Dataset Information

Skeletal regeneration in the brittle star Amphiura filiformis.

ABSTRACT:

Background

Brittle stars regenerate their whole arms post-amputation. Amphiura filiformis can now be used for molecular characterization of arm regeneration due to the availability of transcriptomic data. Previous work showed that specific developmental transcription factors known to take part in echinoderm skeletogenesis are expressed during adult arm regeneration in A. filiformis; however, the process of skeleton formation remained poorly understood. Here, we present the results of an in-depth microscopic analysis of skeletal morphogenesis during regeneration, using calcein staining, EdU labeling and in situ hybridization.

Results

To better compare different samples, we propose a staging system for the early A. filiformis arm regeneration stages based on morphological landmarks identifiable in living animals and supported by histological analysis. We show that the calcified spicules forming the endoskeleton first appear very early during regeneration in the dermal layer of regenerates. These spicules then mature into complex skeletal elements of the differentiated arm during late regeneration. The mesenchymal cells in the dermal area express the skeletal marker genes Afi-c-lectin, Afi-p58b and Afi-p19; however, EdU labeling shows that these dermal cells do not proliferate.

Conclusions

A. filiformis arms regenerate through a consistent set of developmental stages using a distalization-intercalation mode, despite variability in regeneration rate. Skeletal elements form in a mesenchymal cell layer that does not proliferate and thus must be supplied from a different source. Our work provides the basis for future cellular and molecular studies of skeleton regeneration in brittle stars.

SUBMITTER: Czarkwiani A

PROVIDER: S-EPMC4841056 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The brittle star Amphiura filiformis, which regenerates its arms post autotomy, is emerging as a useful model for studying the molecular underpinnings of regeneration, aided by the recent availability of some molecular resources. During regeneration a blastema initially is formed distally to the amputation site, and then a rapid rebuild is obtained by adding metameric units, which will eventually differentiate and become fully functional. In this work we first characterize the developmental process of the regenerating arms using two differentiation markers for muscle and skeletal structures - Afi-trop-1 and Afi-?coll. Both genes are not expressed in the blastema and newly added undifferentiated metameric units. Their expression at different regenerating stages shows an early segregation of muscle and skeletal cells during the regenerating process, long before the metameric units become functional. We then studied the expression of a set of genes orthologous of the sea urchin transcription factors involved in the development of skeletal and non-skeletal mesoderm: Afi-ets1/2, Afi-alx1, Afi-tbr, Afi-foxB and Afi-gataC. We found that Afi-ets1/2, Afi-alx1, Afi-foxB and Afi-gataC are all expressed at the blastemal stage. As regeneration progresses those genes are expressed in a similar small undifferentiated domain beneath the distal growth cap, while in more advanced metameric units they become restricted to different skeletal domains. Afi-foxB becomes expressed in non-skeletal structures. This suggests that they might play a combinatorial role only in the early cell specification process and that subsequently they function independently in the differentiation of different structures. Afi-tbr is not present in the adult arm tissue at any stage of regeneration. In situ hybridization results have been confirmed with a new strategy for quantitative PCR (QPCR), using a subdivision of the three stages of regeneration into proximal (differentiated) and distal (undifferentiated) arm segments.

Project description:BackgroundRegeneration 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.ResultsOur 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.ConclusionsWe 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.