Localization of ecdysone receptor (EcR) protein in larval wings. (A) Ventral surface of forewing (distal section shown) of non-injected individual reared at 19°C with different arrow heads pointing at anterior (trait 3) versus posterior (trait 4) eyespots. Corresponding region of anterior (B) and posterior (C) eyespot fields of developing pupal forewing at 19°C around 6% and 23% of pupal time, respectively. Panel (D) is a detail of the presumptive eyespot center in panel (C). (E) Ventral surface of forewing (distal section) of non-injected individual reared at 27°C with arrow heads pointing at anterior and posterior eyespots. Corresponding region of anterior (F) and posterior (G) eyespot fields of developing pupal forewing at 27°C around 6% and 23% of pupal time, respectively. Panel (H) corresponds to the DAPI (nuclear) stain in panel (G) showing higher density and lack of row-like organization of the cells at the center of the presumptive eyespot. Panel (I) corresponds to EcR expression in larval hindwing and (J) is a detail of (I). (K) Detail of overlap in EcR protein and DAPI from developing forewing at 27°C (around 6% of pupal duration), showing nuclear localization of EcR. (L) Presumptive eyespot center (around 23% of pupal duration at 27°C) expressing EcR’s target gene Broad (core isoform) shows that EcR is active. Yellow arrows indicate veins for reference. All in all, we see EcR-positive cells over the entire wing from larval to late pupal stages, and in higher cell density in the presumptive eyespot centers. These centers are larger for larger eyespots. Scale bar = 100 μm. DAPI, 4′,6-diamidino-2-phenylindole.