Zueva O et al. (2018), The complex simplicity of the brittle star nerv...

ECB-ART-46131

Front Zool 2018 Feb 01;15:1. doi: 10.1186/s12983-017-0247-4.

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The complex simplicity of the brittle star nervous system.

Zueva O , Khoury M , Heinzeller T , Mashanova D , Mashanov V .

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Background: Brittle stars (Ophiuroidea, Echinodermata) have been increasingly used in studies of animal behavior, locomotion, regeneration, physiology, and bioluminescence. The success of these studies directly depends on good working knowledge of the ophiuroid nervous system. Results: Here, we describe the arm nervous system at different levels of organization, including the microanatomy of the radial nerve cord and peripheral nerves, ultrastructure of the neural tissue, and localization of different cell types using specific antibody markers. We standardize the nomenclature of nerves and ganglia, and provide an anatomically accurate digital 3D model of the arm nervous system as a reference for future studies. Our results helped identify several general features characteristic to the adult echinoderm nervous system, including the extensive anatomical interconnections between the ectoneural and hyponeural components, neuroepithelial organization of the central nervous system, and the supporting scaffold of the neuroepithelium formed by radial glial cells. In addition, we provide further support to the notion that the echinoderm radial glia is a complex and diverse cell population. We also tested the suitability of a range of specific cell-type markers for studies of the brittle star nervous system and established that the radial glial cells are reliably labeled with the ERG1 antibodies, whereas the best neuronal markers are acetylated tubulin, ELAV, and synaptotagmin B. The transcription factor Brn1/2/4 - a marker of neuronal progenitors - is expressed not only in neurons, but also in a subpopulation of radial glia. For the first time, we describe putative ophiuroid proprioceptors associated with the hyponeural part of the central nervous system. Conclusions: Together, our data help establish both the general principles of neural architecture common to the phylum Echinodermata and the specific ophiuroid features.

