Skeletal regeneration in the brittle star Amphiura filiformis.
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.
Department of Genetics, Evolution and Environment, University College London, London, UK.
Anna
A
Czarkwiani
Department of Biosciences, University of Milan, Milan, Italy.
Cinzia
C
Ferrario
Department of Genetics, Evolution and Environment, University College London, London, UK ; Centre for Mathematics, Physics and Engineering in the Life Sciences and Experimental Biology, University College London, London, UK ; Present address: Department of Ecology and Evolution & Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.
David Viktor
DV
Dylus
Department of Biosciences, University of Milan, Milan, Italy.
Michela
M
Sugni
Department of Genetics, Evolution and Environment, University College London, London, UK ; Research Department of Genetics, Evolution and Environment, University College London, Room 426, Darwin Building, Gower Street, London, WC1E 6BT UK.
Paola
P
Oliveri
Brittle star; C-lectin; Echinoderms; Proliferation; Regeneration; Skeleton; p19; p58b