Dylus DV et al. (2018), Developmental transcriptomics of the brittle st...

ECB-ART-46155

Genome Biol 2018 Feb 28;191:26. doi: 10.1186/s13059-018-1402-8.

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

Dylus DV, Czarkwiani A, Blowes LM, Elphick MR, Oliveri P.

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BACKGROUND: Amongst the echinoderms the class Ophiuroidea is of particular interest for its phylogenetic position, ecological importance and 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 Strongylocentrotus purpuratus. Furthermore, we provide access to the new data via a web interface: http://www.echinonet.eu/shiny/Amphiura_filiformis/ . RESULTS: We have identified highly conserved genes associated with the development of a biomineralised 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, rather than when cells initiate differentiation. CONCLUSIONS: Our findings indicate that there has been a high degree of gene regulatory network rewiring and clade-specific gene duplication, supporting the hypothesis of a convergent evolution of larval skeleton development in echinoderms.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC115919910 LOC115924597 LOC115925415 LOC581907 LOC583082 mpi msp130

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	Fig. 1. Pipeline used to obtain the A. filiformis developmental transcriptome. a Developmental timepoints used for RNA-seq: 9hpf corresponds to a late cleavage stage, 18hpf to a blastula stage, 27hpf to a mesenchyme blastula stage and 39hpf to a late gastrula stage (arrows point to position where spicules are formed). The brittle star A. filiformis and the sea urchin S. purpuratus pluteus larvae showing general morphological features and the birifrangent extended skeleton (m mouth, St stomach, Sk skeleton). b Assembly pipeline showing the individual steps and the reduction in sequences
	Fig. 2. Gene content in representatives of four echinoderm classes. a Phylogenetic relationships of the four species compared in this study according to the currently most supported phylogeny for the classes these species belong to. b Venn diagram showing the overlaps of genes that were identified using a reciprocal tBLASTx (e-value 1e-6) strategy. The different numbers in each overlap field indicate the species that was used as reference for the BLAST search. Afi Amphiura filiformis, Pmi Patiria miniata, Ame Antedon mediterranea, Spu Strongyloncetrotus prupuratus, Echi Echinoderm core (overlap of all four classes)
	Fig. 3. Conservation of gene functional classes in echinoderms. Sea urchin functional classes are based on S. purpuratus [21] and show proportions identified in the other three echinoderms. Average and standard deviation are calculated between Afi, Pmi and Ame and are normalised based on the sea urchin. Afi Amphiura filiformis, Pmi Patiria miniata, Ame Antedon mediterranea, Spu Strongylocentrotus prupuratus, Echi Echinoderm core (overlap of all four classes)
	Fig. 4. Homologs of sea urchin skeletogenic genes identified in other echinoderms and expression patterns for selected candidates. Venn diagram showing the overlap of genes involved in sea urchin skeletogenesis with homologs found in other echinoderms; 494/901 are shared between four classes of echinoderms, which is a higher proportion than a set of random genes (Additional file 1: Figure S7). Whole mount in situ expression patterns in two important brittle star developmental stages for several selected candidates from different regions of overlap reveals an association with cells associated with skeleton formation. In the top right corner is depicted the currently most supported phylogeny for these four species. Schematics representing mesenchyme blastula and early gastrula stages are in the bottom right corner (in purple are shown the mesenchymal cells that will give rise to skeleton). Afi Amphiura filiformis, Pmi Patiria miniata, Ame Antedon mediterranea, Spu Strongylocentrotus prupuratus, Echi Echinoderm core (overlap of all four classes). MBl mesenchyme blastula, G gastrula
	Fig. 5. Global A. filiformis gene expression and comparison of larval regulatory states. a Fuzzy clustering of 39,000 ECs in 27 clusters of four developmental time points sorted in four distinct modes of expression (EARLY, LATE, INTERMEDIATE, BI-MODAL). Each line represents the expression of a single gene, and the grey intensity indicates the normalised expression. b Comparison of TFs in the four modes of expression between sea urchin (SPU) and brittle star (AFI). The majority of TFs show differences in expression
	Fig. 6. Scenario of larval skeleton evolution. A simplified phylogeny of echinoderms with representative larval stages (skeleton in red), which illustrates the position of major transitions in the evolution of the larval skeleton. Specifically, at the base of echinoderms are shown common features for the evolution of the adult skeleton and at the class level are depicted specific features for ophiuroids and echinoids

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