Introduction

The extracellular matrix (ECM) plays a crucial role in cell differentiation by providing ligands that interact with receptors on the cell surface. The cuticle functions as a specialized form of ECM. In butterflies, wing patterns are formed by the precise arrays of scales. During the early pupal stage, the cuticle layer forms in close apposition to the epithelial cells that will later differentiate into scale cells. In the blue pansy butterfly (Junonia orithya), the prospective eyespot pattern is pre-determined early in the pupal stage. The center of the future eyespot is recognized as a developmental organizer. Our laboratory discovered that the physical contact of the pupal wing epithelium with the cuticle plays an essential role in eyespot formation. Based on these observations, we hypothesized that the physical interactions between the epithelial cells and the cuticle contributes to the spatial regulation of eyespot pattern development.

Methods

We utilized three types of microscopes: a standard light microscope, a transmission electron microscope (TEM), and a confocal laser scanning microscope (CLSM). We mainly examined the width of the intervening spaces between the epithelial cells and the cuticle as an indicator of the physical interactions between them.

Results

We found that the width of the intervening space, cuticle thickness, and cuticle surface structures differed between the pupae of 6 and 12 h post-pupation in the forewings, based on the light microscopy and TEM observations. Furthermore, using a CLSM, we found that the center of the prospective hindwing eyespot is different from its surrounding regions in the width of intervening space in the pupae of 11–13 h post-pupation.

Conclusion

The width of the intervening space between the epithelial cells and the cuticle on the prospective eyespot center may be an indication of the behavior of the eyespot organizer for the determination of eyespot patterns.