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Genes referenced: amn gli3 LOC100892350 LOC115917880 LOC115919910 pou1f1 sqle tefl tubgcp2
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	Fig. 1. Brittle star arm anatomy. a An individual of O. brevispinum. b Scanning electron micrograph of the oral arm surface of A. kochii. c and c’ Cross section of the arm of A. kochii. c The arm was embedded into epoxy resin and sectioned transversely. The resin was then removed, and the cut surface was imaged with a scanning electron microscope. c’ The corresponding plastic section stained with methylene blue. d and d’ Longitudinal paragsagittal sections through the arm in A. kochii stained with methylene blue. The skeletal elements are not visible in any of the micrographs, because they were removed during tissue processing. Abbreviations: ac – arm coelom; am – aboral intervertebral muscle; as – aboral shield; il – intervertebral ligament; ljn – lateral juxtaligamental node; om – oral intervertebral muscle; os – oral shield; pd – podium; pmn – hyponeural proximal muscle nerve; rnc – radial nerve cord; s – spine; ts – tentacle scale; wvc – water-vascular canal; v – vertebral ossicle
	Fig. 2. Transition between the radial nerve cord and the circumoral nerve ring in A. kochii. Semi-thin sections stained with methylene blue. a Radial section (parallel to the long axis of the arm). b Horizontal section (i.e., orthogonal with respect to the oral-aboral axis) through the nerve ring. Abbreviations: nr – nerve ring; rnc – radial nerve cord
	Fig. 3. Simplified diagram of the anatomy of the nervous system in the arm segment. The aboral side is up. The distal and proximal lateral hyponeural nerves are not shown for simplicity. Abbreviations: am – aboral intervertebral muscle; amn – aboral mixed nerve;hin – horizontal intermuscular hyponeural nerve; ljn – lateral juxtaligametal node; ojn – oral juxtaligamental node; om – oral intervertebral muscle; pd – podium; pg – podial ganglion; pmn – proximal muscle nerve; rnc – radial nerve cord; rwc – radial water-vascular canal; s – spine; sn – spine nerve; sg – spine ganglion
	Fig. 4. Three-dimensional reconstruction of the radial nerve cord and peripheral nerves in the arm segment in A. kochii. Other anatomical structures are provided for reference, when indicated. a Aboral view of ectoneural system (green). Components of the water-vascular system are shown in red. b Distal view of the ectoneural system. c Side view of muscles (brown) and the hyponeural system (magenta). d Oblique side view of the hyponeural system (magenta). e Proximal view of the complete nervous system. The muscles (brown) and the water-vascular system (red) are also shown. f Distal view of the complete nervous system. The orientation of the projections is indicated by axes on each image: a – aboral; d – distal; o – oral; p – proximal. Abbreviations: am – aboral intervertebral muscle; amn – aboral mixed nerve; dln – distal lateral hyponeural nerve; hin – horizontal intermuscular hyponeural nerve; ljn – lateral juxtaligametal node; mhn – median hyponeural nerve; ojn – oral juxtaligamental node; om – oral intervertebral muscle; pd – podium; pg – podial ganglion; pln – proximal lateral hyponeural nerve; pmn – proximal muscle nerve; pn – podial nerve; rnc – radial nerve cord; rwc – radial water-vascular canal; sg – spine ganglion; sn – spine nerve
	Fig. 5. Representative semithin sections from the series that was used to generate the 3D models shown in Fig. 4. The series was cut in the proximal-to-distal direction. The Z-coordinate (the position of the section in the series) is indicated in the top left corner. Abbreviations: ac – arm coelom; am – aboral intervertebral muscle; amn – aboral mixed nerve; dln – distal lateral hyponeural nerve; ec – epineural canal; ee – epineural epithelium; en – ectoneural part of the radial nerve cord; hin – horizontal intermuscular hyponeural nerve; hl – hemal lacuna; hn – hyponeural part of the radial nerve cord; il – intervertebral ligament; ljn – lateral juxtaligametal node; mhn – median hyponeural nerve; ojn – oral juxtaligamental node; om – oral intervertebral muscle; omn – oral mixed nerve; pd – podium; pg – podial ganglion; pln – proximal lateral hyponeural nerve; pmn – proximal muscle nerve; pn – podial nerve; rnc – radial nerve cord; rwc – radial water-vascular canal; s – spine; sg – spine ganglion; sn – spine nerves; tsn – tentacle scale nerve
	Fig. 6. Radial glia in the ectoneural neuroepithelium of the radial nerve cord in A. kochii. Transmission electron microscopy. a Low magnification view of the ectoneural neuroepithelium. b Intercelluar junctions between apicolateral surfaces of two adjacent radial glial cells. c Basal endfoot of a radial glial cell. d Attachment of the apical surface of a radial glial cell to the epineural cuticle. Abbreviations: bl – basal lamina; bp – basal process of a radial glial cell; c – epineural cuticle; ec – epineural canal; if – intermediate filaments; ne – neuropil; rg – radial glia. Arrowheads show hemidesmosomes
