Fig. 3.

Smo phosphorylation site variants show transdominant effects in vitro and in vivo. (A) Overexpressed untagged and GFP-tagged Smo variants cause transdominant effects on reporter activity. Either pAcSV- or pUAST-based plasmids expressing the Smo variants shown were transfected in the presence of endogenous Smo. The results of a representative ptc-Luc reporter assay are shown. (B) Overexpression of GFP-tagged Smo-Glu in embryos leads to expansion of Wg expression. Immunofluorescence using anti-GFP (green) and anti-Wg (red) Abs is shown for dorsal views at ×10 and ×25 of embryos at extended germ-band stage that are either wild-type (WT; w¹¹¹⁸) or expressing UAS GFP-tagged wild-type (Smo), III,V,VI Ala (Ala), or III,V,VI Glu (Glu) forms of Smo using the prd-GAL4 driver. P ↔ A, orientation of the posterior/anterior axis. (C) Overexpression of Smo variants leads to wing patterning alterations. Wings collected from adult flies that are either wild-type (WT) or heterozygous for the ptc-GAL4 (Upper) or 71B GAL4 (Lower) driver and expressing GFP-Smo variants as in B are shown. Longitudinal veins 1–5 are labeled. Asterisk, proximal L3 and L4 fusion for the GFP-Smo-Ala variant overexpressed with ptc-GAL4; arrowheads, ectopic veination near the proximal end of L3 when GFP-Smo is overexpressed (black) and proximal L3 and L4 fusion when GFP-Smo-Ala is overexpressed (white) using 71B GAL4. Flies expressing GFP-Smo-Glu die as pupae with ptc-GAL4 and as pharate adults with 71B GAL4. A severely defective wing from an escaper from the GFP-Smo-Glu 71B GAL4 line is shown (Lower Right).