dorsal sides of the wings with anterior up. Scale bars are 150 μm for main panels and 75 μm for insets. (T, U) The length and number of wing hairs per unit area (hair density) were measured using the ImageJ plug in Fiji-wing. (T) Number of wing hairs per area from the L3-L4 intervein region divided by that in the L2-L3 + L4-L5 intervein regions of the same wings (N = three wings, with four L3-L4 areas and two L2-L3 + two L4-L5 areas per wing, **P ≤ 0.01, *P ≤ 0.05). (U) Length of wing hairs in the L3-L4 intervein region divided by that in the L2-L3 + L4-L5 intervein regions of the same wings (N = three wings with n = 20 hairs for L3-4 and 10 hairs for L2-L3 + 10 hairs for L4-L5 per wing. ** = P ≤ 0.01, * = P ≤ 0.05, n.s. = not significant, by Student’s t-test).

Figure 3

Immunofluorescent analysis of the effect of gene knockdown on ploidy of wing imaginal discs. (A-D’) Confocal images of wandering third instar wing discs labeled with antibodies against mRFP and the nuclear DNA dye DAPI, from UAS-mRFP / +; dpp-GAL4 / + controls (A,A’), or after knockdown of stg (B, B’), lin-52 (C,C’) or βNactes6 (D,D’). The red UAS-mRFP reporter expression indicates those cells that express dpp-GAL4, which is demarcated by red outlines in A’, B’, C’, and D’, with DAPI labeled nuclei shown in black and white. (E) Quantification of the nuclear size and DAPI fluorescence of shRNA expressing cells (RFP+) were measured and normalized to cells outside of the dpp-GAL4 domain in the wing pouches of same wing disc. lin-52 and βNactes6 knockdown resulted in significantly increased nuclear size and DNA content, whereas stg knockdown had increased nuclear size but not DNA content (N = two discs, with a 20-40 RFP+ and 20-40 RFP- cells scored per disc, ** = P ≤ 0.01 by Student’s t-test).