A microscopic and molecular overview of collagen during echinoderm arm regeneration

Echinoderms are well known for their remarkable regenerative capabilities. The extracellular matrix (ECM) plays a major role during the regenerative process. Collagen is the main ECM component which provides a fundamental structural support; however, little is known about its involvement during regeneration in armed echinoderms. In the present study, adult specimens of the starfish Echinaster sepositus (Retzius, 1783) and of the brittle star Amphiura filiformis (O.F. Müller, 1776) were employed as experimental models to explore from a microscopic anatomy and a molecular perspective the role of collagen in arm regeneration, particularly during the first repair phase, following traumatic amputation (p.a.). Light and electron microscopy were employed to characterize ECM (mainly collagen) during the repair phase. In situ hybridisation and antibody staining for different types of collagen and for a key enzyme of its biosynthesis (prolyl-4-hydroxylase; P4H) were performed at both early and advanced regenerative stages. Our results indicate that immediately after injury the re-epithelialisation of the wound occurs within 24-48 hours p.a. in both E. sepositus and A. filiformis. An oedematous area, composed by a heterogeneous cell population embedded in a disorganized collagen/ECM matrix, is present in starfish 72 hours p.a. Collagen in this area becomes more organised in fibrils and fibres within one week p.a. Instead, in the brittle star an oedema is not present and small collagen fibrils are already present at the early stages under the new epidermis. Not all the A. filiformis selected collagen genes are expressed at early stage, being detectable in different structures at advanced stages, thus indicating diverse spatial and temporal contribution in collagen deposition and maturation throughout the regenerative process. In both starfish and brittle star P4H appears to be expressed in the epidermis at early stage, suggesting the potential role of epidermic cells in collagen biosynthesis. These results seem consistent with the preliminary collagen/P4H antibody stainings performed on both experimental models. Deeper ultrastructural and molecular studies are necessary in order to reach a complete comprehension of the role of collagen and ECM-related molecules during echinoderm arm regeneration.