FGF signalling plays similar roles in development and regeneration of the skeleton in the brittle star Amphiura filiformis

Regeneration as an adult developmental process is in many aspects similar to embryonic development. Although many studies point out similarities and differences, no large-scale, direct and functional comparative analyses between development and regeneration of a specific cell type or structure in one animal exist. Here, we use the brittle star Amphiura filiformis to characterise the role of the FGF signalling pathway during skeletal development in embryos and arm regeneration. In both processes, we find ligands expressed in ectodermal cells flanking underlying skeletal mesenchymal cells, which express the receptors. Perturbation of FGF signalling showed inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes. Differential transcriptome analysis finds mostly differentiation genes rather than transcription factors to be downregulated in both contexts. Moreover, comparative gene analysis allowed us to discover brittle star specific, differentiation genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as in other deuterostomes and provide evidence for the re-deployment of a developmental gene regulatory module during regeneration.

Species interactions and environmental context affect intraspecific behavioural trait variation and ecosystem function

Functional trait-based approaches are increasingly adopted to understand and project ecological responses to environmental change; however, most assume trait expression is constant between conspecifics irrespective of context. Using two species of benthic invertebrate (brittlestars Amphiura filiformis and Amphiura chiajei ), we demonstrate that trait expression at individual and community levels differs with biotic and abiotic context. We use PERMANOVA to test the effect of species identity, density and local environmental history on individual (righting and burrowing) and community (particle reworking and burrow ventilation) trait expression, as well as associated effects on ecosystem functioning (sediment nutrient release). Trait expression differs with context, with repercussions for the faunal mediation of ecosystem processes; we find increased rates of righting and burial behaviour and greater particle reworking with increasing density that are reflected in nutrient generation. However, the magnitude of effects differed within and between species, arising from site-specific environmental and morphological differences. Our results indicate that traits and processes influencing change in ecosystem functioning are products of both prevailing and historic conditions that cannot be constrained within typologies. Trait-based study must incorporate context-dependent variation, including intraspecific differences from individual to ecosystem scales, to avoid jeopardizing projections of ecosystem functioning and service delivery.

FGF signalling plays similar roles in development and regeneration of the skeleton in the brittle star Amphiura filiformis

Regeneration is an adult developmental process considered to be an epiphenomenon of embryonic development. Although several studies have shown that various embryonic genes are expressed during regeneration, there have been no large-scale, direct and functional comparative studies between the development and regeneration of a specific structure in one animal. Here, we use the brittle star Amphiura filiformis to characterise the role of the FGF signalling pathway during skeletal development and regeneration. In both processes, we find the ligands expressed in ectodermal cells flanking underlying mesodermal cells, and the receptors expressed specifically by these skeletogenic cells. Perturbation of FGF but not VEGF signalling during skeletogenesis completely inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes like cell migration or proliferation. Transcriptome-wide differential analysis identified a highly similar cohort of skeletogenic differentiation genes downstream of the FGF signalling pathway, whereas upstream transcription factors involved in the initial specification of the skeletogenic lineage where unaffected. Comparison to the sea urchin indicated that many of the affected genes are associated with differentiation. Moreover, several genes showed no homology to a cohort from other species, leading to the discovery of brittle star specific, downstream skeletogenic genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as it is in other deuterostomes, and for the first time provide evidence for the re-deployment of a gene regulatory module during both regeneration and development.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae , Amphiura filiformis and Ophiopsila aranea , has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales.

Developmental transcriptomics of the brittle star Amphiura filiformis reveals gene regulatory network rewiring in echinoderm larval skeleton evolution

ABSTRACTAmongst the echinoderms the class Ophiuroidea is of particular interest for its phylogenetic position, ecological importance, developmental and regenerative biology. However, compared to other echinoderms, notably echinoids (sea urchins), relatively little is known about developmental changes in gene expression in ophiuroids. To address this issue we have generated and assembled a large RNAseq data set of four key stages of development in the brittle star Amphiura filiformis and a de novo reference transcriptome of comparable quality to that of a model echinoderm - the sea urchin Strongyloncentrotus purpuratus. Furthermore, we provide access to the new data via a web interface: http://www.echinonet.eu/shiny/Amphiura_filiformis/. With a focus on skeleton development, we have identified highly conserved genes associated with the development of a biomineralized skeleton. We also identify important class-specific characters, including the independent duplication of the msp130 class of genes in different echinoderm classes and the unique occurrence of spicule matrix (sm) genes in echinoids. Using a new quantification pipeline for our de novo transcriptome, validated with other methodologies, we find major differences between brittle stars and sea urchins in the temporal expression of many transcription factor genes. This divergence in developmental regulatory states is more evident in early stages of development when cell specification begins, than when cells initiate differentiation. Our findings indicate that there has been a high degree of gene regulatory network rewiring in the evolution of echinoderm larval development.Data DepositionsAll sequence reads are available at Genbank SRR4436669 - SRR4436674. Any sequence alignments used are available by the corresponding author upon request.