MOLECULAR AND CELLULAR ASPECTS OF ARM REGENERATION IN BRITTLE STARS.

Abstract Regeneration processes are very complex developmental phenomena, occurring in adult and larval organisms, which require not only new cell formation but also detailed information to specify the identity of tissues to be generated at the wound site. Regeneration after injury requires several changes in terms of activation of cellular mechanisms, activities and behavior and regenerating tissues initiate diverse processes such as wound healing, programmed cell death, dedifferentiation/transdifferentiation, stem (or progenitor) cell proliferation, differentiation. Regeneration, in which lost or damage tissue are re-grown, requires specification of the identity of new tissues to be made at specific positions. In general, it is not yet understood whether this process relies only on intrinsic regulative properties of regenerating tissues or whether positional information provides input into tissue re-patterning. However, there are still few established experimental systems that enable the study of this issue in regenerating animals, especially considering animals closely related to vertebrates, the echinoderms, which are a deuterostomian, group phylogenetically related to chordates. Larval and adult echinoderms from each of the five classes present a natural and rapid regeneration potential. Among echinoderms, ophiuroids (or brittlestars) are well known for possessing a remarkable regeneration plasticity, in many species expressed by the ability to rapidly and completely regenerate arms lost following self-induced (autotomy) or traumatic amputation. In spite of the well documented regenerative phenomena, there is still a huge lack of studies providing large-scale identification of genes involved in the molecular architecture of ophiuroid wonderful regeneration capabilities. Therefore, this thesis focuses on the study of cellular and molecular aspects of arm regeneration in ophiuroid models. As far as the cellular aspects are concerned different approaches were followed. The first one followed the overall arm regeneration process in Ophioderma longicauda model. Microscopic analysis allowed the characterization and reconstruction of the main phases in its morphogenetic and histogenetic events from self-induced amputation event up to 12 weeks regrowth. This results confirmed that arm regeneration in ophiuroids is achieved through a combination of morphallactic and epimorphic processes involved both in recycling and reorganization of old tissues and blastema formation. In addition, a rough comparison with the regeneration phenomena in other close species, Ophioderma appressa and Ophioderma cinerea, was provided in order to confirm the general aspects of the process. The second approach was focussed on another model species, Amphiura filiformis, whose arm regeneration process has been previously studied in detail. Hence, the selected experimental model was the explant, i.e. an isolated arm fragment obtained by a double amputation of the arm, proximal and distal maintained in living conditions up to 39 days. Here the morphogenetic and histogenetic events leading to the regrowth of a regenerating arm from the distal explant end were reconstructed in order to complement the previous results of the normal arm. The explant regeneration following double amputation in A. filiformis demonstrated a clear developmental polarity expressed by regeneration of the arm on the distal end and by cicatrization on the proximal end. As far as the molecular aspects, the work was addressed by trying to identify misregulated genes during early regenerative processes in arm regeneration of O. longicauda. An SSH library resulted in the identification of a fragment of the cDNA encoding the DNAJ, subfamily C member 7-like (DNAJC7) protein from regenerating arms (24 and 48 hours). This 341 bp cDNA clone matches to the 3`UTR region of a Strongylocentrotus purpuratus DNAJ ortholog (79% identity), a member of the DNAJ heat shock proteins (HSP 40) family. The optimized set up of in-situ hybridization (ISH) on the whole animal arm showed a clear expression in the radial water canal system (RWC) of both the freshly cut and the regenerating arm at 24 h and 48 h. A quantitative qRT-PCR disclosed a marked up regulation of this gene during regeneration. Based on the above-mentioned evidences we hypothesized a potential involvement of DNAJC7 like in arm regeneration in the ophiuroid O. longicauda. Keywords: Echinoderms, ophiuroids, arm regeneration, explants, molecular aspects, misregulated genes, DNAJ