Francesco Bonasoro

Cellular and molecular mechanisms of arm regeneration in crinoid echinoderms: the potential of arm explants

Abstract Crinoid echinoderms can provide a valuable experimental model for studying all aspects of regenerative processes from molecular to macroscopic level. Recently we carried out a detailed study into the overall process of arm regeneration in the crinoid Antedon mediterranea and provided an interpretation of its basic mechanisms. However, the problem of the subsequent fate of the amputated arm segment (explant) once isolated from the animal body and of its possible regenerative potential have never been investigated before. The arm explant in fact represents a simplified and controlled regenerating system which may be very useful in regeneration experiments by providing a valuable test of our hypotheses in terms of mechanisms and processes. In the present study we carried out a comprehensive analysis of double-amputated arm explants (i.e. explants reamputated at their distal end immediately after the first proximal amputation) subjected to the same experimental conditions as the regenerating donor animals. Our results showed that the explants undergo similar regenerative processes but with some significant differences to those mechanisms described for normal regenerating arms. For example, whilst the proximal-distal axis of arm growth is maintained, there are differences in terms of the recruitment of cells which contribute to the regenerating tissue. As with normal regenerating arms, the present work focuses on (1) timing and modality of regeneration in the explant; (2) proliferation, migration and contribution of undifferentiated and/or dedifferentiated/transdifferentiated cells; (3) putative role of neural growth factors. These problems were addressed by employing a combination of conventional microscopy and immunocytochemistry. Comparison between arm explants and regenerating arms of normal donor adults indicates an extraordinary potential and regenerative autonomy of crinoid tissues and the cellular plasticity of the phenomenon.

Pattern of bromodeoxyuridine incorporation in the advanced stages of arm regeneration in the feather star Antedon mediterranea

Abstract.The overall process of arm regeneration in the crinoid Antedon mediterranea is a typical epimorphic process (blastemal regeneration). This can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase. The crucial problem of the identification of cell lineages responsible for both repair and regenerative processes has been approached by monitoring cell proliferation during the advanced regenerative phase using light-microscopic and ultrastructural immunocytochemical methods to detect the incorporation of the thymidine analogue bromodeoxyuridine (BrdU) into regenerating tissues. Various treatment protocols and BrdU incubation times have been employed and provided information not only on the sources, sites of proliferation, and recruitment times of the new cells, but also on the cell lineage involved and subsequent fate (differentiation and/or migration) of the labelled cells. Our results are consistent with the following conclusions: (1) The arm regeneration process is due to a massive intervention of active proliferating cells identifiable as migratory, morphologically undifferentiated cells (amoebocytes and coelomocytes). (2) The preferential proliferation sites of these cells are the terminal blastema, the coelomic epithelium, and the brachial nerve of both the regenerating arm and the stump, even far from the amputation. (3) The two main cell components contributing to the regenerate have different origins: the blastemal cells and all the cell lineages derived from the amoebocytes; the coelomic cells from the migratory coelomocytes, in their turn derived from proliferation of the coelomic epithelium. (4) The blastemal regeneration of Antedon is due to a combined recruitment of pluripotent elements, implying the intervention of presumptive stem cells (amoebocytes) and the transdifferentiation/dedifferentiation of differentiated elements of the coelomic epithelium.

Identification of particular epithelial areas and cells that transport polypeptide-coated nanoparticles in the nasal respiratory mucosa of the rabbit

The active transcytosis of many different polypeptides (either presented free or adsorbed on latex nanoparticles), found in the respiratory mucosa of the upper nasal concha, has previously been shown to be proportional to the total volume of the lymphoid aggregates present in the tissue. By combining the use of fluorescent nanoparticles, flux measurements, confocal and scanning electron microscopy and conventional histology, it is shown in this paper that: (i) the areas of epithelium overlying lymphoid aggregates are the only transporting polypeptides; (ii) the respiratory epithelium in these areas consists mainly of non-ciliated microvillar cells, with numerous ciliated cells and rare mucous goblet cells at the periphery of the area only; (iii) non-ciliated microvillar cells are distinguishable in cells with well developed finger-like microvilli and cells with an irregularly pleated apical membrane, similar to that of intestinal and bronchial antigen-sampling M-cells; (iv) groups of polypeptide-coated nanospheres are found bound to this latter type of cells, demonstrating that these are the transporting cells, detected at the first stage of the transcytotic cycle.

The compass depressors ofParacentrotus lividus (Echinodermata, Echinoida): ultrastructural and mechanical aspects of their variable tensility and contractility

SummaryThe compass depressors are bands of soft tissue which connect the compass ossicles of the echinoid lantern to the inner edge of the test. They are essentially ligaments with on one side a thin layer of muscle cells. The ligamentous component consists mainly of a parallel array of collagen fibrils with interspersed 12 nm microfibrils. The most notable cellular constituents are granule-containing cell bodies and their processes which resemble the juxtaligamental cells that have been found in all echinoderm mutable collagenous tissues and which may control the tensility of these tissues. The muscle cells occupy about 8% of the total cross-sectional area of the compass depressor and are located in a richly innervated pseudostratified myoepithelium. When subjected to constant low loads in creep tests the compass depressor stretches to a fixed length beyond which there is no further extension. The length at this creep limit coincides with the maximum length to which the compass depressor is stretched by natural movements of the intact lantern. Stress-strain tests show that treatment with 1 mM acetylcholine or 100 mM K+ ions can increase reversibly the stiffness of the compass depressor to an extent that cannot be due to contraction of the myoepithelium, suggesting that the mechanical properties of the ligament are under physiological control. Tension-length data on the myoepithelium suggest that it generates a maximum active tension when the compass depressor is stretched to the creep limit. The implications of these results for the function of the compass depressors are discussed.

