Mashanov VS et al. (2009), The central nervous system of sea cucumbers (Ec...

ECB-ART-41190

Front Zool 2009 Jun 18;6:11. doi: 10.1186/1742-9994-6-11.

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The central nervous system of sea cucumbers (Echinodermata: Holothuroidea) shows positive immunostaining for a chordate glial secretion.

Mashanov VS , Zueva OR , Heinzeller T , Aschauer B , Naumann WW , Grondona JM , Cifuentes M , Garcia-Arraras JE .

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BACKGROUND: Echinoderms and chordates belong to the same monophyletic taxon, the Deuterostomia. In spite of significant differences in body plan organization, the two phyla may share more common traits than was thought previously. Of particular interest are the common features in the organization of the central nervous system. The present study employs two polyclonal antisera raised against bovine Reissner''s substance (RS), a secretory product produced by glial cells of the subcomissural organ, to study RS-like immunoreactivity in the central nervous system of sea cucumbers. RESULTS: In the ectoneural division of the nervous system, both antisera recognize the content of secretory vacuoles in the apical cytoplasm of the radial glia-like cells of the neuroepithelium and in the flattened glial cells of the non-neural epineural roof epithelium. The secreted immunopositive material seems to form a thin layer covering the cell apices. There is no accumulation of the immunoreactive material on the apical surface of the hyponeural neuroepithelium or the hyponeural roof epithelium. Besides labelling the supporting cells and flattened glial cells of the epineural roof epithelium, both anti-RS antisera reveal a previously unknown putative glial cell type within the neural parenchyma of the holothurian nervous system. CONCLUSION: Our results show that: a) the glial cells of the holothurian tubular nervous system produce a material similar to Reissner''s substance known to be synthesized by secretory glial cells in all chordates studied so far; b) the nervous system of sea cucumbers shows a previously unrealized complexity of glial organization. Our findings also provide significant clues for interpretation of the evolution of the nervous system in the Deuterostomia. It is suggested that echinoderms and chordates might have inherited the RS-producing radial glial cell type from the central nervous system of their common ancestor, i.e., the last common ancestor of all the Deuterostomia.

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Genes referenced: gli3 LOC100887844 LOC115919910 LOC752072

