Fig. 5. Wingless sets up a region competent to express Slouch and is required later to specify the fate of cluster II. (A) Late stage 11 wg^CX4 mutant embryos fail to maintain Twist at high levels. (B) Ectopic Twist expression in wg^CX4 mutant embryos maintains Twist expression through stage 11. (C) Late stage 11 wg^CX4 mutant embryo showed loss of all mesodermal Slouch, although some expression remained in the CNS. cI (black arrowhead) and cII (white arrowhead) are indicated in both the panel and the inset. Black arrows denote the midline in the panel; black arrows in C',D' and white arrows in C'',D'' show Slouch-expressing CNS cells in the insets. (C) Immunocytochemical staining of a single hemisegment shows that both cII and cI are missing. (C'') Confocal micrographs of embryos stained with antibodies to Slouch (green/FITC) and Kr (red/Cy3). No co-localization of Kr and Slouch is detected in the mesoderm. White arrow indicates Slouch CNS expression. (D) wg^CX4,twiGAL4>UAStwi embryos showed rescue of mesodermal Slouch expression in positions corresponding to cI. (D') Immunocytochemical staining shows that cII is absent but cI is present (black arrowhead). (D'') Confocal micrograph of a single hemisegment shows that Slouch staining is absent from the normal position of cII, and Slouch (green/FITC) does not co-localize with Kr (red/Cy3), supporting the identity of this cluster as cI (black arrowhead). We note that the amount of Twist maintained in these cells after specification is detrimental. While Twist is necessary for the specification of the Slouch clusters, maintained elevated expression can lead to repression of these clusters (V.T.C. and M.K.B., unpublished). (B) Quantification of Slouch cluster rescue in wg mutant embryos that overexpress twist. Graphs show percentage of hemisegments in which cI (blue) or cII (red) are present under conditions listed at the bottom of the graph. n, number of hemisegments counted.