expression. This phenotype is consistent with a localized increase in Wingless signaling activity in the region around eve-stripe 3. The same however was not observed for stripe 7 (posterior end of the embryo), perhaps due to differential expression of GAL4 in stripe 3 versus stripe 7. Found at: doi:10.1371/journal.pone.0006129.s003 (3.11 MB EPS) Figure S3 (A) Knockdown of dBili with a second dsRNA, ds11848_2, revealed only ,30% knockdown of endogenous message compared to the original dsRNA (DRSC1.0-CG11848) which robustly knocked down message levels by .50%. Primer sequence used for the generation of ds11848_2 PCR is as follows: Forward primer: 59-GTAATACGACTCACTATAGGGAGA GAAGATA- CAAGTGAGGCATTC-39 and Reverse primer: 59GTAATAC- GACTCACTATAGGGAGAGGCAAATAAAATATCTGATG- GGTGCGTGG-39 (B) Knockdown of dBili with ds11848_2 displayed a modest increase (,35–40%) in dTF12 (Wg-reporter) activity. (C & D) Expression of dBili-HA strongly inhibits dTF12- reporter activity when the pathway is activated by Wg overexpres- sion. The inhibitory effect of dBili is comparable to that of Axin expression in both S2R+ (C) and clone8 (D) cells. (E) dBili protein (in GREEN) is localized in a concentric ring, just inside and abutting Arm protein (in RED) at the membrane of cells in the drosophila embryo. (E’) Magnitifed view of boxed region in panel E. Found at: doi:10.1371/journal.pone.0006129.s004 (5.47 MB EPS) Figure S4 The zebrafish homolog of Bili (zfBili) is expressed during embryogenesis. In situ hybridization using a sense (left column) and antisense probes (right column) were used to detect mRNA. (A) zfBili is expressed maternally as it is detected in the animal pole 4 h post-fertilization. (B) zfBili continues to be expressed ubiquitously at 50% epiboly. (C) 24 hpf zfBili remains ubiquitous but shows specific staining in the otic vesicle (arrow head). (D) Weak Bili expression in the tail at 36 hpf. (E) A second morpholino targeting zfBili (bilimo2) but not a control morpholino (como) enhances the Wnt8 overexpression phenotype (n = 50 for each condition). Embryos were scored as wt (green), small eyes (yellow), no eyes (orange) or severe (red). Data shown is representative from four independent experiments. Found at: doi:10.1371/journal.pone.0006129.s005 (10.82 MB EPS) Figure S5 Bili negatively regulates Wnt/{capital b-catenin signaling in mammalian cells. (A) siRNA mediated knockdown of hBili enhances Wnt1 mediated induction of a b-catenin responsive reporter. HEK293T cells were transfected with siRNA targeting control, b-catenin, or hBili. Cells were then transfected with WNT1 References 1. Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480. 2. Kimelman D, Xu W (2006) beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene 25: 7482–7491. 3. Bilic J, Huang YL, Davidson G, Zimmermann T, Cruciat CM, et al. (2007) Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. Science 316: 1619–1622. 4. Zeng X, Tamai K, Doble B, Li S, Huang H, et al. (2005) A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438: 873–877. 5. Davidson G, Wu W, Shen J, Bilic J, Fenger U, et al. (2005) Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438: 867–872. 6. Lee E, Salic A, Kruger R, Heinrich R, Kirschner MW (2003) The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol 1: E10. 7. He X, Semenov M, Tamai K, Zeng X (2004) LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 131: 1663–1677. 8. Bejsovec A (2006) Flying at the head of the pack: Wnt biology in Drosophila. Oncogene 25: 7442–7449. Bili Inhibits Wnt Signaling cDNA and a b-catenin responsive reporter and assayed the following day. (B) hBili siRNA effectively knocked down hBili transcripts in HEK293T as measured by qRT-PCR. (C) hBili siRNA effectively decreased the expression of a hBili-Venus fusion protein as assayed by fluorescence measurement. hBili siRNA had no effect on Venus expression. (D) A second siRNA (Bili #2) targeting hBili enhanced WNT1 mediated activation of a b-catenin responsive reporter. (E) Bili #2 siRNA also effectively decrease expression of a hBili-Venus fusion protein with no effect on Venus alone. (F) hBili knockdown in HEK293T cells with hBili siRNA enhanced the WNT3A mediated transcriptional induction of endogenous AXIN2 transcripts as assayed by qRT-PCR. Error bars represent STDEV (*p,0.05, **p,0.005, ***p,0.0005). Found at: doi:10.1371/journal.pone.0006129.s006 (1.48 MB EPS) Figure S6 Bili does not affect control reporters or CREB responsive reporters. (A) HEK293T cells were transfected with FUBAR, a control reporter not responsive to Wnt/b-catenin signaling, renilla luciferase normalization, and control or hBili siRNA. FUBAR was not responsive to WNT3A conditioned media (right two bars) and hBili siRNA had no effect. (B) HEK293T cells were transfected with FOPFlash, another control reporter not responsive to Wnt/b-catenin signaling, renilla luciferase normalization, and GFP cDNA or increasing doses of hBili cDNA or control siRNA or hBili siRNA. Neither hBili cDNA control nor hBili siRNA had an effect on the reporter. (C) hBili siRNA HEK293T cells stably expressing a CREB responsive reporter were transfected with renilla luciferase normalization, and control, hBili, or hBili#2 siRNA and treated with DMSO, 1 uM forskolin, or 10 uM forskolin. Error bars represent STDEV. Found at: doi:10.1371/journal.pone.0006129.s007 (1.14 MB EPS) Acknowledgments We thank Jason Berndt, Ben Major, Charlotte Hubbert, and Andy Chien for reading the manuscript. We gratefully acknowledge Dr. Norbert Perrimon for critically reading the manuscript and for providing the Drosophila RNAi screening library through DRSC, Boston, where the primary screen was performed. We also thank Dr. Stephen Small for fly stocks. Author Contributions Conceived and designed the experiments: LK TB AK RD RTM. Performed the experiments: LK BC TB WHC RD. Analyzed the data: LK TB RD RTM. Contributed reagents/materials/analysis tools: TB. Wrote the paper: LK TB RTM. 9. Friedman A, Perrimon N (2007) Genetic screening for signal transduction in the era of network biology. Cell 128: 225–231. 10. DasGupta R, Kaykas A, Moon RT, Perrimon N (2005) Functional genomic analysis of the Wnt-wingless signaling pathway. Science 308: 826–833. 11. Liu G, Bafico A, Harris VK, Aaronson SA (2003) A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor. Mol Cell Biol 23: 5825–5835. 12. Louvet-Vallee S (2000) ERM proteins: from cellular architecture to cell signaling. Biol Cell 92: 305–316. 13. Hendriksen J, Fagotto F, van der Velde H, van Schie M, Noordermeer J, et al. (2005) RanBP3 enhances nuclear export of active (beta)-catenin independently of CRM1. J Cell Biol 171: 785–797. 14. Bejsovec A, Martinez Arias A (1991) Roles of wingless in patterning the larval epidermis of Drosophila. Development 113: 471–485. 15. Bejsovec A, Wieschaus E (1993) Segment polarity gene interactions modulate epidermal patterning in Drosophila embryos. Development 119: 501–517. 16. Wodarz A, Nusse R (1998) Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14: 59–88. 17. Kelly GM, Greenstein P, Erezyilmaz DF, Moon RT (1995) Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways. Development 121: 1787–1799. PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6129