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

Molecular characterization of skeletal regeneration in the brittle star amphiura filiformis

Echinoderms are well known for their extensive regenerative abilities, but have been neglected in the field due to the lack of available molecular tools and resources [1]. Recently, developmental [2] and adult transcriptomes [3, 4] of the brittle star Amphiura filiformis have been sequenced, which opened up this species for molecular investigations of its rapid arm regeneration process. We use this brittle star as a model to understand the cellular and molecular aspects of skeletogenesis during adult arm regeneration and the potential role of the FGF signalling pathway in this process. Ultimately, we compare the molecular network driving regeneration of the skeleton to that underlying embryonic skeleton development [5]. Following a characterization of the anatomy and development of the skeleton during arm regeneration in A. filiformis [6], we established methods for spatio-temporal expression analysis [7] and pharmacological treatments to characterise genes involved in adult arm regeneration. We found that 18 embryonic skeletogenic mesoderm genes (transcription factors, signaling receptors and downstream differentiation genes) are also expressed in the dermal layer of the adult regenerating arm, where skeletal spicules form. FGF signalling perturbation using the SU5402 inhibitor interferes with skeleton formation during both embryonic development and adult regeneration of this brittle star. A large-scale comparison of genes affected by SU5402 in adult arm regeneration and during embryonic development revealed a conservation of network components downstream of FGF signalling between those two developmental modes. Acknowledgements: We thank the staff at the Sven Lov\ue9n Centre for Marine Sciences in Kristineberg, especially Olga Ortega-Martinez and Sam Dupont, for assistance during animal and sample collection. References: 1. Dupont S, Thorndyke M (2007) Bridging the regeneration gap: insights from echinoderm models. Nat Rev Genet 8:8\u201310 2. Delroisse J, Ortega-Martinez O, Dupont S, Mallefet J, Flammang P (2015) De novo transcriptome of the European brittle star Amphiura filiformis pluteus larvae. Mar Genomics. doi: 10.1016/j.margen.2015.05.014 3. Purushothaman S, Saxena S, Meghah V, Swamy CVB, Ortega-Martinez O, Dupont S, Idris M (2014) Transcriptomic and proteomic analyses of Amphiura filiformis arm tissue-undergoing regeneration. J Proteomics 1\u201312 4. Delroisse J, Mallefet J, Flammang P (2016) De Novo Adult Transcriptomes of Two European Brittle Stars: Spotlight on Opsin-Based Photoreception. PLoS One 11:e0152988 5. Dylus DV, Czarkwiani A, St\ue5ngberg J, Ortega-Martinez O, Dupont S, Oliveri P (2016) Large-scale gene expression study in the ophiuroid Amphiura filiformis provides insights into evolution of gene regulatory networks. Evodevo 7:2 6. Czarkwiani A, Ferrario C, Dylus D V., Sugni M, Oliveri P (2016) Skeletal regeneration in the brittle star Amphiura filiformis. Front Zool 13:18 7. Czarkwiani A, Dylus D V., Oliveri P (2013) Expression of skeletogenic genes during arm regeneration in the brittle star Amphiura filiformis. Gene Expr Patterns 13:464\u201347

Extracellular matrix gene expression during arm regeneration in Amphiura filiformis

Extracellular matrix (ECM) plays a dynamic role during tissue development and re-growth. Body part regeneration efficiency relies also on effective ECM remodelling and deposition. Among invertebrates, echinoderms are well known for their striking regenerative abilities since they can rapidly regenerate functioning complex structures. To gather insights on the involvement of ECM during arm regeneration, the brittle star Amphiura filiformis was chosen as experimental model. Eight ECM genes were identified and cloned, and their spatio-temporal and quantitative expression patterns were analysed by means of whole mount in situ hybridisation and quantitative PCR on early and advanced regenerative stages. Our results show that almost none of the selected ECM genes are expressed at early stages of regeneration, suggesting a delay in their activation that may be responsible for the high regeneration efficiency of these animals, as described for other echinoderms and in contrast to most vertebrates. Moreover, at advanced stages, these genes are spatially and temporally differentially expressed, suggesting that the molecular regulation of ECM deposition/remodelling varies throughout the regenerative process. Phylogenetic analyses of the identified collagen-like genes reveal complex evolutionary dynamics with many rounds of duplications and losses and pinpointed their homologues in selected vertebrates. The study of other ECM genes will allow a better understanding of ECM contribution to brittle star arm regeneration