Sakaguchi DS et al. (1989), Growth cone interactions with a glial cell line...

XB-ART-26669

Dev Biol 1989 Jul 01;1341:158-74. doi: 10.1016/0012-1606(89)90086-9.

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Growth cone interactions with a glial cell line from embryonic Xenopus retina.

Sakaguchi DS , Moeller JF , Coffman CR , Gallenson N , Harris WA .

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We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.

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Species referenced: Xenopus laevis
Genes referenced: ag1 fn1 gopc mt-co2 nob1 prss1 tes vim zic1
???displayArticle.antibodies??? Glia Ab1 Muller Glia Ab1 Ncam1 Ab2 Nervous Ab2 Neuronal Ab2 Neuronal Ab3

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	FIG. 1. Antibody labeling of XRl cells by XAG2 (A, B) and XAN3 (C, D). (A, C) Phase photomicrographs of the fluorescence photographs in B and D. (A, B) mAb XAG2 labels XRl cells. In contrast, XAN3 does not label the nonneuronal XRl cells (C, D). Calibration bar, 50 pm
	FIG. 2. Immunofluorescence labeling of Xenopus tadpole tissue sections with mAb XAGB. (A, C, E) Nomarski images of the fluorescence photographs in B, D, and F, respectively. (A, B) Section of retina labeled with XAG2. In B one sees the fluorescent processes of Miiller cells (arrows) as they stretch toward the brightly labeled ILM. Their cell bodies appear to be labeled in the INL, whereas the somas of the retinal ganglion cells are unlabeled. Abbreviations: OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cell layer; ILM, inner limiting membrane. (C, D) Section of optic nerve labeled with XAGZ. (D) XAG2 labels the sheath of the optic nerve as well as lightly labeling astrocytic processes among the optic fibers. (E, F) Section of tadpole optic tectum labeled with mAB XAGZ. Monoclonal antibody XAG2 labels the ventricular zone and pial surface of the tectum as well as a multitude of fine radially oriented processes. In addition, larger radially oriented profiles, probably blood vessels, are also labeled with this antibody. Calibration bar, 50 pm.
	FIG. 3. Immunofluorescence labeling of fixed and permeabilized XRl cells. (A) R5 monoclonal antibody. (B) anti-vimentin. (C, D) Double-labeling with anti-GFAP (C) and XAG2 (D), respectively. Calibration bar, 50 pm.
	FIG. 4. Immunofluorescence labeling of tissue sections with monoclonal antibody R5. (A, C, E) Nomarski images of fluorescence photographs B, D, and F, respectively. (A, B) R5 labels the Mtiller cells in the Xenopus retina, in particular their endfeet. (C, D) In the optic nerve R5 labels thin processes among the optic fibers but does not label either the optic fibers or the sheath. (E, F) R5 labels radial processes, but not cell somata in the tadpole tectum. Abbreviations as in Fig. 2.
	FIG. 5 Nel collagen ‘(0 confine t hem explants cull bar, 100 pm. uril ;e outgrowth from retinal explants cultured on collagen (A), on a confluent layer of XRl cells (B), and on XRl COII lditioned In e ach photomicrograph the explanted retina is located at the left of the photograph. (A) Neurites growing from the I explant seb VC !s to flat cells which grow out from the explanted tissue and seldom venture onto the collagen substrate. (B, C) N( euri tes from :urf :d on XRl cells (B) or on collagen conditioned by XRl cells (C) show extensive outgrowth away from the exl Jlant. Cal libration
