The coelomic epithelium and coelomocytes of the starfish Marthasterias glacialis (Linnaeus, 1758) in non-regenerating ARM-TIP: microscopic anatomy and proteomics characterization

In echinoderms the coelomic epithelium (CE) is hypothesized to be the source of circulating coelomocytes, echinoderms' immune cells, and to play a key role during arm regeneration as source of stem cells. In this context, we decided to characterize the starfish CE and free wandering coelomocytes (CO) in non-regenerating conditions by means of a multidisciplinary approach combining microscopy and proteomics analyses. This was done in an attempt to address the above mentioned issues and provide a fundamental basis for further regeneration studies. For this purpose, we used Marthasterias glacialis as model system. For microscopy analyses, CE and circulating CO were collected and processed for standard protocols of light and transmission electron microscopy (TEM). For proteomics analysis, the CE was removed from amputated arms and processed for the identification of soluble proteins by Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) analysis. Microscopy results confirmed that M. glacialis CE presents the same complex multi-layered structure described for other asteroids. However, we observed the presence of a never described layer of flagellated cells, filled by swollen RER cisternae. TEM images indicated that the peritoneocytes are actively involved in apocrine-like secretion, especially in the distal most arm-tip. Among the CO, we identified two main subpopulations: a thrombocyte-like cytotype, characterized by numerous electron-lucent vesicles and several long filopodia, and a macrophage-like cytotype (immunocytes), characterized by a less electron-dense cytoplasm, phagosomes and short cytoplasmic processes. No presumptive stem cells were found among the circulating CO. Proteomics results indicated that the CE contains proteins involved in the rearrangement of the cytoskeleton, related to phagocytosis and endocytosis, proteins involved in the immunity response, and in apocrine secretion. Overall, our findings suggested that M. glacialis CE and CO are involved in several physiological functions: active secretion of protein material, some of this possibly needed for the distal arm growth; immune-related functions, such as pathogen or endogenous cell removal; haemostatic function. Shared ultrastructural features between CE and CO suggested that at least one of the CO subpopulations might derive from the CE. Further integrated studies on normal and regenerating arms are necessary to deeply understand the role of CE and CO in starfish physiology.