	Fig. 7. Neurons in the ectoneural neuroepithelium of the radial nerve cord in A. kochii. Transmission electron microscopy. a Sub-apical neuron. a’ Cilium in a subapical neuron (n). b Neuron (n, colored) that reaches the lumen of the epineural canal (ec). c – f’ Ectoneural neuropil. c and d show the transversal and longitudinal sections, respectively, of the neuropil area containing processes of giant neurons (gn). e Process of the neurosecretory-like cell (nsc). f and f’ Synapses (arrows) in the neuropil. Abbreviations: bp – basal process of a radial glial cell; ec – epineural canal; ee – epineural epithelium (roof of the epineural canal); gn – giant neuronal processes; n – neuron; np – neural process; nsc – neurosecretory-like cell; rg – radial glial cell
	Fig. 8. Roof of the epineural canal (epineural epithelium) in A. kochii. Transmission electron microscopy. a Flattened glial cell (gc) of the epineural epithelium. b Hemidesmosomes (arrowheads) anchoring a glial cell of the epineural epithelium to the basal lamina (bl) and to the epineural cuticle (c). c The epineural epithelium occasionally contains basiepithelial neural processes (np), but not neuronal perikarya. Abbreviations: bl – basal lamina; c – epineural cuticle; ec – epineural canal; gc – flattened glial cell; if – intermediate filaments; ne – ectoneural neuroepithelium; np – neuronal processes
	Fig. 9. Organization of the hyponeural part of the radial nerve cord (RNC) in A. kochii. Transmission electron microscopy. a and b Hyponeural system in the interganglionic region of the RNC. a Low-magnification view of the hyponeural cord. b Part of the oral wall (floor) of the interganglionic hyponeural cord formed by flattened glial cells with no neuronal elements. c–g Hyponeural system in the ganglionic swelling of the RNC. C Secretory radial glia. d Neurosecretory-like cell in the aboral wall (roof) of the hyponeural cord. e and f Abundant neurons in the oral wall (floor) of the hyponeural cord. f Giant neuron. g High-magnification view of a muscle bundle integrated in the lateral region of the hyponeural neuroepithelium. Abbreviations: bp – basal process of radial glial cell; en – ectoneural neuroepithelium; gc – flattened glial cell; gn – “giant” neuron; hc – lumen of the hyponeural canal; hl – hemal lacuna; hn – hyponeural neuroepithelium; m – bundle of muscle cells; np – neuronal processes; nsc – neurosecretory-like cell; rg – radial glia
	Fig. 10. The hyponeural neuroepithelium makes direct contacts with the ectoneural epithelium (a and a’) and with the intervertebral muscles (b and b’) in A. kochii. Transmission electron microscopy. The colorized neuron in a and a’ has its cell body within the hyponeural neuroepithelium and sends a process into the ectoneural neuropithelium. a’ and b’ show detail views of the boxed areas in a and b, respectively. Abbreviations: en – ectoneural neuroepithelium; hn – hyponeural neuroepithelium; n – neuron; np – neural processes; om – oral intervertebral muscle. White arrowheads indicate the basal lamina
	Fig. 11. Organization of the spine ganglion in A. kochii. Transmission electron microscopy. a Peripheral part of the spine ganglion with cell bodies of neurons and two types of neurosecretory-like cells. b The central neuropil area of the spine ganglion with nerve processes of spine nerve passing through. c Synapses (arrows) between a neural process and processes of neurosecretory-like cells. d Basal body of a cilium in a neurosecretory cells. e Glial cells at the periphery of the ganglion. Abbreviations: gc – glial cell; n – neuron; nsc I – neurosecretory-like cell type I; nsc II – neurosecretory-like cell type II
	Fig. 12. Organization of the hyponeural peripheral nerves in A. kochii. Transmission electron microscopy. a Proximal muscle nerve. b, c Median hyponeural nerve. b Bundles of neuronal processes (asterisk) branching off the median hyponeural nerve and entering the oral intervertebral muscle. The inset in b shows a neuromuscular synapse (arrow). c Processes of neurosecretory-like cells passing through an opening in the basal lamina. Abbreviations: bl – basal lamina; gc – glial cell; il – intervertebral ligament; m – myocyte; np – neuronal processes
	Fig. 13. Organization of the mixed peripheral nerves and ganglia in A. kochii. Transmission electron microscopy. a, b Aboral mixed nerve. c–e Lateral juxtaligamental node. f–h Oral juxtaligamental node. a Neuronal cell body in the wall of the arm coelom. b Neurosecretory-like cell in the wall of the arm coelom. c Low magnification view of the lateral juxtaligamental node. d Coelomic epithelial cell separating neurosecretory cells from the lumen of the coelom. e Synapse (arrow) between an axon and a process of a neurosecretory cell in the neuropil area of the lateral juxtaligamental node. f General view of the oral juxtaligamental node. g Processes of neurosecretory-like cells leaving the oral juxtaligamental node and entering the collagenous connective tissue. h Synapse (arrow) between an axon and a process of a neurosecretory-like cell. Abbreviations: ce – coelomic epithelial cell; cl – lumen of the coelom; ec – epineural canal; ee – epineural epithelium; en – ectoneural neuroepithelium; n – neuron; np – neural processes; nsc – neurosecretory cell
