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Expression of Tgf-beta in the regenerative process of crinoid echinoderms
No full textFor many years, we focused our studies on the remarkable power of regeneration exhibited by crinoid echinoderms. Current investigations are exploring the aspects related to the 'putative' growth factors crucial for the regenerative process in crinoids. Due to its importance in wound healing phenomena of vertebrates and invertebrates, both in the embryo and in adults, the present study concentrates on the possible presence/expression and roles of TGF-β in arm regeneration processe of the crinoid Antedon mediterranea. This problem has been explored by employing 1) a biochemical and immunocytochemical approach, which allowed us to detect the presence of TGF-β1 and its putative changes of pattern distribution during the regeneration process; 2) a molecular approach, thanks to which we cloned a BMP2/4 homologue from crinoids (AnBMP2/4) confirming the possible presence of different genes of the TGF-β superfamily. Expression studies indicate an important role for AnBMP2/4 during the more advanced stages of regeneration at a time when new tissues are being established and the brachial nerve is extending into the regenerate. This expression in an ‘adult’ regenerating system shows remarkable parallels with recent investigations in sea urchin embryos where a BMP2/4 homologue is involved in the regulation of the ectoderm/endoderm boundary and epidermal/non epidermal fate decisions.
Our results suggest in particular that AnBMP2/4 plays an important role in crinoid skeletogenesis as well as in neurogenesis and support the idea of an evolutionary developmental programme where essential gene families are conserved throughout phylogeny both in terms of expression and function. The future employment of techniques such as RNA interference or bead implantation, in adult echinoderms, will be of fundamental importance to understand the function of AnBMP2/4 and other related genes
PCB-induced environmental stress and the regenerative response in crinoids.
No full textSome persistent and ubiquitous pollutants (PCBs) which can affect the natural environment because of their bio-accumulation in organisms exert their effects by acting as “endocrine disrupters”. In this respect they interact with members of the nuclear receptor family and can induce dramatic effects on gene expression, reproductive competence and development. This paper focuses on the impact of such compounds on natural arm regeneration in crinoids. Since crinoids are benthic and microfilter-feeding animals they are particularly susceptible to the presence of micropollutants in marine sediments. We used the regenerative response of crinoids to monitor and evaluate chronic toxicity by means of tests performed in the laboratory under controlled conditions of known environmental parameters and contamination levels. Exposure to Aroclor 1260, a commercial cocktail of chlorinated congeners, have been performed in static conditions. The concentration of the pollutant has been analysed in both the water and in the whole animal during and at the end of tests. Since the phenomena of tissue renewal involves substantial cell cycle activity and proliferation, the crinoids regeneration bio-test represents an ideal bioindicator of stress at the cellular and molecular level due to persistent pollutants, particularly endocrine disrupters. On the basis of the present results it is quite evident that regenerative response is especially sensitive and that exposure to these types of toxic chemical can induce significant variations in times and modalities of regenerative developmental compared to normal regeneration
Mechanical adaptability of a sponge extracellular matrix: evidence for cellular control of mesohyl stiffness in Chondrosia reniformis
No full textOccurrence and expression of a novel transforming growth factor beta homologue in crinoids
No full textInvertebrates have frequently been used to help understand the complexities of regulatory gene function and evolution. The bone morphogenetic proteins (BMPs) are a highly conserved group of secreted regulatory factors that play an important part in early embryonic patterning. In the present study we have used the remarkable regenerative potential of crinoid echinoderms to explore the BMPs' site of expression in an adult developmental programme. Our results suggest that a crinoid BMP2/4 homologue is actively involved during the early stages of blastemal regeneration at a time when fundamental patterns are being established. This supports the idea of an evolutionary developmental programme where essential gene families are conserved throughout phylogeny in terms of both expression and function
Mechanical adaptability of a sponge extracellular matrix: evidence for cellular control of mesohyl stiffness in Chondrosia reniformis
No full textAnbmp2/4 is a new member of the transforming growth factor-beta superfamily isolated from a crinoid and involved in regeneration
No full textInvertebrates have frequently been used to help understand the complexities of regulatory gene function and evolution. The bone morphogenetic proteins (BMPs) are a highly conserved group of secreted regulatory factors that play an important part in early embryonic patterning. In the present study we have used the remarkable regenerative potential of crinoid echinoderms to explore the BMPs' site of expression in an adult developmental programme. Our results suggest that a crinoid BMP2/4 homologue is actively involved during the early stages of blastemal regeneration at a time when fundamental patterns are being established. This supports the idea of an evolutionary developmental programme where essential gene families are conserved throughout phylogeny in terms of both expression and function
GROWTH FACTORS, HEAT-SHOCK PROTEINS AND REGENERATION IN ECHINODERMS
No full textThe study of regeneration in armed echinoderm species, including crinoids, ophiuroids and asteroids, is attracting increasing attention. Recent interest has focused on the presence and potential role of growth factors, including members of the nerve growth factor (NGF) and transforming growth factor-beta (TGF-beta) families, in the regenerative process and their possible relationship to the normal developmental (ontogenetic) regulatory cascade. In addition, the expression patterns of the heat-shock family of stress proteins (Hsps) during regeneration are also important. Their role forms part of a normal stress response to the trauma of autotomy in combination with a putative function in tissue remodelling and associated protein turnover during regeneration. The temporal dynamics of the stress response may also be strongly indicative of environmentally adaptive pressures operating on these systems
Stress and neuronal regeneration in echinoderms.
