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

Abstract

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.

Self-Similarity, Distortion Waves, and the Essence of Morphogenesis: A Generalized View of Color Pattern Formation in Butterfly Wings

“…These spots and marks are likely produced by organizing cells for adult eyespots. The epithelial distortion structures of the prospective elements have also been confirmed by in vivo imaging of the living tissue (Ohno and Otaki 2015a;Iwasaki et al 2017). The association of the organizing centers with distortion structures may be called the distortion rule for organizing centers.…”

Contact-Mediated Eyespot Color-Pattern Changes in the Peacock Pansy Butterfly: Contributions of Mechanical Force and Extracellular Matrix to Morphogenic Signal Propagation

“…Butterfly wings exhibit extreme diversity of color patterns based on developmental and evolutionary modifications of the nymphalid groundplan [6][7][8][9][10][11]. The butterfly wing system is largely a two-dimensional entity as depicted in the nymphalid groundplan, but strictly speaking, it is three-dimensional; organizers for color patterns are located at the bottom (or top) of an indentation (or a bump) of the wing epithelium in the pupal stage, and this epithelial structure is reflected as pupal cuticle spots [12][13][14]. Furthermore, this three-dimensionality is reflected in adult wings [13].…”

Synergistic Damage Response of the Double-Focus Eyespot in the Hindwing of the Peacock Pansy Butterfly

“…It is speculated that this slow-moving signal is waves of mechanical distortion [25]. Importantly, organizing centers are present as physical bumps or indentations [32,37]. These organizing centers can be identified as the pupal cuticle spots in pupae [38,39].…”

“…Although there are some classical histological studies on developing wing tissues of butterflies and moths [34,43,44], real-time live imaging studies have just begun on developing wing tissues and organizing cells [30,37,45]. The distortion hypothesis should be tested in the future in light of the importance of mechanical forces in development [46,47].…”