(A) GT insertions in LRP6 (top histogram) and AXIN2 (bottom histogram) mapped for the sorted cells from the WNT positive regulator, low stringency screen (Figure 1E). The histograms depict the number of GT integrations in the sense (blue) or antisense (red) orientation, relative to the coding sequence of the gene, within consecutive 500 base pair (bp) intervals along the length of each gene. Due to the directionality of the splice acceptor in the GT, typically only sense GT insertions in introns disrupt the gene, whereas GT insertions in exons generally disrupt the gene regardless of orientation. RefSeq gene tracks for LRP6 and AXIN2 are shown beneath each histogram following the University of California, Santa Cruz (UCSC) genome browser display conventions: coding exons are represented by thick blocks, UTRs by thin blocks, and introns by horizontal lines connecting the blocks. Both genes are displayed with their 5’ ends to the left, and encompass chromosome 12, bps 12267499–12116000 for LRP6, and chromosome 17, bps 65561999–65528500 for AXIN2 (hg18). (B) Schematic representation of the human AXIN2 protein drawn to scale in the horizontal dimension. Amino acid numbers are indicated below, and arrows show the sites at which truncations were made by CRISPR/Cas9-mediated genome editing in the indicated cell lines. Known domains, regions and motifs (based on UniProt annotation) are depicted in gray (TB, tankyrase-binding motif). Exon 11, eliminated by GT insertions found in cells sorted during the WNT screens for positive regulators, is delineated by a thinner white block. (C) Fold-induction in WNT reporter (median EGFP fluorescence from 20,000 cells) following treatment with 50% WNT3A CM or 10 μM of the GSK3 inhibitor CHIR-99021. Each circle represents a unique clonal cell line (determined by genotyping, Supplementary file 2), and the average of three to four independent clones for each genotype is indicated by a horizontal line. For each treatment, percentage reporter activation relative to WT cells is also indicated above each group of circles to facilitate comparisons. Significance was determined by one-way ANOVA, and is indicated as ****(p<0.0001) or ns (not significant). (D) Fold-induction in soluble CTNNB1 protein (average CTNNB1 intensity normalized to ACTIN intensity from duplicate immunoblots) following treatment with 50% WNT3A CM or 10 μM CHIR-99021. Each circle represents a unique clonal cell line, and the average of two independent clones for each genotype is indicated by a horizontal line. Significance was determined by unpaired t-test with Welch’s correction. Representative immunoblots used for quantification of CTNNB1 and ACTIN are shown in Figure 3—figure supplement 1C. (E) Nuclear CTNNB1 (average nuclear fluorescence per unit area from three fields of view) in single clonal cell lines of the indicated genotypes was quantified as described in Materials and methods. Cells were treated with 50% WNT3A CM where indicated. For each cell line, the fold-increase in CTNNB1 nuclear accumulation following treatment with WNT3A, expressed as percentage of WT, is also indicated above the bars to facilitate comparisons. For WNT3A-treated cells, differences in nuclear CTNNB1 between WT and mutant cells were not statistically significant as determined by one-way ANOVA. Examples of confocal sections used for quantification of nuclear CTNNB1 are shown in Figure 3—figure supplement 2B–2D. (F) WNT reporter activity (median EGFP fluorescence from 10,000 cells), soluble CTNNB1 protein (average CTNNB1 intensity normalized to ACTIN intensity from duplicate immunoblots), and nuclear CTNNB1 protein (average nuclear fluorescence per unit area from 2 to 3 fields of view), depicted as percentage of WT, for cells treated with 50% WNT3A CM. Each circle represents a unique clonal cell line, and where applicable the average of two independent clones is indicated by a horizontal line. (G) Adult D. melanogaster wings expressing Axin-V5 (top images) or AxinΔC-V5 (bottom images) under the control of the c765-Gal4 driver. Loss of sensory bristles and tissue at the wing margin, indicative of impaired Wg signaling, is shown (arrow) in the higher magnification view of the delineated area. While loss of Notch signaling can also result in wing margin defects due to a requirement of Notch signaling for Wg expression at the dorso-ventral boundary in the wing imaginal disc (Diaz-Benjumea and Cohen, 1995), we ruled out that possibility by confirming intact Wg expression in the wing imaginal disc of flies expressing AxinΔC-V5 (Figure 3—figure supplement 3B). Scale bars = 20 μm. (H) Percentage of flies with wing margin defects. 4.3% of flies expressing Axin-V5 exhibited loss of bristles at the wing margin, but no loss of wing tissue; 46.6% of flies expressing AxinΔC-V5 exhibited loss of bristles at the wing margin as well as loss of wing tissue.