Figure 6.

Altering expression of yellow, tan, and ebony in D. melanogaster alters pigmentation. For comparison to the effects of knocking down TFs with unknown effects, we examined the pigmentation phenotypes caused by altering genes required for pigment synthesis with our dissection and imaging protocols. Specifically, we examined the effects of altering the yellow, ebony, and tan genes, which are required for pigment synthesis (Massey and Wittkopp 2016). Changes in abdominal pigmentation were caused by loss-of-function mutations as well as Gal4-driven increases and decreases in expression for all three genes. The Gal4 driver used for this work was pnr-Gal4, which activates expression along the dorsal midline, as shown by a UAS-GFP construct in (A). Note that pnr-Gal4 activates expression in a subset of the dorsal abdominal cuticle [outlined in red in (A)], allowing pigmentation outside of the pnr-Gal4 expression domain to be used as an internal control within each cuticle. Wild-type D. melanogaster abdominal cuticle from females (B) and males (C) are also shown for comparison. (D–F) Loss-of-function mutations in yellow (D), ebony (E), and tan (F) altered pigmentation throughout the abdominal cuticle, with yellow mutants showing decreased black pigments, ebony mutants showing decreased yellow pigments, and tan mutants showing decreased brown pigments (G–I) Reducing activity of yellow (G), ebony (H), and tan (I) using RNAi caused similar changes in pigmentation, but only in the pnr-GAL4 expression domain. (J–L) Increasing activity of yellow (J), ebony (K), and tan (L) in the pnr-Gal4 expression domain had opposite effects on pigmentation. Images shown in this panel were taken on multiple days, causing imaging conditions to vary among genotypes.