BackgroundIn various ascidian species, circulating stem cells have been documented to be involved in asexual reproduction and whole-body regeneration. Studies of these cell population(s) are mainly restricted to colonial species. Here, we investigate the occurrence of circulating stem cells in the solitary Styela plicata, a member of the Styelidae, a family with at least two independent origins of coloniality.ResultsUsing flow cytometry, we characterized a population of circulating putative stem cells (CPSCs) in S. plicata and determined two gates likely enriched with CPSCs based on morphology and aldehyde dehydrogenase (ALDH) activity. We found an ALDH + cell population with low granularity, suggesting a stem-like state. In an attempt to uncover putative CPSCs niches in S. plicata, we performed a histological survey for hemoblast-like cells, followed by immunohistochemistry with stem cell and proliferation markers. The intestinal submucosa (IS) showed high cellular proliferation levels and high frequency of undifferentiated cells and histological and ultrastructural analyses revealed the presence of hemoblast aggregations in the IS suggesting a possible niche. Finally, we document the first ontogenetic appearance of distinct metamorphic circulatory mesenchyme cells, which precedes the emergence of juvenile hemocytes.ConclusionsWe find CPSCs in the hemolymph of the solitary ascidian Styela plicata, presumably involved in the regenerative capacity of this species. The presence of proliferating and undifferentiated mesenchymal cells suggests IS as a possible niche.

Ascidians are interesting neurobiological models because of their evolutionary position as a sister-group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3-acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non-neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal βIII-tubulin were observed. In contrast, the expression of caspase-3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians.

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