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.

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.

Large-scale gene expression study in the ophiuroid Amphiura filiformis provides insights into evolution of gene regulatory networks.

BACKGROUND: The evolutionary mechanisms involved in shaping complex gene regulatory networks (GRN) that encode for morphologically similar structures in distantly related animals remain elusive. In this context, echinoderm larval skeletons found in brittle stars and sea urchins provide an ideal system. Here, we characterize for the first time the development of the larval skeleton in the ophiuroid Amphiura filiformis and compare it systematically with its counterpart in sea urchin. RESULTS: We show that ophiuroids and euechinoids, that split at least 480 Million years ago (Mya), have remarkable similarities in tempo and mode of skeletal development. Despite morphological and ontological similarities, our high-resolution study of the dynamics of genetic regulatory states in A. filiformis highlights numerous differences in the architecture of their underlying GRNs. Importantly, the A.filiformis pplx, the closest gene to the sea urchin double negative gate (DNG) repressor pmar1, fails to drive the skeletogenic program in sea urchin, showing important evolutionary differences in protein function. hesC, the second repressor of the DNG, is co-expressed with most of the genes that are repressed in sea urchin, indicating the absence of direct repression of tbr, ets1/2, and delta in A. filiformis. Furthermore, the absence of expression in later stages of brittle star skeleton development of key regulatory genes, such as foxb and dri, shows significantly different regulatory states. CONCLUSION: Our data fill up an important gap in the picture of larval mesoderm in echinoderms and allows us to explore the evolutionary implications relative to the recently established phylogeny of echinoderm classes. In light of recent studies on other echinoderms, our data highlight a high evolutionary plasticity of the same nodes throughout evolution of echinoderm skeletogenesis. Finally, gene duplication, protein function diversification, and cis-regulatory element evolution all contributed to shape the regulatory program for larval skeletogenesis in different branches of echinoderms.