Fig. 2. Slouch-expressing cells in cluster II require more Wingless than Slouch cluster I. (A-D) In this and all subsequent figures ventral views of late stage 11 abdominal segments 5-8 are shown. Insets depict one representative hemisegment at a higher magnification. Black arrowheads mark cI; white arrowheads mark cII. Black arrows denote the midline in panels; black arrows in A'-D' and white arrows in A''-D'' indicate Slouch-expressing CNS cells. (A) Wild-type embryo shows stereotypic repeating pattern of Slouch-positive clusters. (A') Immunocytochemistry of a single hemisegment reveals that cII (white arrowhead) aligns with the CNS, while cI (black arrowhead) localizes just posterior to cII. (A'') Confocal micrograph of a wild-type hemisegment; in cII (white arrowhead) Kr (red/Cy3) and Slouch (green/FITC) co-localize (yellow), while in cI (black arrowhead) Slouch (green/FITC) alone is expressed. Slouch is also expressed in the CNS (white arrow). Kr is also expressed in the CNS. Arrowheads and colors are maintained for all figures. (B) wg^PE6 embryo at the nonpermissive temperature (25°C) lost Slouch expression. (B') Immunocytochemistry reveals an example in which both cII and cI are missing. (B'') Confocal micrograph of a single hemisegment shows an example in which Slouch staining is absent from the position where cII is normally found, but is present for cI. Slouch expression does not overlap with Kr. The identity of these Kr-positive cells is unknown. (C) wg^PE6 at the permissive temperature (18°C) showed expression of cI and cII in some hemisegments. (C') Immunocytochemistry reveals an example where both cII (white arrowhead) and cI (black arrowhead) are present. (C'') Confocal micrograph shows another example in which Slouch staining is absent from the normal cII position, and Slouch does not co-localize with Kr in cI (black arrowhead). The identity of these Kr-positive cells is unknown. (D) twiGAL4; twiGAL4>UASNTcf embryos in which dominant-negative Tcf is expressed pan-mesodermally; this construct specifically repressed expression of cII and did not affect cI. (D') Immunocytochemistry shows that cII is missing but cI (black arrowhead) is present. (D'') Confocal micrograph of a single hemisegment shows that Slouch staining is absent from its normal position of cII, and Slouch expression does not overlap with Kr in cI (black arrowhead). (E) Quantification of Slouch cluster loss in Wg pathway partial loss-of-function embryos. Graphs show percentage of hemisegments in which cI (blue) or cII (red) are present. n, number of hemisegments counted.