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Butterfly wing eyespot patterns are determined in pupal tissues by organisers located at the centre of the prospective eyespots. Nevertheless, organiser cells have not been examined cytochemically  in vivo , partly due to technical difficulties. Here, we directly observed organiser cells in pupal forewing epithelium via an  in vivo  confocal fluorescent imaging technique, using 1-h post-pupation pupae of the blue pansy butterfly,  Junonia orithya . The prospective eyespot centre was indented from the plane of the ventral tissue surface. Three-dimensional reconstruction images revealed that the apical portion of “focal cells” at the bottom of the eyespot indentation contained many mitochondria. The mitochondrial portion was connected with a “cell body” containing a nucleus. Most focal cells had globular nuclei and were vertically elongated, but cells in the wing basal region had flattened nuclei and were tilted toward the distal direction. Epithelial cells in any wing region had cytoneme-like horizontal processes. From 1 h to 10 h post-pupation, nuclear volume increased, suggesting DNA synthesis during this period. Morphological differences among cells in different regions may suggest that organiser cells are developmentally ahead of cells in other regions and that position-dependent heterochronic development is a general mechanism for constructing colour patterns in butterfly wings.

Butterfly eyespot organiser: in vivo imaging of the prospective focal cells in pupal wing tissues