Interestingly, only through the introduction of IFC we were able to detect a third potentially new spherical-cell morphotype in P2 that exhibited heterogeneous cytoplasm and nuclei (Figure 4C4). we could not Fondaparinux Sodium rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other Fondaparinux Sodium morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations. (Linnaeus, 1758), commonly known as the spiny starfish, is a member of the class Asteroidea (phylum Echinodermata). Members of this class (i.e., Asteroids) are known for their remarkable regenerative abilities (1, 2), which allow the animals to restore and regrow lost body parts after injury, providing an ecological advantage in environments shared with predators. The regenerative process involves the mobilization of many cell types to the area of injury to perform two major processes: 1) monitor and clearing environmental pathogens that have gained sudden access to internal fluids, and 2) healing the wound and subsequently rebuilding the missing structures. It has been known for some time that coelomocytes, the circulating cells present in the coelomic cavities, play a critical role in all these processes (3). Coelomocytes are mostly known as effectors of the immune response and are thus key contributors to the monitoring of pathogens and restoring of missing tissues (4). How these processes occur remains a matter of debate, due in great part to the still limited knowledge of coelomic fluid composition and of the mechanistic/molecular basis of coelomocytes functions. In echinoderms, the coelomic fluid completely fills the Fondaparinux Sodium coelomic spaces of the body, including the Fondaparinux Sodium perivisceral coelomic cavities, the water vascular system and the perihemal systems (5C7). Coelomocytes perform diverse immune functions, such as cellular clots formation, phagocytosis, encapsulation, and clearance of bacteria and other foreign materials (7, 8). After starfish experience traumatic injury or autotomy, coelomocytes rapidly aggregate in the damaged area, forming a clot that seals the inner environment from the external RGS20 milieu, thus preventing the loss of coelomic fluid and ensuring hemostasis (8, 9). Recently, the pharynx and axial organs were proposed as the echinoid analogues of hematopoietic organs (10). Despite such hypotheses, the specific tissue where starfish coelomocytes originate remains unknown. Researchers have proposed that these cells originate in the axial organ (11), the Tiedemanns bodies (12) or the perivisceral coelomic epithelium (CE) (13, 14). This final view is currently considered the most plausible, supported by several experimental results that show release of cells from the CE in response to injury or to exposure to foreign particles (13, 15, 16). While the main activities of the coelomocytes are known (most relate to immune response as stated above), the number, types and specific physiological contribution of each morphotype are still poorly understood. Indeed, due to the diversity in morphological descriptions offered in the literature, use of different names for similar cellular phenotypes, changes in experimental outcomes arising from alternative coelomocytes handling protocols and the diversity of characterization methods, these circulating cells are subjected to various classification schemes and therefore cannot yet be systematically identified. The main result of this is an intrinsic difficulty in homologizing cell types within and across different echinoderm classes. For example, Smith et al. (7) recognized only three categories of coelomocytes in sea urchins (phagocytes, spherule cells or amoebocytes, and vibratile cells). In the mean time, using May-Grunwald/Giemsa staining, Taguchi et al. (17) tentatively classified coelomocytes of the sea cucumber into up to twelve types, including several granulocytes (17). In the starfish Asteroidea) coelomocytes has not yet been reached, likely Fondaparinux Sodium also due to a combination of?the?intrinsic heterogeneity of morphotypes, poor taxonomic?sampling and, while already mentioned variability in experimental protocols. In order to contribute to a greater understanding of the cellular components present in the coelomic fluid of an asteroid, we carried out a comprehensive analysis and the characterization of coelomocytes of the starfish were collected at low tide within the west coast of Portugal.

Interestingly, only through the introduction of IFC we were able to detect a third potentially new spherical-cell morphotype in P2 that exhibited heterogeneous cytoplasm and nuclei (Figure 4C4)