	FIG. 6. (A) Neurite extension onto various noncellular substrates. The vertical axis shows the percentage of retinal explants with neurites extending onto the substrate after 8 days in culture. The different substrates and conditions are on the horizontal axis. C, collagen (number of explants, n = 46); CPO, collagen-coated with polyornithine (n = 42); CON A, conconavalin A (n = 38); fibronectin at 1 pg/ml (n = 20), 10 rg/ml (n = 20), and 50 pg/ml (n = 20); Laminin at 1 fig/ml (n = 24), 10 pg/ml (n = 57), and 40 @g/ml (n = 33). (B) Neurite outgrowth onto various cell line derived substrates. CM, collagen with XRl conditioned medium (n = 56); XRl, XRl conditioned collagen (n = 125); XRS, XR8 conditioned collagen (n = 24); XR9, XR9 conditioned collagen (n = 99); A6, A6 conditioned collagen (n = 79); XTC, XTC conditioned collagen (n = 48).
	FIG. 7. Scanning electron micrographs of Xenopus retinal neurons cultured on various substrates. (A) Neurons grown on concanavalin A substrates usually have flattened growth cones and extensive neurite extensions (arrow). (B, C) The growth cones of neurons cultured on a confluent layer of XRl cells (B) or on collagen conditioned by XRl cells (C) are less flat and their neurites possess fewer branches, resembling those found in viva. Calibration bar, 10 pm
	FIG. 8. Neurite outgrowth on XRl conditioned collagen substrates. (A) Chemical extractions and enzymatic treatments. CONT., untreated control (n = 40); TRIT., substrate treated with 3% Triton X-100 (n = 56); NH,OH, substrate treated with 0.02 M NH,OH (n = 40); NEUR., substrate treated with 0.2 units/ml neuraminidase (n = 38); HEP., substrate treated with 5 units/ml heparinase (n = 42); TRYP., substrate treated with trypsin (n = 32); COLL., substrate treated with collagenase (n = 45). (B) Neurite outgrowth on XRl conditioned collagen substrates treated with antibodies. Untreat. Cont., untreated control (n = 48); mAb XAG2, substrate treated with monoclonal antibody XAGZ (n = 42); anti-N-CAM, substrate treated with 0.5 mg/ml anti-N-CAM antibody (n = 48); INO, substrate treated with 0.8 mg/ml IN0 antibody (n = 30); NOB1 IgG, affinity-purified IgGs from polyclonal antiserum produced in a rabbit immunized with XRl cells and membrane (n = 32). Cont. IgG, IgG fraction of preimagainst mune sera (n = 32); NOB1 Fab, Fab fragments from NOB1 IgGs (n = 68); NOB1 (-)Fab, Fab fragments from affinity-depleted NOB1 (n = 42); Cont. Fab, Fab fragments from preimmune IgGs (n = 47). (C) Neurite outgrowth on laminin. CONT., laminin (10 pg/ml) (n = 57); anti-LN, laminin treated with anti-laminin polyclonal sera (n = 44); NOB1 Fab, laminin treated with NOB1 Fab fragments (n = 24). Neurite outgrowth on XRl conditioned collagen substrates. CONT., XRl conditioned collagen (n = 125); NOB1 Fab, XRl conditioned substrates treated with NOB1 Fabs (n = 68); anti-LN, XRl conditioned substrate treated with anti-laminin (n = 40
	FIG. 9. NOB1 antibody labeling. (A, B) NOB1 labeling of XRl cells (A) and A6 cells (B). (C, D) Labeling of XRl cells (C) and A6 cells (D) using NOB1 antiserum preabsorbed against intact A6 cells. (E) Immunodepleted NOB1 labeling of an XRl conditioned collagen substrate. (F) Scanning electron micrograph of an XRl conditioned collagen substrate. Calibration bar, A-E, 25 pm; F, 5 pm.
	FIG. 10. Immunoblot analysis using immunodepleted NOB1 antiserum (lanes 1-5) and anti-laminin antibodies (lanes 6-8). Lane 1 (XRl), XRl whole cell homogenate sample. Lane 2 (A6), A6 whole cell homogenate sample. Lanes 3 and 6 (C-XRl), Triton-treated XRl conditioned collagen sample. Lanes 4 and 7 (C-A6), Triton-treated A6 conditioned collagen sample. Lanes 5 and 8, laminin sample. Molecular weight markers are at the left (kDa).