Bili Inhibits Wnt Signaling Figure 1. dBili (cg11848) is a negative regulator of Wg signaling. (A) Secondary reporter assay in clone 8 cells showing gfp, armadillo (arm), ran and dBili dsRNA effects on wingless (wg) (white bars) and DNLRP6 (gray bars) activation of b-catenin reporter, dTF12. Individual dsRNA effects on reporter activity in the absence of wg or DNLRP6 (inset). (B) Expression pattern of dBili mRNA by in situ hybridization. Embryos are oriented with anterior on the left and posterior on the right. (a & b) Stages 5–6 showing dBili expression in the neuroectoderm and cells adjacent to the ventral midline (black arrows) with high levels of expression in cells adjacent to the ventral furrow (white arrowhead). (c–e). At stages 13–17 the expression of dBili in the developing central nervous system (panel c & e) and the ventral epidermis in stripes (e & e’). At the end of embryogenesis (stage 16/17), the majority of dBili expression is restricted to the CNS (panel d). (C) (a & b) Wing imaginal discs. Panel a, shows dBili expression in the wing pouch (black arows) and in the notum/hinge area (red arrow). Panel b, staining for Wg protein in wing pouch and notum/hinge (blue arrow). (b & c) Leg imaginal discs. Panel c shows dBili staining in the ventral wedge (black arrows), identical to Wg expression (white arrow, panel b) (d & e) Expression in the eye disc of dBili (black arrows, d) and Wg (white arrows, e). Expression in the antenna disc of dBili (d, red arrow) and Wg (e, blue arrow). doi:10.1371/journal.pone.0006129.g001 and the ventral epidermis at stage 14 (onset of head involution and dorsal closure) have approximately two rows of cells expressing En (Fig. 2, F and G). These regions were expanded to approximately four rows of cells in the presence of shdBili (Fig. 2, H and I). These data further support the conclusion that dBili is an inhibitor of Wg signaling during Drosophila embryonic development. PLoS ONE | www.plosone.org 3 July 2009 | Volume 4 | Issue 7 | e6129