(A) GFP signals in wing imaginal disc mosaic for the ubi-GFP transgene detected simultaneously by native GFP fluorescence (green) and by anti-GFP antibody (magenta). (B) Quantification of native GFP signal and anti-GFP antibody signal in (A), showing their linearity to the gfp gene dose (N = 4). Panels (C–D) show mosaic imaginal disc tissues obtained after mitotic recombination of heterozygous genotypes (see text). Homozygous da and emc mutant clones are negatively marked by GFP (green) or βGal (blue), respectively, within the same wing disc. Reciprocal twin spots are brightly labeled while unrecombined heterozygous cells show intermediate labeling. (C) Anti-Da labeling (red) in cells with different da and emc gene copies. Note that da-/+ emc+/+ (red arrowheads) and da-/+; emc-/+ (yellow arrowheads) cells have indistinguishable levels of Da protein. Cells with higher da gene dose have more Da protein (green arrowheads). (D) Anti-Emc labeling (red) in cells with different da and emc gene copies. Note that da-/+; emc-/+ (red arrowheads) and da-/+ emc+/+ (yellow arrowheads) have similar Emc protein levels, while da+/+; emc-/+ (green arrowheads) cells express higher levels of Emc than da-/+; emc-/+ (red arrowheads) cells do. (E–F) Quantification of Da (E) and Emc (F) antibodies fluorescence intensities. Mean ± SEM is shown (N = 7). X-axes represent the endogenous da gene dose and different colors represent different emc gene dose (E–F). In panel (E), the Da level in da+/-; emc+/+ cells appears greater than half that in to da+/+; emc+/+ cells but this was not reproduced in other studies (see panel G and Figure 2—figure supplement 1C). The Da level in da+/-; emc+/+ cells was not statistically different from that in da+/-; emc+/- cells. Remarkably, da+/+; emc-/+ cells have higher Emc protein levels than da-/+ emc+/+ cells do (p=0.00068, two-tailed t-test). (G) Quantification of Da antibody labeling from mosaic wing discs where clones vary the copy number of the endogenous da locus from 0–2 and independently vary the copy number of an unlinked genomic rescue transgene from 0 to 2 (see text and Figure 2—figure supplement 1E). X-axis represents the endogenous da gene dose and different colors represent da rescue transgene dose. Dashed horizontal lines represent mean Da levels for 1,2,3 and 4 gene copy genotypes. Note that the genomic transgene consistently expresses more Da protein than the endogenous locus. Mean ± SEM is shown,(N = 10). (H–I) Random flip-on clones overexpressing emc using a UAS-emc line are marked by GFP (green). Emc (red) over-expression abolishes Da (blue) upregulation in the morphogenetic furrow of the eye disc (H, yellow arrowheads) and the presumptive wing margin in the wing disc (I, yellow arrowheads), but has no discernible effect elsewhere. Genotypes: (A) hsFLP; Ubi-GFP FRT40/FRT40, (C–D) hsFLP; da3 FRT40/Ubi-GFP FRT40; emcAP6 FRT80/arm-LacZ FRT80, (H–I) hsFLP; UAS-emc 5.3/+; act > CD2>Gal4, UAS-GFP/+.