Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.