Ultrastructural and molecular analysis of the origin and differentiation of cells mediating brittle star skeletal regeneration

Abstract

Background Regeneration is the ability to re-grow body parts or tissues after trauma, and it is widespread across metazoans. Cells involved in regeneration can arise from a pool of undifferentiated proliferative cells or be recruited from pre-existing differentiated tissues. Both mechanisms have been described in different phyla; however, the cellular and molecular mechanisms employed by different animals to restore lost tissues as well as the source of cells involved in regeneration remain largely unknown. Echinoderms are a clade of deuterostome invertebrates that show striking larval and adult regenerative abilities in all extant classes. Here, we use the brittle star Amphiura filiformis to investigate the origin and differentiation of cells involved in skeletal regeneration using a combination of microscopy techniques and molecular markers. Results Our ultrastructural analyses at different regenerative stages identify a population of morphologically undifferentiated cells which appear in close contact with the proliferating epithelium of the regenerating aboral coelomic cavity. These cells express skeletogenic marker genes, such as the transcription factor alx1 and the differentiation genes c-lectin and msp130L, and display a gradient of morphological differentiation from the aboral coelomic cavity towards the epidermis. Cells closer to the epidermis, which are in contact with developing spicules, have the morphology of mature skeletal cells (sclerocytes), and express several skeletogenic transcription factors and differentiation genes. Moreover, as regeneration progresses, sclerocytes show a different combinatorial expression of genes in various skeletal elements. Conclusions We hypothesize that sclerocyte precursors originate from the epithelium of the proliferating aboral coelomic cavity. As these cells migrate towards the epidermis, they differentiate and start secreting spicules. Moreover, our study shows that molecular and cellular processes involved in skeletal regeneration resemble those used during skeletal development, hinting at a possible conservation of developmental programmes during adult regeneration. Finally, we highlight that many genes involved in echinoderm skeletogenesis also play a role in vertebrate skeleton formation, suggesting a possible common origin of the deuterostome endoskeleton pathway.

Molecular Paleobiology of the Echinoderm Skeleton

“…Adult echinoderm skeletogenic cells have been most thoroughly characterized in echinoids, where skeletal cells have been classified into at least two distinct groups, the sclerocytes and the odontoblasts (Märkel et al, 1986, Märkel et al, 1989. Additionally, sclerocytes have been identified and characterized in regeneration ophiuroid arms (Piovani et al, 2021). In echinoids, sclerocytes are the skeleton-secreting cells which secrete biomineral throughout the majority of the animal, while odontoblasts are those skeletogenic cells which are responsible for biomineralization of the continuously growing teeth of the Aristotle's lantern (Märkel et al, 1986, Märkel et al, 1989.…”

“…In growing echinoderm skeletal elements, such as the margins of interambulacral plates of the echinoid test, there is a higher density of skeletal cells in areas of active skeletogenesis (Shimizu, 1997). Like the skeletogenic cells of the larvae, sclerocytes, are responsible for biomineralization within cytoplasmic sheaths or, when multiple cells have merged, a syncytial vacuole (Märkel et al, 1986, Smith, 1990, Märkel et al, 1989, Dubois and Jangoux, 1990, Ameye et al, 1999, Heatfield and Travis, 1975, Stricker, 1986, Piovani et al, 2021. Within the cytoplasmic sheath of this syncytial vacuole, the skeleton is surrounded by a matrix coat, comprised of polysaccharides and proteins (Märkel et al, 1989, Ameye et al, 1999.…”

“…While echinoderm skeletogenic cells were first identified and characterized based upon their function and morphology, most recent work has begun to understand these cells in the context of the suites genes they express (Rafiq et al, 2014, Piovani et al, 2021, and the role of these genes in building the skeleton. The expression and activity of these genes is the product of the molecular interactions encoded in the genome, and characterized by the skeletogenic gene regulatory network (Shashikant et al, 2018, Oliveri et al, 2008.…”

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology

“…Besides providing new circulating coelomocytes, the coelomic epithelium can also provide progenitor cells for the reconstruction of the regenerating tissue as reported in crinoids (116). This can occur thanks to a dedifferentiation process followed by epithelial-mesenchymal transition, allowing migration of scarcely differentiated cells in the underlying developing connective tissue (117)(118)(119)(120). After arm amputation, trauma-stressed sea stars (Asterias rubens) show an increase both in the total number of the circulating coelomocytes and in the levels of Hsp70 stress protein within the cells.…”

Regeneration in Echinoderms: Molecular Advancements

“…Transcription factors have become key targets in recent studies, being crucial for processes intrinsic to regeneration such as cellular differentiation. Other studies focused on the presence (or absence) of extracellular matrix (ECM) genes (Ferrario et al, 2020), skeletogenic genes (Piovani et al, 2021) and glucosaminoglycans (Ramachandra et al, 2014;Ramachandra et al, 2017) as possible mediators of the arm regeneration process.…”