Extracellular matrix gene expression patterns in Amphiura filiformis arm regeneration

The brittle star Amphiura filiformis (Afi) is becoming a well-established experimental model to study echinoderm arm regeneration from both cellular [1, 2] and molecular point of view [2, 3]. Many tissues are involved during this process, among which connective tissue, whose main extracellular matrix (ECM) component is collagen; however, little is still known about ECM role in the regeneration process of this species. In the present study, we employed whole mount in situ hybridisation (WMISH) in order to explore gene expression patterns of collagen and other ECM-related molecules in Afi arm re-growth after traumatic amputation. Adult specimens were collected at the Sven Lovén Centre for Marine Sciences (Kristineberg, Sweden). 39 genes, among which 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 Afi transcriptome available in Oliveri’s lab. 11 genes were successfully cloned and tested, along with positive controls, by colorimetric WMISH at early (stage 2) [2], intermediate (stage 4) [2] and late (50% and 95%) regenerative stages [2, 3]. Our results indicate that selected collagen genes are expressed in different structures at intermediate and late stages, whereas they are not expressed at early stage and in the distal undifferentiated arm-tip at late stages; some are not expressed in any considered regenerative stage, thus suggesting their possible diverse spatial and temporal contribution in ECM deposition and maturation throughout the regenerative process. Laminin is expressed at the tip of the spines, in both epidermis and dermis and in the radial nerve cord at different regenerative stages, whereas ficolin is expressed at the level of the vertebrae only at late stages, therefore suggesting their importance in nervous system and skeletal element regeneration. No expression pattern is detectable for the selected TIMP at the examined stages. Further analyses on ECM-related molecule and collagen genes are necessary in order to deeply understand the role of the connective tissue during Amphiura filiformis arm regeneration. Acknowledgements: the authors want to thank the Sven Lovén Centre for Marine Sciences in Kristineberg (Sweden), especially Olga Ortega-Martinez and Sam Dupont, for help during animal and sample collection. References: [1] Biressi, ACM, Zou, T, Dupont, S, Dahlberg, C, Di Benedetto, C, Bonasoro, F, Thorndyke, M, Carnevali, MDC. (2010) Wound healing and arm regeneration in Ophioderma longicaudum and Amphiura filiformis (Ophiuroidea, Echinodermata): comparative morphogenesis and histogenesis. Zoomorphology, 129, 1-19. [2] Czarkwiani, A, Ferrario, C, Dylus, DV, Sugni, M, Oliveri, P. (2016) Skeletal regeneration in the brittle star Amphiura filiformis. Frontiers in Zoology, 13, 18. [3] Dupont, S, Thorndyke, MC. (2006) Growth or differentiation? Adaptive regeneration in the brittle star Amphiura filiformis. Journal of Experimental Biology, 209, 3873-81.