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Fig. 1. A. filiformis non-regenerating arm skeletal elements and ultrastructure of cell types. A 3D schematic representation of a non-regenerating arm showing the main anatomical structures colour coded as in legend on the left. B SEM of the skeletal elements present in a single metameric unit and displayed in their corresponding anatomical position. AP, aboral arm plate; LP, lateral arm plate; OP, oral arm plate; S, spine; V, vertebra. C SEM micrograph of a cell in the stroma of the skeleton of the vertebra: arrowhead indicates the nucleus, white arrow points at fibrils with morphology typical of collagen, magnified in C’. D TEM micrograph of a sclerocyte in the vertebra: white arrows indicate fibrils in cross section likely of collagen, magnified in D’. E TEM micrograph of a presumptive pigment cell in the oral arm plate with an evident nucleolus and spindle-shaped electron-dense structures typical of pigment granules, magnified in E’. F TEM micrograph of a phagocyte in the aboral arm plate characterized by the presence of phagosome, magnified in F’. G TEM micrograph of a granulocyte present in the vertebra: red arrowheads highlight fibrils, present in both cross and longitudinal sections; arrows point at large electron-dense granules of different shapes and sizes, magnified in G’. H TEM micrograph of a nerve cell in the aboral arm plate characterized by a nerve process magnified in H’; white arrow indicates the small electron-dense roundish vesicles generally containing neurotransmitters or other neuro-signalling molecules. Scale bars = 2 μm. In all images: asterisks indicate the presence of biomineralized skeletal tissue (stereom calcareous elements or trabeculae) now empty, i.e. electron-transparent due to fixation and decalcification processes; white arrowhead shows the nucleus with hetero- and euchromatin; and red dotted box indicates magnified part of the figure
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Fig. 2. The main cell population of the dermis at early regenerative stages shows a gradient of cellular differentiation. Schematics (top) and TEM micrographs (a–f). a Mesenchymal cells at stage 4/5 located at the distal tip near the axial structures look rather undifferentiated. Main features are a secondary boundary layer (always highlighted in red in the schematics), a few vesicles, and a patchy nucleus. b Mesenchymal cells at stage 2/3 in the area next to where the ACC and RNC are adjacent on either side of the RWC. Cells show large RER, many vesicles, and a cytoplasmic pocket containing fibrils likely of collagen. c Mesenchymal cells at stage 4/5 at the very distal tip of the regenerate. Cells show large RER, vesicles, and a more electron-transparent cytoplasmic pocket. d Mesenchymal cells at stage 4/5 at the distal-most tip of the regenerate show a pocket syncytium. e, f Mesenchymal cells at stage 4/5 right under the epidermis show growing spicules. The main cellular features are indicated in the schematic the first time they appear. Red arrows indicate collagen fibrils, capital N indicates nucleus, and asterisks indicate growing spicules. ACC, aboral coelomic cavity; EPI, epidermis; St., stage. Scale bars = 2 μm
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Fig. 3. Regenerating mesenchymal cells express skeletogenic gene markers and appear to detach from the ACC epithelium. a Semi-thin sections of whole mount ISH for skeletal genes: Afi-c-lectin (embedded in resin), Afi-alx1 and Afi-msp130L (embedded in wax). Red arrows point at areas where gene expression (dark blue/purple staining) is present, as schematized on the right. Yellow, aboral coelomic cavity; blue, radial water canal; pink, radial nerve cord; CR, cross section; SAG, sagittal section. Crossed arrows further indicate the orientation of the section: A, aboral; O, oral; R, right; L, left; P, proximal; D, distal. Scale bars = 10 μm. b TEM micrographs of cells likely detaching from the ACC epithelium. (1’) and (2’) are details of (1) and (2), respectively. Yellow, ACC; pink, RNC. Scale bars = 2 μm
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Fig. 4. Gene expression at early stages of regeneration (stage 2/3 and stage 4/5). a Colour coded sagittal scheme of stage 2/3 and frontal schemes of stages 2/3 and 4/5. Crossed arrows near schematic indicate the axis of the section. Schematics of gene expression in b and c follow the sagittal scheme as this view allows clear distinction of all tissues. Pictures of gene expression in whole mount are taken in frontal view from the aboral side after orienting the samples, unless otherwise specified. b Whole mount ISH at two regenerative stages (stage 2/3 and stage 4/5) as indicated at the top of the columns using antisense probes for transcription factors. Probe name is indicated on the left of the summary schematics. c Whole mount ISH at two regenerative stages (stage 2/3 and stage 4/5) as indicated at the top of the columns using antisense probes for known differentiation genes. Probe name is indicated on the left of the summary schematics. In b and c, summary schematics of expression are based on several images of different focal plane observations of multiple samples; however, only one focal plane is shown here. A, aboral; O, oral; R, right; L, left; P, proximal; D, distal. Dark blue/purple indicates probe-specific signal. Scale bars = 100 μm
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Fig. 5. Gene expression at late stages of regeneration. a Sagittal schematic of regenerating arm at late stages of regeneration with proximal (Prox.) and distal (Dist.) schematics of cross sections. Purple, epidermis; grey, skeleton; yellow, ACC; blue, RWC; pink, RNC. b Whole mount ISH (proximal and distal position as indicated at the top of the columns), using antisense probes for transcription factors. c Whole mount ISH (proximal and distal position as indicated at the top of the columns), using probes for known differentiation genes. For b and c, probe name is indicated at the bottom of the proximal image. On the left of each pair of ISH pictures, there is a summary of expression (blue) in cross schematics for the proximal (Prox.) and distal (Dist.) arm. Summary schematics of expression are based on focal plane observations of multiple samples; however, only one focal plane is shown here. In the distal figures, dotted squares indicate the differentiated terminal structure (terminal ossicle and podium) and black lines indicate the proliferating area. The summary expression data in the schematic is taken from the proliferating area. Scale bars = 50 μm
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Fig. 6. Summary of the origin of skeletogenic cells and the evolution of the molecular signature in deuterostomes. a Model of main hypothesis of sclerocyte origin and differentiation at early stages of regeneration (stage 2/3 and 4/5): new sclerocyte precursors detach from the aboral coelomic cavity (ACC) epithelium and differentiate while moving towards the epidermis together with summary of regulatory (red), differentiation (black), and signalling (blue) gene expression of the ACC, the dermis, and the epidermis. Schematics are based on data shown in Fig. 2 and Additional file 1 Fig. S2; gene expression data is based on Figs. 3 and 4 and Additional file 2 Figs. S5 and S6 combined with already published data (References in Table S2). b Summary of molecular signature of different skeletal elements at the proximal side of late stages of regenerating arms. Gene expression data is based on proximal differentiating skeletal elements shown in Fig. 5 and Additional file 2 Figs. S5 and S6 together with published data. AP, aboral arm plate; LP, lateral arm plate; OP, oral arm plate; Sp, spine; V, vertebra. c Phylogenetic tree with conserved role of skeletogenic genes between the brittle star Amphiura filiformis (present work), Strongylocentrotus purpuratus, and vertebrates. Genes encoding for transcription factors are in red, and the differentiation genes are in black. For details and relative references, see Additional file 2 Table S2. For this summary figure, we considered the gene present if we could find data on a member of the gene family; therefore, it is not a strict 1:1 orthology, considering also the two rounds of genome duplication occurred at the base of vertebrate evolution
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