Search

In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. However, its overall functions have been scarcely studied in detail. In this work, we used an integrated approach based on both microscopy (light and electron) and proteomic analyses to investigate the arm CE in the starfish Marthasterias glacialis during different physiological conditions (i.e., non-regenerating and/or regenerating). Our results show that CE cells share both ultrastructural and proteomic features with circulating coelomocytes (echinoderm immune cells). Additionally, microscopy and proteomic analyses indicate that CE cells are actively involved in protein synthesis and processing, and membrane trafficking processes such as phagocytosis (particularly of myocytes) and massive secretion phenomena. The latter might provide molecules (e.g., immune factors) and fluids for proper arm growth/regrowth. No stem cell marker was identified and no pre-existing stem cell was observed within the CE. Rather, during regeneration, CE cells undergo dedifferentiation and epithelial-mesenchymal transition to deliver progenitor cells for tissue replacement. Overall, our work underlines that echinoderm CE is not a “simple epithelial lining” and that instead it plays multiple functions which span from immunity-related roles as well as being a source of regeneration-competent cells for arm growth/regrowth.

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.

In situ hybridisation and immunohistochemistry analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFalpha), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development. Granulocytes with large granules and compartment/morula cells are CiTNFalpha-producing cells in both inflamed pharynx and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the involvement of CiTNFalpha in recruiting lymphocyte-like cells and promoting the differentiation of inflammatory haemocytes. Specific antibodies against a CiTNFalpha peptide have identified a 43-kDa cell-bound form of the protein. Observations of pharynx histological sections (at 4 and 8 h post-LPS inoculation) from naive and medium-inoculated ascidians have confirmed the CiTNFalpha-positive tissue response. Larval histological sections and whole-mount preparations have revealed that CiTNFalpha is expressed by trunk mesenchyme, preoral lobe and tunic cells, indicating CiTNFalpha-expressing cell immigration events and an ontogenetic role.

Glucocorticoid hormone receptors (GR), members of the nuclear hormone receptor superfamily, are ligand-dependent transcription factors expressed in various tissues by binding to specific DNA sequences. Since glucocorticoids have a role in maintaining the homeostatic status in fish, we previously cloned and sequenced a GR (DlGR1) of adult Dicentrarchus labrax; we also showed mRNA expression (in situ hybridization) and tissue immunohistochemical localization of DlGR1 in several organs. This work has now been extended to the examination of the expression, tissue distribution, and cytolocalization of DlGR1 in larval developmental stages by similar methods to those used for the adult organs. The riboprobe included the DlGR1 cDNA transcriptional activation domain (1.0–1,300 nucleotide sequence) showing no significant similarity with a known second GR cDNA sequence of sea bass. The antibody was specific for an opportunely selected peptide sequence of the DlGR1 transcriptional domain. In histological sections of brain, head kidney, gills, liver, anterior intestine, and spleen cells, the riboprobe was mainly located in the cell nucleus. The antibody identified DlGR1 in the head kidney, gills, liver, and anterior intestine, mainly located in the cytosol. These results are in agreement with the receptor location in adult tissues. The greater presence of both the transcript and protein of DlGR1 in the late developmental stages suggests an increasing expression of this receptor. The cytolocalization (nuclear-cytosolic) and presumptive roles of DlGR1-containing tissues are discussed.