Bili Inhibits Wnt Signaling Figure 2. Expansion of wg signaling activity in embryos (ventral view) with diminished levels of dBili. (A) Cuticle preparation of WT control embryo displaying wild type patterning of the embryonic epidermis with uniformly spaced denticle belts. (B) Cuticle preparation of embryo expressing shRNA targeting dBili (shdBili). (B’) WT and shdBili expressing embryos showing a weaker phenotype (black arrows). (C & D) High magnification view of A & B, respectively. D’ & E show boxed region in D. (E–I) Engrailed (En) protein expression. (F & G) Expression of engrailed in WT embryos is restricted to one or two cell-layers in the epidermis during early stage 12 (germband retraction) and in the ventral epidermis at stage 14 (onset of head involution and dorsal closure). (H & I) En expression in shdBili stage-matched embryos (compare with F & G). doi:10.1371/journal.pone.0006129.g002 Bili is conserved in vertebrates and negatively regulates Wnt/b-catenin signaling in zebrafish The Wg signaling pathway is highly conserved throughout evolution. Therefore, we next asked if Bili negatively regulates Wnt signaling in vertebrates. To this end, we asked if Bili function is conserved in zebrafish. Ectopic activation of Wnt/b-catenin signaling in early zebrafish development causes dose-dependent anterior truncations and mesodermal defects (Fig. 3A) [17]. If zfBili functions as a negative regulator of Wnt/b-catenin signaling during zebrafish development, then silencing zfBili expression should exacerbate these phenotypes. Overexpression of Wnt8 results in anterior truncation phenotypes of varying severity (Fig. 3A). Silencing Bili with either of two non-overlapping antisense morpholinos targeting zfBili (bilimo or bilimo2) shifted the Wnt8 induced phenotypes toward increasing severity (Fig. 3C, top panel, Fig. S4E). As controls, we first confirmed that zfBili is expressed throughout early zebrafish development (Fig. S4A–D) and second verified that bilimo, but not a control morpholino (como), repressed translation of zfBili (Fig. 3B). We next investigated the effect of zfBili gain-of-function on zebrafish development. Embryos injected with zfBili RNA, but not control RNA, displayed a slight expansion of anterior structures, which is consistent with diminished Wnt/b-catenin signaling (data not shown) [18]. Importantly, zfBili overexpression rescued Wnt8 gain-of-function phenotypes (Fig. 3C, bottom). Together, these results demonstrate that Bili is an evolutionarily conserved negative regulator of Wnt/b-catenin signaling. Bili negatively regulates Wnt/b-catenin signaling in human cultured cells We next tested whether Bili regulates Wnt signaling in human cells. We performed siRNA knockdown or cDNA overexpression of hBili in RKO colorectal carcinoma cells or human embryonic kidney (HEK293T) cells expressing a b-catenin responsive PLoS ONE | www.plosone.org 4 July 2009 | Volume 4 | Issue 7 | e6129