Mashanov V et al. (2022), A subterminal growth zone at arm tip likely und...

ECB-ART-50380

Front Zool 2022 Apr 12;191:15. doi: 10.1186/s12983-022-00461-0.

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A subterminal growth zone at arm tip likely underlies life-long indeterminate growth in brittle stars.

Mashanov V , Whaley L , Davis K , Heinzeller T , Machado DJ , Reid RW , Kofsky J , Janies D .

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BACKGROUND: Echinoderms are a phylum of marine invertebrates with close phylogenetic relationships to chordates. Many members of the phylum Echinodermata are capable of extensive post-traumatic regeneration and life-long indeterminate growth. Different from regeneration, the life-long elongation of the main body axis in adult echinoderms has received little attention. The anatomical location and the nature of the dividing progenitor cells contributing to adults' growth is unknown. RESULTS: We show that the proliferating cells that drive the life-long growth of adult brittle star arms are mostly localized to the subterminal (second from the tip) arm segment. Each of the major anatomical structures contains dividing progenitors. These structures include: the radial nerve, water-vascular canal, and arm coelomic wall. Some of those proliferating progenitor cells are capable of multiple rounds of cell division. Within the nervous system, the progenitor cells were identified as a subset of radial glial cells that do not express Brn1/2/4, a transcription factor with a conserved role in the neuronal fate specification. In addition to characterizing the growth zone and the nature of the precursor cells, we provide a description of the microanatomy of the four distal-most arm segments contrasting the distal with the proximal segments, which are more mature. CONCLUSIONS: The growth of the adult brittle star arms occurs via proliferation of progenitor cells in the distal segments, which are most abundant in the second segment from the tip. At least some of the progenitors are capable of multiple rounds of cell division. Within the nervous system the dividing cells were identified as Brn1/2/4-negative radial glial cells.

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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. c—arm coelom; en—ectoneural neuroepithelium of the radial nerve cord; hn—hyponeural neuroepithelium of the radial nerve cord; pbe—epidermis of the terminal pit bottom; s—sphincter in the radial water-vascular canal; s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_2$$\end{document}2–s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_5$$\end{document}5—subterminal arm segments 2 thru 5; sp—arm spines; t—terminal segment; tf—tube foot; wvc—radial water-vascular canal
	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} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}μm. a—aboral; c—arm coelom (somatocoel); d—distal; e—epidermis; en—ectoneural system; hn—hyponeural system; l—left; m—intervertebral muscles; o—oral; p—proximal; pd—hydrocoelic lining of the podia (tube feet); r—right; s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_2$$\end{document}2—s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_5$$\end{document}5—subterminal arm segments 2 thru 5; t—terminal segment; wvc—hydrocoelic 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)
	Fig. 3. Organization of the fourth arm segment of A. kochii. Cross sections. (a) Low magnification view. Toluidine blue. (b–e) 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. bl—basal lamina separating the ectoneural and hyponeural neuroepithelia; c—arm coelom; en—ectoneural neuroepithelium of the radial nerve cord; hn—hyponeural neuroepithelium of the radial nerve cord; m—intervertebral muscle; n—neuronal perikaryon; rg—radial glial cell; rnc—radial nerve cord; tf—tube foot; wvc—water-vascular canal
	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. c—arm coelom; en—ectoneural part of the radial nerve cord; hn—hyponeural part of the radial nerve cord; wvc—water-vascular canal. Dashed lines indicate the arm coelom; dotted lines show the boundaries of the hyponeural part of the radial nerve cord
	Fig. 5. Overall organization of the terminal arm segment of A. kochii. (a) Low magnification view of a cross section. Toluidine blue. (b–d) 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). ce—cuticle of the epidermis; cn—epineural cuticle; e—surface epidermis; ec—epineural canal; ee—epineural epithelium; en—ectoneural neuroepithelium of the radial nerve cord; pbe—epidermis of the pit bottom; s—sphincter in the water-vascular canal
	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. ce—epidermal cuticle; cn—epineural cuticle; ec—epineural canal; ee—epineural epithelium; n—neuronal perikaryon
	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 B–D show clusters of immunopositive neuronal cell bodies
	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 4 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} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}μm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \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). (d–f’) 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 d’, e’, f’, respectively. Arrowheads indicate dual-labeled EdU\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^+$$\end{document}+BrdU\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^+$$\end{document}+-cells t—terminal segment; s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_2$$\end{document}2–s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_4$$\end{document}4—arm segments 2 thru 4, as counted from the distal tip of the arm (outlined by dashed lines in d’, e’, f’)
	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 4 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. (b–d”) Detailed views of the radial nerve cord in segment 2. (b–b”) 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. c–d” Dual labeling with the EdU click reaction and neuronal markers Brn1/2/4 (c–c”) and Elav (d–d”). t—terminal segment; s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_2$$\end{document}2–s\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$_7$$\end{document}7—arm segments 2 thru 7, as counted from the distal tip of the arm

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Ben Khadra, An integrated view of asteroid regeneration: tissues, cells and molecules. 2017, Pubmed, Echinobase