No full textWe are presently exploring the links between neuronal regeneration, development and the stress response in echinoderms. During echinoderm development there is known to be an increase in HMW ubiquitin-protein conjugates together with a parallel increase in ubiquinated H2A and H2B histones. This could reflect the high levels of protein turnover experienced during ontogeny. In contrast, the stress response (or "heat-shock" response) induced either by temperature or chemical stress produces an increase in ubiquinated HMW proteins, induction of heat-shock proteins (hsp72 and hsp90) and a decrease in ubiquinated histones. We have analysed these parameters in crinoids and asteroids during various phases of arm regeneration. Normal arms of Antedon mediterranea, Antedon bifida and Asterias rubens were compared with regenerating arms at different stages in both standard and conditions of environmental stress. In normal arms we detect significant quantities of ubiquinated HMW proteins and histones, perhaps indicating rapid cellular growth and protein turnover. By western blot analysis and immunocytochemical labelling of neurones, using monoclonal antibodies against ubiquitin and the inducible form of hsp70, we found significant ubiquitin changes at early stages of regeneration. In particular a decrease in ubiquitinated histones for both crinoids and asteroids with different timing of deubiquitination was observed. No other significant changes were noticed during the rest of the regenerative period. The expression of hsp70, instead, was induced also in advanced phases of regeneration confirming the chaperon role of this molecule. In conclusion, it is clear that ubiquitin dynamics are important in regeneration and furthermore hsp 70 is an important component of regeneration rather than a response to stress alone. (Supported by a Thomas Holloway studentship, RHUL strategy fund, University of Milan, The British Council and the University of London Central Research Fund)
Stress and regeneration in crinoids and asteroids
No full textComplete and functional regrowth of arms in echinoderms depends on several factors. Probably the most crucial are the site of amputation along the proximal-distal axis of the arm, particularly important in crinoids (Candia Carnevali et al 1995), and the ambient temperature, as seen in the ophiuroid A. filiformis (Mallefet et al 2000). Indeed, a traumatic amputation that does not follow the natural autotomy plane may involve more complex reparative/regenerative mechanisms and therefore may be slower compared to the non traumatic (Candia Carnevali et al 1995). Whatever the selected regenerative ‘pathway’, massive tissue rearrangement and strong up-regulation of cell proliferation/differentiation was detected in all the species investigated in this thesis. However, the time course of these events does vary dependent upon the regenerative ‘pathways’.
During the arm regeneration process, these animals might experience stress which may be accompanied by a large turnover of protein. Generally, when an organism is subject to metabolic and environmental stressors, a common protective mechanism, known as the stress response, is activated (Srinivas and Swamynathan 1996; Morimoto 1998). This results in the expression of heat-shock proteins (Hsps). It is known that Hsps, which are encoded by highly conserved families of genes, play key roles not only in the correct folding of proteins (and hence repair processes following damage) but also during normal development (Becker and Craig 1994). One specific example occurs in Drosophila where small increases in Hsp70 expression during development enhances thermotolerance (Feder 1999) but if overexpression of the Hsp70 gene is induced, larval mortality increases and development slows down (Krebs and Feder 1997).
Amongst invertebrates, echinoderms are well known for their extensive capacity for regeneration following natural predation-induced trauma or as a part of reproductive strategy (Candia Carnevali et al 1998). Echinoderm classes with arms are often subject to frequent arm loss. Clearly such autotomy followed by subsequent repair and regeneration is likely to represent a stressful event.
Ubiquitin is a small, (76 amino acids) highly conserved phylogenetically, protein and is present in all eukaryotes (Finley and Chau, 1991; Hochstrasser, 1996). Although ubiquitin occurs free in the cell, it is most commonly found covalently conjugated to a wide range of target proteins. This conjugation is a reversible post-translational modification, which has been implicated, in numerous biological processes. Ubiquitin plays important roles in a range of cellular functions including the cell cycle, DNA replication, DNA repair and signal transduction (Deshaise 1995; Muller and Schwartz 1995; King et al 1996). One important and well-known function of ubiquitination is to target proteins for rapid degradation by the 26S proteasome, a protease complex present in both the cytoplasm and the nucleus (Arrigo et al 1988). In this ATP-dependent pathway, a protein is tagged with poly-ubiquitin chains via isopeptide bonds, which are formed between the carboxyl terminal glycine of ubiquitin molecules and the ε-amino groups of lysine residues in other ubiquitin molecules. This ubiquitinating reaction (Fig. 1) is catalysed by sequential actions of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and often E3 (ubiquitin-ligase). When cells are exposed to heat shock, many aberrant proteins are produced and the ubiquitin-dependent proteolytic pathway (the ubiquitin-proteasome system) is believed to play a key role in rapid degradation of these abnormal proteins (Schwartz and Ciechanover 1999).
In contrast, some proteins, such as the histones H2A and H2B, are ubiquitinated but not subsequently degraded. It is still unclear whether these ubiquitin molecules are attached to histones via the poly-ubiquitin chain or not. Thus, it is still uncertain why and how ubiquitinated histones are deubiquitinated in response to heat-shock, during chromosome condensation in the mitotic cell cycle, in apoptosis and during neuronal differentiation in PC12h cells (Bond et al 1988; Takada et al 1994; Marushige and Marushige 1995)
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