Expression pattern of extracellular matrix genes during arm regeneration in the brittle star Amphiura filiformis (O.F. Müller, 1776)

Arm regeneration in Ophiuroids (Echinodermata) is a topic of interest from morphological, cellular, molecular and evolutionary perspective. During this process, different tissues are involved, among which connective tissue, whose main extracellular matrix (ECM) component is collagen. Despite its importance during both repair and regenerative phases, little is still known about ECM role in this process. In the present study, we employed the burrowing brittle star Amphiura filiformis in order to explore gene expression patterns of collagen and other ECM-related molecules during arm regeneration after traumatic amputation. Different types of collagen, matrix metalloproteinases (MMPs), tissue inhibitor of MMPs (TIMPs), proteoglycans (PGs), proteins (e.g. ficolin) and glycoproteins (e.g. laminin) were selected starting from EchinoBase database with BLASTX search in the A. filiformis transcriptome available in Oliveri\u2019s lab. From the 39 selected genes, 11 were successfully cloned and tested, along with positive controls, by colorimetric whole mount in situ hybridisation at early (stage 2), intermediate (stage 4) and advanced regenerative stages. Our results indicate that selected collagen genes are expressed in different structures at intermediate and advanced stages, whereas they are not expressed at early stage and in the distal undifferentiated arm-tip at advanced stages; some are not expressed in any considered regenerative stage, thus indicating their diverse spatial and temporal contribution in ECM deposition and maturation throughout the regenerative process. Ficolin is expressed only at advanced stages at the level of the vertebrae, whereas laminin is expressed at the tip of the spines, in both epidermis and dermis and in the radial nerve cord at different regenerative stages, therefore suggesting their importance in skeleton and nervous system regeneration. No expression pattern is detectable at the examined stages for the selected TIMP. Further molecular analyses on ECM-related molecule and collagen genes, together with ultrastructural and immunohistochemical investigations, are necessary in order to better understand the role of the connective tissue during Amphiura filiformis arm regeneration