Fig. 4. rdx acts in a feedback loop to reduce levels of Ci. Wing imaginal discs are oriented with anterior towards the left and genotypes in the upper right-hand corner. (A-D) rdx expression is detected by ß-gal (green) in the enhancer trap line rdx²/+ (rdxZ). In wild type (A,B), rdx is in a strip of approximately five cells along the compartment border. En expression (red in A) marks the posterior compartment, with additional expression extending approximately three cells into the anterior compartment. rdx expression overlaps the anterior edge of the En expression (inset in A). The anterior edge of Ci155 expression (red in B,E,F,L) marks the compartment border. The anterior edge of rdx coincides with that of Ci155 (inset in B), so rdx expression abuts the compartment border. The Hh-dependence of rdx expression (green) is demonstrated by its expansion when Hh is overexpressed (C); or by its loss when Ptc overexpression suppresses Hh responses (D). rdx⁶ (E) and rdx⁵ (F) loss-of-function clones are marked by loss of GFP (green). Clones within six to ten cells of the compartment border (arrowheads near white line) accumulated Ci155 (red), while clones farther from the border (arrow) did not. Filling wings with unmarked rdx⁵ clones caused a slight overgrowth of the anterior compartment (G) compared with wild type (H); the arrowhead indicates the compartment border (just anterior to vein 4) while the outline of a wild type wing is superimposed for comparison. RdxA (I,K-M) or MycRdxA (J) was mis/overexpressed using the Gal4 line MS1096. The resulting expression of Rdx is demonstrated by Myc (red in J): highest in the dorsal wing pouch (large bracket), low in part of the notum (small bracket) and off in the remainder of the disc. This reduced levels of Ci155 (red in L) and prevented Hh-dependent En induction (blue in L) in the anterior compartment (marked by the edge of Ci155 expression). It reduced levels of Ptc (green in J) near the compartment border, but broadened expression of dpplacZ (K). The resulting wings (I) had small anterior compartments and ectopic veins anterior to vein 3. Western blots (10 wing discs/lane) demonstrate a modest reduction (approximately halved) in both Ci155 and Ci75 when Rdx expression is driven by MS1096 (M). (N-P) Levels of Hh, Ptc, rdx, Ci155, CiR across the anteroposterior axis (x-axis) of wing imaginal discs, with levels indicated on the y-axis. The vertical broken lines indicate the border between the anterior (Ant) and posterior (Post) compartments. In wild type (N), rdx is expressed adjacent to the compartment border, where Hh activates Ci; there it reduces Ci155. With rdx loss (O), Ci155 is elevated adjacent to the compartment border, though this has little effect on Hh target gene expression. Ectopic Rdx (P) reduces both Ci155 and CiR, resulting in dysregulation of Hh target gene expression.