Fig. 4

Skeletogenesis during early and late regeneration stages. a) DIC (top panels) and fluorescent images (bottom panels) of calcein-labeled spicules at stages 3, 4 and 5. White arrows show individual spicules, which can be compared in both images. Insets show detail of single spicules. b) High magnification DIC images showing single spicules localized in the dermal layer. c) 50 % differentiated arm showing the formation of the skeletal elements (reflective structures). The highly calcified distal cap (terminal ossicle) forms at the distal-most end of the regenerating arm. The spicules, which will form the lateral shields and spines, appear in the first metameric unit (see inset for detail). Proximally, eight metameric units later, first vertebral spicules can be observed (see inset for detail). At the proximal end of the regenerate skeletogenesis is already very advanced and forms the individual stereomic skeletal elements including the oral, aboral and lateral shields, spines and vertebrae (see insets for detail). d) Differentiated skeletal elements in an adult non-regenerating arm. Arrows - calcein-labeled spicules, asterisk - terminal ossicle, E - epidermis, De - dermis. L - left, R - right, P - proximal, D - distal. Red dashed line - amputation plane