Immunodetection of the Optix protein in Heliconius erato forewings. (a) Western blot on protein extractions from H. erato petiverana and H. melpomene rosina forewing using the anti-Optix serum. The predicted size of the Heliconius spp. Optix protein is 30 kDa. (b) Detection of optix by mRNA in situ hybridization in a H. erato petiverana forewing. (c) Immunofluorescence using the anti-Optix serum reproduces the mRNA pattern. Blue DAPI is a nuclear counterstain. Arrowheads (b-c): forewing blade expression. (d) Organization of a ventral H. erato petiverana forewing featuring two red patterns (forewing band and forewing bar) and the posterior field of wing coupling, spear-shaped scales. (e) Sectional visualization of Optix expression in the wing coupling scale cell precursors. (f, g) Magnified view of the adult forewing bar, as a small ventral pattern situated at the base of the wing costa. (h, i) Optix detection in the corresponding region marks the precursor scale cells of the presumptive red forewing bar. Arrowheads: Optix-positive, small interstitial cells of the wing blade. (j-m) Side-specific visualization of Optix expression on two different confocal plans exposes dorso-ventral offsets in forewing bar boundaries. The dotted line highlights the relative position of the dorsal pattern boundary on ventral side focal plans. In panel l, ventral and mirror-imaged dorsal views are super-imposed using vein landmarks. Scale bars: d, 20 μm; h, j, k, 500 μm.