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Fig. 1. Overall morphology of the arm tip of A. kochii. (a, b) Scanning electron microscopy. (a) Low magnification view of the oral side. (b) Detailed view of the terminal pit. (c) Parasagittal semi-thin section. Toluidine blue. câarm coelom; enâectoneural neuroepithelium of the radial nerve cord; hnâhyponeural neuroepithelium of the radial nerve cord; pbeâepidermis of the terminal pit bottom; sâsphincter in the radial water-vascular canal; s\documentclass[12pt]{minimal}
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\begin{document}$$_5$$\end{document}5âsubterminal arm segments 2 thru 5; spâarm spines; tâterminal segment; tfâtube foot; wvcâradial water-vascular canal
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Fig. 2. Three-dimensional reconstruction of the arm tip of A. kochii. The following anatomical structures are shown: epidermis (semitransparent violet), ectoneural part of the nervous system (green), hyponeural part of the nervous system (magenta), water-vascular system (red), arm coelom (yellow), and intervertebral muscles (brown). (a) Oral view. (b) Aboral view. (c) Side view. (d) Oblique side view, arm coelom not shown. The edge length of the scale cube (grey) is 25 \documentclass[12pt]{minimal}
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\begin{document}$$\mu$$\end{document}μm. aâaboral; câarm coelom (somatocoel); dâdistal; eâepidermis; enâectoneural system; hnâhyponeural system; lâleft; mâintervertebral muscles; oâoral; pâproximal; pdâhydrocoelic lining of the podia (tube feet); râright; s\documentclass[12pt]{minimal}
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\begin{document}$$_5$$\end{document}5âsubterminal arm segments 2 thru 5; tâterminal segment; wvcâhydrocoelic water-vascular canal. The 3D video animation of this anatomical model is available in Additional file 2. The original Blender model used to produce this figure and the video is available in Zenodo (https://doi.org/10.5281/zenodo.5762494)
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Fig. 3. Organization of the fourth arm segment of A. kochii. Cross sections. (a) Low magnification view. Toluidine blue. (bâe) Transmission electron microscopy. (b) Low magnification transmission electron micrograph of the radial nerve cord, water-vascular canal, arm coelom, and intervertebral muscles. (c) High magnification view of the intervertebral muscle. Arrowhead shows the cytoskeletal filaments of the contractile apparatus. (d) Neuronal perikaryon in the ectoneural neuroepithelium. (e) Hyponeural part of the radial nerve cord. blâbasal lamina separating the ectoneural and hyponeural neuroepithelia; câarm coelom; enâectoneural neuroepithelium of the radial nerve cord; hnâhyponeural neuroepithelium of the radial nerve cord; mâintervertebral muscle; nâneuronal perikaryon; rgâradial glial cell; rncâradial nerve cord; tfâtube foot; wvcâwater-vascular canal
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Fig. 4. Organization of the second arm segment of A. kochii. Cross sections. (a) Low magnification view. Toluidine blue. (b, c) Transmission electron microscopy. (b) Overview of the radial nerve cord, water-vascular canal, and arm coelom. (c) High magnification view of the ectoneural neuropil. câarm coelom; enâectoneural part of the radial nerve cord; hnâhyponeural part of the radial nerve cord; wvcâwater-vascular canal. Dashed lines indicate the arm coelom; dotted lines show the boundaries of the hyponeural part of the radial nerve cord
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Fig. 5. Overall organization of the terminal arm segment of A. kochii. (a) Low magnification view of a cross section. Toluidine blue. (bâd) Transmission electron microscopy. (b) Parasagittal section. Two opposing arrows indicate the level where the fusion between the ectoneural neuroepithelium and the epidermis takes place. (c) Cross section through the sphincter in the water-vascular canal at a level indicated by a dotted line in (b). (d) Detailed view of a parasagittal section showing the fusion between the epineural cuticle (cn) and the cuticle of the surface epidermis (ce). It approximately corresponds to the boxed area in (A). ceâcuticle of the epidermis; cnâepineural cuticle; eâsurface epidermis; ecâepineural canal; eeâepineural epithelium; enâectoneural neuroepithelium of the radial nerve cord; pbeâepidermis of the pit bottom; sâsphincter in the water-vascular canal