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	Figure 1. Simplified diagrams of the anatomical organization of the holothurian nervous system. A. Lateral view of an adult sea cucumber, oral end to the right. The dashed line corresponds to the cross section at the mid-body level shown in (B). C. Higher magnification of the boxed area in (B) showing the relationship between the radial nerve cords and other radial organs. ce, coelomic epithelial lining of the body cavity; ctl, connective tissue layer of the body wall; en, ectoneural part of the radial nerve cord; hn, hyponeural part of the radial nerve cord; lm, longitudinal muscle band; nr, nerve ring; rc, radial canal of the water-vascular system; rnc, radial nerve cord; tn, tentacular nerve. Not to scale.
	Figure 2. Overview of the central nervous system (E. fraudatrix) on paraffin sections stained with Heindenhein's azan. A. Transverse section showing the radial nerve cord (rnc) and other radial organs, including the longitudinal muscle band (lm), radial water-vascular canal (rc), and the hemal lacuna (h). B. Higher magnification of the radial nerve cord shown in (A). Basal processes of glial supporting cells can be clearly seen in the neuroepithelia (arrowheads). C. Sagittal section through the pharyngeal bulb, showing the nerve ring. en, ectoneural neuroepithelium. The oral end of the animal is to the right. cec, circular epineural canal of the nerve ring; ec, epineural canal; en, ectoneural neuroepithelium; hc, hyponeural canal; hn, hyponeural neuroepithelium; re, non-neural roof epithelium; t, hydrocoelic lining of the tentacle. Scale bars = 100 μm in (A) and (B), 50 μm in (C).
	Figure 3. RS-like immunoreactivity in the ectoneural part of the radial nerve cord, transverse sections. Dashed line shows the border between the ectoneural (en) and hyponeural (hn) parts of the radial nerve cord. A, B Low-magnification view of the radial nerve cord of H. glaberrima showing (A) Immunofluorescent microscopy with the AFRU antiserum (green) and nuclei stained with Hoechst (blue) and (B) the immunofluorescence combined with a Hoffman DIC image. Note the intensely labelled immmunoreactive material overlaying the apical surface of the ectoneural neuroepithelium. C. High magnification view of the radial nerve cord of H. glaberrima with a locally dilated lumen of the epineural canal (ec). AFRU-positive labelling (green) is clearly associated with the apical surface of the ectoneural neuroepithelim (en) and with the non-neural epineural roof epithelium (re). Note also immunoreactive cell bodies (arrowheads) and dotted labelling in the neural parenchyma. The inset shows a high magnification view of an immunoreactive cell body within the ectoneural parenchyma. D, E. Peroxidase-antiperoxidase immunohistochemistry with the RS-K10 antiserum on paraffin sections of the radial nerve cord of E. fraudatrix. D. Labelling associated with the apical region of the ectoneural neuroepithleium and with epineural roof epithelium (re). Note the deposition of the immunopositive material on the apical surface of the neuroepithelium (arrows). E. Labelled cell bodies (arrowheads) and processes (double arrowheads) within the ectoneural parenchyma. Scale bars = 100 μm in (A) and (B), 50 μm in (C), 10 μm in (C inset), (D), and (E).
	Figure 4. RS-like immunoreactivity in the hyponeural part of the radial nerve and in the nerve ring. Dashed line shows the basal border of the hyponeural (hn) part of the radial nerve cord. A. Peroxidase-antiperoxidase immunohistochemistry with the RS-K10 antiserum on a transverse section of the radial nerve cord of E. fraudatrix showing immunopositive cells (arrowheads) in the hyponeural neuroepithelium (hn) and in the non-neural roof epithelium (re). Note that there is no immunoreactive material neither on the apical surfaces of the epithelia, nor in the lumen of the hyponeural canal (hc). B. Dotted labelling (green) in the lateral region of the hyponeural neuroepithelium (asterisk) of H. glaberrima visualized by immunostaning with the AFRU antiserum. Nuclei are stained with Hoechst (blue). C. Sagittal section through the pharyngeal bulb (the oral end of the animal is to the right) of H. glaberrima showing the intense AFRU-positive immunoreactivity (arrows) associated with the apical surface of the ectoneural neuroepithelium (en) and with the epineural roof epithelium. D. High magnification view of a region of the nerve ring with a locally dilated lumen of the circular epineural canal (cec) showing the AFRU-positive labelling (green) in the apical region of the ectoneural neuroepithelium and in the epineural roof epithleium (re). Nuclei are stained with Hoechst (blue). Scale bars = 10 μm in (A), 50 μm in (B), 15 μm in (C), 20 μm in (D).
	Figure 5. Ultrastructural immunolocalization of the RS-like material in the holothurian nervous system. For an explanation of the difference in grain size (smaller grains in A, B inset, C and D and larger grains in B, E, and F), refer to the Methods section. A. Apical surface of a supporting glial cell of the ectoneural neuroepithelium covered with the RS-K10-positive secretion. Note also labelling of the electron-translucent vesicles in the apical cytoplasm of the cell. B. RS-K10-positive labelling of the apical surface of the epineural roof epithelium. The inset shows a labelled vacuole in the cytoplasm of a flattened glial cell. C, D. Apparent release of the immunoreactive material from secretory vacuoles into the lumen of the epineural canal by supporting glial cells (C) and cells of the roof epithelium (D), as revealed by immunogold reactions with the RS-K10 (C) and AFRU (D) antisera. E, F. Immunoreactive cell bodies (RS-K10-positive) within the ectoneural parenchyma. ec, epineural canal; if, bundle of intermediate filaments; v, vacuole. Scale bars = 1 μm in (A), (B), (F), 0.5 μm in (B inset), (C), (D), and (E).
	Figure 6. RS-like immunoreactivity in the epidermis of sea cucumbers. A. Peroxidase-antiperoxidase immunohistochemistry with the RS-K10 antiserum showing strong immunolabelling of the apical region of tentacle epidermis in E. fraudatrix. B. Immunofluorescent microscopy with the AFRU antiserum reveals intense immunolabelling (green) of the apical surface of the podial epidermis in H. glaberrima. Nuclei are stained with Hoechst (blue). C. Immunoelectron (RS-K10 antiserum) micrograph of an epidermial cell of E. fraudatrix showing strong labelling of the apical surface (double arrowhead) and less prominent labelling of the lateral surfaces (arrowhead) of the cell. er, cisternae of the endoplasmic reticulum. Scale bars = 50 μm in (A) and (B), 1 μm in (C).
	Figure 7. Negative control sections (see Methods). A. Peroxidase-antiperoxidase immunocytochemistry, transverse paraffin section of the radial nerve cord of E. fraudatrix. B. Immunofluorescent microscopy; transverse cryosection of the radial nerve cord of H. glaberrima. Nuclei are stained with Hoechst (blue). Dashed line shows the border between the ectoneural (en) and hyponeural (hn) parts of the radial nerve cord. C. Immunoelectron microscopy; the apical region of the ectoneural neuroepithelium of the radial nerve cord (E. fraudatrix). Note a single grain representing unspecific staining (arrowhead). ec, epinueral canal; re, non-neuronal roof epithelium. Scale bars = 20 μm in (A), 50 μm in (B), 5 μm in (C).

References [+] :

Adoutte, The new animal phylogeny: reliability and implications. 2000, Pubmed