Figure 7
RNAi-mediated knockdown of vari in the wing imaginal disc. (A) Gal4/UAS-mediated overexpression of a vari-RNAi transgene along the antero-posterior boundary of a third instar wing imaginal disc with ptcGal4 significantly reduces the amount of Vari protein (white arrows). (B) Close-up view on imaginal disc cells from the wing imaginal disc shown in A. In the overexpression region (dotted white line in B) Vari (green) is no longer detectable at the SJ. Crb (red) still localises to the SAR, demonstrating that apico-basal polarity of the wing imaginal disc epithelial cells is unaffected. (C) Wing blades from adult wild-type flies (C) and flies in which the vari-RNAi transgene was expressed with sdGal4; Gbe+Su(H)lacZ. Overexpression of the transgene affects wing shape and margin formation to different degrees. Bristles and hairs are lost from the posterior and, more rarely, anterior wing margin, respectively (arrows in C', C"). The wings of adult sdGal4; Gbe+Su(H)lacZ>UAS vari-RNAi flies normally appear 'collapsed', but unfold again during the embedding procedure. (D) Anti-Wg antibody staining of sdGal4; Gbe+Su(H)lacZ (D) and sdGal4; Gbe+Su(H)lacZ>UAS vari-RNAi (D', D") third instar wing imaginal discs. In comparison to wild-type (D) vari knockdown leads to a lighter and discontinuous Wg expression at the prospective wing margin (arrows in D', D"). (E) X-Gal staining of sdGal4; Gbe+Su(H)lacZ (E) and sdGal4; Gbe+Su(H)lacZ>UAS vari-RNAi (E') third instar wing imaginal discs to visualize activity of the Notch signaling pathway. Notch activation at the prospective wing margin appears to be more diffuse and sometimes interrupted (arrow in E'). (F) Western blot analysis of protein lysates from wild-type wing and eye imaginal discs, wild-type ovaries as well as wild-type and Df(2L)DS6 embryos, probed with an anti-Vari antibody that recognises both Vari-long and Vari-short. Whereas in wing and eye imaginal discs both Vari-long and Vari-short can be detected, wild-type ovaries express only Vari-short. The protein amount loaded per lane equals 10 wing or eye imaginal discs, 1 ovary and 5 embryos, respectively. The lysates from wild-type and Df(2L)DS6 embryos serve as controls and the unspecific, cross-reacting band slightly below 72K as a loading control. In A, B, D and E anterior is left and dorsal is up. In C anterior is up.