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Fig. 6. Ectoneural epithelium and terminal pit epidermis in the terminal arm segment of A. kochii. Transmission electron microscopy. (a) Cross-section of the ectoneural neuroepithelium of the radial nerve cord before its fusion with the epidermis of the terminal pit. (b) Sagittal section through the epidermis of the terminal pit. Inset shows the cilium and microvilli on the apical surface of the pit epidermis. ceâepidermal cuticle; cnâepineural cuticle; ecâepineural canal; eeâepineural epithelium; nâneuronal perikaryon
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Fig. 7. Organization of the nervous system in the arm tip of O. brevispinum. Maximum intensity Z-projections of confocal image stacks. (A) Acetylated tubulin. Filled arrow shows the terminal aboral loop formed by the hyponeural part of the radial nerve cord, which gives off a number of short tracts (open arrows) running towards the tip. (B) Elav-positive neurons. (C) GFSKLYFamide (GFS)-positive neuronal elements. (D) Expression of the transcription factor Brn1/2/4. Note that this particular antibody, besides specifically binding to the antigen in the nucleus, also non-specifically bind to the cuticle that covers the surface of the epidermis (asterisk)White arrowheads in BâD show clusters of immunopositive neuronal cell bodies
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Fig. 8. Quantification of cell proliferation in the four terminal arm segments O. brevispinum. (a) Dual pulse labeling paradigm. The animals were first pulsed with EdU, followed by a 7 day chase period, then pulsed with BrdU and processed for microscopic analysis. Both pulses lasted 4Â h. (b, c) Boxplots showing individual data points and pairwise P values from the Wilcoxon-Mann-Whitney test. (b) Abundance of dividing (BrdU-incorporated) cells (per \documentclass[12pt]{minimal}
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\begin{document}$$^3$$\end{document}3). (c) Relative abundance of repeatedly proliferating cells (i.e., those that have incorporated both thymidine analogs) measured as the percentage of proliferating cells undergoing the second round of cell division (i.e., the cells that have incorporated both EdU and BrdU). (dâfâ) Representative micrographs showing distribution of labeled cells in the arm tip. Maximum intensity Z-projections of confocal stacks. Aboral-oral view. Boxed areas in d, e, and f indicated regions showed at higher magnification in dâ, eâ, fâ, respectively. Arrowheads indicate dual-labeled EdU\documentclass[12pt]{minimal}
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\begin{document}$$^+$$\end{document}+-cells tâterminal segment; s\documentclass[12pt]{minimal}
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\begin{document}$$_4$$\end{document}4âarm segments 2 thru 4, as counted from the distal tip of the arm (outlined by dashed lines in dâ, eâ, fâ)
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Fig. 9. Distribution of dividing cells across the tissues and cell types in the arm tip segments O. brevispinum. The animals were pulsed with EdU for 4Â h and processed for immunocytochemical staining. All micrographs are sagittal cryosections with the distal end to the right. (a) Low-magnification view of a sagittal section through the arm tip showing seven segments. (bâdâ) Detailed views of the radial nerve cord in segment 2. (bâbâ) Dual labeling with the EdU click reaction and ERG1 antibodies. The latter labels radial glial cells in the echinoderm central nervous system. Arrowhead points to a representative dual-labeled cell. câdâ Dual labeling with the EdU click reaction and neuronal markers Brn1/2/4 (câcâ) and Elav (dâdâ). tâterminal segment; s\documentclass[12pt]{minimal}
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\begin{document}$$_7$$\end{document}7âarm segments 2 thru 7, as counted from the distal tip of the arm
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