Abstract
Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via 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.
Highlights
Marthasterias glacialis (Linnaeus, 1758), commonly known as the spiny starfish, is a member of the class Asteroidea
The flow cytometry analysis of the cells isolated from M. glacialis fluid revealed two clearly distinguishable cell populations, termed P1 and P2 in this study (Figure 1A)
A histogram plot of DRAQ5 staining for both populations showed that the median fluorescence intensity (MFI) of DRAQ5 measured by imaging flow cytometry (IFC) for P2 was approximately twice the MFI value measured for P1 (Figure 1F), consistent with the results obtained via conventional flow cytometry (Figure 1C), confirming that P1 and P2 represented the same respective cell populations when analyzed using the two FC techniques
Summary
Marthasterias glacialis (Linnaeus, 1758), commonly known as the spiny starfish, is a member of the class Asteroidea (phylum Echinodermata). 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]. 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
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