Dynamic structure of the mesohyl in the sponge Chondrosia reniformis (Porifera, Demospongiae)

Abstract The common demosponge Chondrosia reniformis possesses the capacity to undergo an unusual creep process which results in the formation of long outgrowths from the parent body. These shape changes, which have been interpreted as adaptive strategies related to environmental factors, asexual reproduction or localised locomotor phenomena, are due mainly to the structural and mechanical adaptability of the collagenous mesohyl. This contribution describes the morphological correlates of mesohyl plasticisation in C. reniformis. The microscopic anatomy of the mesohyl was examined when it was in different physiological conditions: (1) standard ”resting” condition, (2) ”stiffened” condition and (3) dynamic ”creep” condition. In this last case four representative regions of the sponge body were analysed: the parent region, the elongation region, the transition region and the propagule region. The results show that the histological modification of the sponge mesohyl during plasticisation is limited and localised. The most significant structural changes involve mainly cytological features of specific cellular components characterised by granule inclusions (i.e. the spherulous cells) and the arrangement and density of the collagenous extracellular framework, though the integrity of the collagen fibrils themselves is not affected. Morphological and functional aspects of mesohyl plasticisation invite comparison with the mutable collagenous tissue of echinoderms. Possible functional analogies between these two tissues are hypothesised.

Visceral regeneration in the crinoid Antedon mediterranea: basic mechanisms, tissues and cells involved in gut regrowth

Crinoids are able to regenerate completely many body parts, namely arms, pinnules, cirri, and also viscera, including the whole gut, lost after self-induced or traumatic mutilations. In contrast to the regenerative processes related to external appendages, those related to internal organs have been poorly investigated. In order to provide a comprehensive view of these processes, and of their main events, timing and mechanisms, the present work is exploring visceral regeneration in the feather star Antedon meditteranea. The histological and cellular aspects of visceral regeneration were monitored at predetermined times (from 24 hours to 3 weeks post evisceration) using microscopy and immunocytochemistry. The overall regeneration process can be divided into three main phases, leading in 3 weeks to the reconstruction of a complete functional gut. After a brief wound healing phase, new tissues and organs develop as a result of extensive cell migration and transdifferentiation. The cells involved in these processes are mainly coelothelial cells, which after trans-differentiating into progenitor cells form clusters of enterocytic precursors. The advanced phase is then characterized by the growth and differentiation of the gut rudiment. In general, our results confirm the striking potential for repair (wound healing) and regeneration displayed by crinoids at the organ, tissue and cellular levels.

TISSUE DISTRIBUTION OF MONOAMINE NEUROTRANSMITTERS IN NORMAL AND REGENERATING ARMS OF THE FEATHER STAR ANTEDON MEDITERRANEA

Abstract.Crinoid echinoderms can completely and rapidly regenerate arms lost following self-induced or traumatic amputation. Arm regeneration in these animals therefore provides a valuable experimental model for studying all aspects of regenerative processes, particularly with respect to the nervous system and its specific contribution to regenerative phenomena. Taking into account the primary role of the nervous system in regeneration in other invertebrates, we have investigated the specific involvement of neural factors, viz. the monoamine neurotransmitters dopamine and serotonin, in arm regeneration of Antedon mediterranea. In the present work, the presence of classical monoamines has been revealed by employing specific immunocytochemical and histofluorescence tests in association with biochemical detection by means of high pressure liquid chromatography. The distribution pattern of these neurohumoral molecules at standard regenerative stages has been compared with that of normal non-regenerating arms. Results indicate that both dopamine and serotonin dramatically change in both their distribution and concentration during the repair and regenerative processes. Their remarkably enhanced pattern during regeneration and widespread presence at the level of both nervous and non-nervous tissues indicates that they are important neural growth-promoting factors in crinoid arm regeneration.

The mechanically adaptive connective tissue of echinoderms: Its potential for bio-innovation in applied technology and ecology

Echinoderms possess unique connective tissues, called mutable collagenous tissues (MCTs), which undergo nervously mediated, drastic and reversible or irreversible changes in their mechanical properties. Connective tissue mutability influences all aspects of echinoderm biology and is a key-factor in the ecological success of the phylum. Due to their sensitivity to endogenous or exogenous agents, MCTs may be targets for a number of common pollutants, with potentially drastic effects on vital functions. Besides its ecological relevance, MCT represents a topic with relevance to several applied fields. A promising research route looks at MCTs as a source of inspiration for the development of novel biomaterials. This contribution presents a review of MCT biology, which incorporates recent ultrastructural, biomolecular and biochemical analyses carried out in a biotechnological context.

Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence.

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor–β (TGF–β) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF–β–like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF–β–like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non–neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF–β–like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.

Production, Characterization and Biocompatibility of Marine Collagen Matrices from an Alternative and Sustainable Source: The Sea Urchin Paracentrotus lividus

Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30–400 nm and mesh < 2 μm) and display remarkable mechanical properties in term of stiffness (146 ± 48 MPa) and viscosity (60.98 ± 52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.