[9]. Our goal was to identify new proteins that regulate Wnt/b- catenin signaling at the level of the receptor complex. While Wnt and Frizzled proteins can signal independent of b-catenin, signaling through b-catenin is dependent on LRP5/6 co-receptors [7]. Therefore, we modified a Drosophila dsRNA screen [10] by using a constitutively active LRP6 mutant (DNLRP6) [11] to activate a fly-optimized b-catenin luciferase reporter, dTF12 [10]. In a high-throughput RNAi screen, clone 8 (cl8) Drosophila cells were transfected in a 384-well plate format with individual dsRNAs, dTF12, DNLRP6 and a Renilla luciferase control for cell viability and transfection efficiency. The cells were incubated for four days to allow knockdown of target RNAs and normalized reporter luminescence was then measured as an indicator of Wnt pathway activity. Approximately 0.5% of the library (,100 dsRNAs) modified the activation of the reporter by DNLRP6 in a manner validated by low throughput confirmation of the primary screen. dsRNA targeting eighteen genes with known human orthologs increased reporter activation two-fold or greater (Table S1). Seven of the eighteen amplicons that had potential off-target effects according to the publicly available Drosophila RNAi Screening Center (DRSC, flyrnai.org) database were not pursued further. Bili is a conserved FERM-domain protein Of the 11 remaining candidate negative regulators, we chose to focus on CG11848 (dBili) due to its predicted plasma membrane localization and the specificity of its effect on the Wg pathway. More specifically, of the 12 other screens in the DRSC database, dBili was identified only as a weak hit in a MAPK kinase screen (Friedman et. al., 2007). Bili, annotated as CG11848 (fly) and FRMD8/FKSG44 (human), also has orthologs in worms, zebrafish and mice (Fig. S1A). In human tissue samples, we found Bili mRNA expression highest in heart and spleen (Fig. S1B). Bili is a predicted 464 amino acid protein with a Band4.1/FERM domain in its N-terminus (amino-acids, 26–272) (Fig. S1C). FERM domains (4.1 Ezrin Radixin Meosin) are highly conserved protein domains important for directing plasma membrane association and, in some cases, linking integral membrane proteins to the actin cytoskeleton and modulating signaling [12]. Given that no prior Band4.1 domain proteins have been implicated in Wnt signaling, we focused on Bili to determine whether it functioned in a unique capacity in the Wnt/b-catenin pathway. Bili dsRNA validates in low throughput assays dsRNA targeting either dBili or Ran, a previously identified negative regulator of Wg signaling [13], enhanced the activation of dTF12 by either DNLRP6 or Wg (Fig. 1A). As expected, dBili dsRNA decreased endogenous dBili transcripts compared to control dsRNA (Fig. S3A). A second independent dsRNA targeting dBili, dsBili_2, also decreased dBili transcript levels and enhanced DNLRP6-activated reporter activity (Fig. S3A and B). Additionally, overexpressing dBili in S2R+ or Clone8 Drosophila cell lines inhibits Wg-dependent reporter activation (Fig. S3C and D). These data support the conclusion that dBili is a novel negative regulator of Wg signaling. Bili is coexpressed with Wg during Drosophila development If dBili is a component of the Wg pathway in Drosophila, then it should be co-expressed with other genes involved in Wg signaling. To test this hypothesis we performed in situ hybridization of dBili in Drosophila embryos (Fig. 1B) and larval imaginal discs (Fig. 1C). At early stages (stages 5–6) dBili RNA is uniformly expressed in the early embryonic ectoderm at low levels (data not shown), and Bili Inhibits Wnt Signaling more highly expressed in the neuroectoderm (Fig. 1B, a and b). Additionally, dBili is expressed in cells adjacent to the ventral midline just prior to initiation of the invaginating ventral furrow (black arrows, Fig. 1B, a and b). Interestingly, at later stages (stages 13–17), the expression of dBili is evident in the developing central nervous system (CNS, Fig. 1B, c and e) and the ventral epidermis in stripes (black arrows, Fig. 1B, e and e’) similar to the expression of Wg (Riggleman et al. 1990). At the end of embryogenesis (stage 16/17), the majority of dBili expression is restricted to the CNS (Fig. 1B, d). dBili and Wg expression patterns also overlap in wing, leg, and the eye-antennal imaginal discs in third instar larvae (Fig 1C). In the wing imaginal disc, dBili is expressed broadly in the wing pouch (black arrows, Fig. 1C, panel a) and in the notum (red arrow, Fig. 1C, panel a). The expression of dBili in the notum and that of Wingless (Wg) protein (blue arrow, Fig. 1C, panel b) appears to be non-overlapping, however they do abut each other. In the leg disc, dBili is expressed in a small discrete domain in the posterior leg disc (red arrow, Fig. 1C, panel c) as well as in the ventral wedge (black arrows, Fig. 1C, panel c) in a pattern slightly broader yet nearly identical to Wg expression (white arrow, Fig. 1C, panel b). In both dorsal and ventral compartments of the eye disc, dBili expression (black arrows, Fig. 1C, panel d) partially overlaps with the lateral edge expression of Wg (white arrows, Fig. 1C, panel e). In the antennal disc, dBili is expressed as a dorsal wedge (red arrow, Fig. 1C, panel d) which also overlaps with that of Wg (blue arrow, Fig. 1C, panel e). We conclude that the overlapping and adjacent expression of dBili and Wg are consistent with dBili functioning in the Wg pathway. dBili negatively regulates Wg signaling during Drosophila embryogenesis We next investigated the function of dBili during Drosophila embryogenesis. Wg has a well-established role in patterning the ventral epidermis and has been shown to be involved in cell fate determination [14,15]. Epidermal cells with active Wg signaling secrete naked-cuticle, whereas cells lacking Wg signaling activity secrete denticle belts. If dBili is a negative regulator of Wg signaling, reduced Bili function should resemble Wg gain of function phenotypes including ectopic naked cuticle. dBili levels were reduced during embryogenesis by expressing a short-hairpin RNA (shdBili) in the paired domain (prdGA- L4.UAS-shdBili). Quantitative real time-PCR (qRT-PCR) anal- ysis following expression of dBili shRNA under the control of prd- GAL4 in the Drosophila embryo revealed .75% knockdown of dBili mRNA levels compared to prd-GAL4 control embryos (Fig. S2A). As a control for the specificity of shdBili we also quantified levels of armadillo mRNA and found no change (Fig. S2A). Importantly, the expression of shdBili led to the expansion of Wg signaling activity as demonstrated by the secretion of mostly naked cuticle with very few cells secreting cuticle with trichomes (denticles) (Fig. 2A and 2B). Furthermore, driving dBili shRNA expression with even skipped stripe 3/7-GAL4, disrupted denticle stripe 2/3 (Fig. S2B). Thus, depleting dBili in the Drosophila embryo phenocopies hyperactivated Wg signaling, suggesting that dBili is a negative regulator of the Wg pathway in Drosophila embryos. Next we assessed the molecular consequences of depleting dBili in the paired domain. During embryonic segmentation, Wg activates and maintains the expression of engrailed (En) and defines para-segment boundaries [16]. Consistent with Wg gain-of- function, immunohistochemistry for En protein revealed a marked and uniform expansion of its expression in shdBili embryos (Fig. 2E–I). Wild type embryos at stage 12 (germ band retraction) PLoS ONE | www.plosone.org 2 July 2009 | Volume 4 | Issue 7 | e6129