	Fig. 14. Synaptotagmin B (SynB) in the nervous system of the arm in O. brevispinum. Whole-mount preparation. Maximum intensity Z-projection of a confocal stack. a Low-magnification view of two segments of the radial nerve cord and associated peripheral nerves. a’ and a” show high-magnification views of the corresponding boxed areas in a. Abbreviations: om – oral intervertebral muscle; pg – podial ganglion; rnc – radial nerve cord; sn – spine nerve. Arrowheads in a” show neuronal cell bodies
	Fig. 15. Synaptotagmin B (SynB) in the nervous system of the arm in O. brevispinum. Longitudinal sections. a Radial nerve cord stained with the anti-SynB antibody and with the glial marker ERG1. The SynB staining is shown by itself in a’. The inset in a shows a low-magnification view of the radial nerve cord. b Aboral intervertebral muscle and ligament. Note the immunopositive bundles of nerve fibers in the muscle (arrows). c Detailed view of innervation of the aboral muscle by SynB-immunopositive neurons. d Innervation of the intervertebral ligament. Abbreviations: am – aboral muscle; en – ectoneural neuroepithelium; hn – hyponeural neuroepithelium; il – intervertebral ligament. Arrowheads in a, a’ and d how the cell bodies of SynB-immunopositive neurons
	Fig. 16. Distribution of the neuropeptide GFSKLYFamide (GFS) in the arm nervous system of O. brevispinum. a and a’ Low magnification view of a RNC segment. Whole-mount specimen, Z-projection of a confocal stack. b and c A pair of stereotypically positioned neurons in two different arm segments. Whole-mount specimen, volume-rendered confocal stack. d and d’ A branch of the longitudinal immunopositive tract contributing to the podial nerve. e and e’ Longitudinal section through the ectoneural neuroepithelium of the RNC. e shows triple staining with the anti-GFS antibodies, the ERG1 glial marker and the DRAQ5 nuclear stain, whereas e’ shows only the GFS-positive cells in a separate channel. f and f’ Cross section through the ectoneural epithelium of the RNC. f shows staining with the anti-GFS antibodies, ERG1 antibodies, and DRAQ5 nuclear dye, whereas f’ shows only anti-GFS immunostaining in a separate channel. Abbreviations: ec – epineural canal; pg – podial ganglion; open arrowhead – longitudinal immunopositive tracts; asterisk – median network of immunopositive fibers between the longitudinal tracts; filled arrowheads – a pair of stereotypically positioned neurons; filled arrow – a bundle of processes contributing to the podial nerve; open arrow – a bipolar neuron
	Fig. 17. ELAV in the arm nervous system of O. brevispinum. a and a’ Low magnification view of a RNC segment. Whole-mount specimen. Z-projection of a confocal stack. b and b’ Low-magnification view of a longitudinal section of the RNC. The insets show higher magnification of the proximal muscle hyponeural nerve. c and c’ Cross section of the RNC at the ganglionic swelling level. d – d”’ High-magnification view of a longitudinal section of the RNC showing that there is no co-labeling of the same cells with the ERG1 and anti-ELAV antibodies. Abbreviations: en – ectoneural neuroepithelium; hn – hyponeural neuroepithelium; pd – podium; pmn – proximal muscle hyponeural nerve; rnc – radial nerve cord
	Fig. 18. Expression of the transcription factor Brn1/2/4 in the radial nerve cord (RNC) of O. brevispinum. a and a’ Whole-mount preparation showing the oral view of two RNC segments. Maximum intensity Z-projection of a confocal stack. b A low-magnification view of a sagittal section through the RNC. c–c”’ High-magnification view of a longitudinal section of the RNC co-labeled with anti-Brn1/2/4 antibody and the ERG1 glial marker
	Fig. 19. Acetylated tubulin in the radial nerve cord (RNC) of O. brevispinum. a Low-magnification aboral view. Maximum intensity Z-projection of a confocal image stack. b High-magnification view of neuronal fibers in the RNC. Arrowheads indicate commissural bundles crossing into the contralateral regions of the RNC. c Sagittal section through the ganglionic swelling of the RNC. Dual labeling with anti-acetylated tubulin (magenta) and anti-ELAV (green) antibodies. The nuclei are stained with DRAQ. Note a “giant” neuron (gn) in the distal region of the ganglionic region co-labeled with ELAV and acetylated tubulin. Abbreviations: gn – “giant” neuron; pmn – proximal muscle nerve; rnc – radial nerve cord; sn – spine nerve
	Fig. 20. Acetylated tubulin in the peripheral nerves of the arm in O.brevispinum. a Cross section showing the radial nerve cord, horizontal intermuscular hyponeural nerve, and lateral juxtaligamental node. b Longitudinal section showing the oral juxtaligamental node and the bundles of immunopositive processes that originate from this ganglion and innervate the adjacent collagenous connective tissue (arrowheads). c Longitudinal section showing immunopositive neural processes (arrows) innervating the aboral muscle. d Cross section through the podial ganglion and spine nerves. sn. Abbreviations: am – aboral muscle; hin – horizontal intermuscular hyponeural nerve; ljn – lateral juxtaligamental node; ojn – oral juxtaligamental node; om – oral muscle; pd – podium; pg – podial ganglion; pmn – proximal muscle nerve; rnc – radial nerve cord; sn – spine nerves

References [+] :

Astley, Getting around when you're round: quantitative analysis of the locomotion of the blunt-spined brittle star, Ophiocoma echinata. 2012, Pubmed, Echinobase