Pollock NS et al. (2002), Expression of voltage-dependent potassium chann...

XB-ART-6463

J Comp Neurol 2002 Oct 28;4524:381-91. doi: 10.1002/cne.10401.

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Expression of voltage-dependent potassium channels in the developing visual system of Xenopus laevis.

Pollock NS , Ferguson SC , McFarlane S .

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Accumulating evidence suggests that voltage-dependent potassium (Kv) channels have important and varied roles in the development of neuronal and non-neuronal cell types. They have been implicated in processes such as proliferation, cell adhesion, migration, neurite outgrowth, and axon guidance. In this study, we used antibodies against several electrically active Kv channel alpha-subunits (Kv1-4) to describe the spatial and temporal expression patterns of Kv channel subunits in Xenopus laevis retinal ganglion cell (RGC) somata, axons, and growth cones. We found that RGCs express Kv1.3-, Kv1.5-, Kv3.4-, and Kv4.2-like subunits. Each subunit displayed unique cellular and subcellular distributions. Moreover, the expression patterns changed considerably over the major period of Xenopus retinal cell genesis and differentiation. Weak or no immunoreactivity was observed with antibodies against Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv3.2 subunits in RGCs or other retinal cell types. In support of our previous pharmacologic evidence implicating Kv channels in RGC axon outgrowth, we found that Kv1.5-, Kv3.4-, and Kv4.2-like proteins, but not Kv1.3-like subunits, are abundantly expressed in RGC growth cones.

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Species referenced: Xenopus laevis
Genes referenced: actb actl6a isl1 kcna1 kcna3 kcna4 kcnc4 kcnd2 kcnd3 slc12a3 zic1
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	Fig. 1. Voltage-dependent potassium 1.1 (Kv1.1)–like protein is expressed at low levels in the Xenopus retina. Immunolabelling of transverse sections through the eye and brain of a stage 40 Xenopus embryo was done with the anti-mouse Kv1.1 antibody (A,B) and the Kv1.1 antibody preincubated with a control Kv1.1 peptide antigen (C). Migrating neural crest cells are immunopositive (A), but only weak Kv1.1-like immunoreactivity is observed in the retina (B). D, dorsal; E, eye; Hb, hindbrain; NCC, neural crest cells; L, lens; OPL, outer plexiform layer; V, ventral. Scale bar 200 m for A and 100 m for B and C.
	Fig. 2. Voltage-dependent potassium 1.3 (Kv1.3)–like subunits are expressed in the Xenopus retina. Transverse sections through the Xenopus retina at different developmental stages were immunostained with a rabbit antibody against the C-terminus of the human Kv1.3 subunit. A: At stage 32, cells in the neuroepithelium of the retina and brain are immunopositive. B: At stage 37/38, retinal ganglion cells (RGCs) and their processes along the vitreal surface are labelled, as are the inner and outer plexiform layers (IPL and OPL) and the ciliary marginal zone (CMZ). C,E: By stage 40, the optic nerve head and optic nerve are brightly labelled. D: Almost no labelling is observed when the Kv1.3 subunit antibody is preincubated with a control Kv1.3 subunit peptide antigen. Similar low power sections are shown in C and D. Br, brain; D, dorsal; L, lens; NP, neuropil; on, optic nerve; onh, optic nerve head; PE, pigmented epithelium; RGCL, retinal ganglion cell layer; V, ventral; vs, vitreal surface. Scale bar 50 m for A, B, and E and 100 m for C and D.
	Fig. 3. Voltage-dependent potassium (Kv) subunit-like immunoreactivity in developing retinal ganglion cells (RGCs) and their axons. A: Double labelling of a stage 33/34 retina with anti-Kv1.3 (green) and mouse anti-bromodeoxyuridine (BrdU; red) antibodies shows that in the central retina Kv1.3-like immunopositive cells (arrows) are not BrdU positive. B: Double labelling (yellow) with antibodies against the rat Kv4.2 subunit and islet-1 at stage 33/34. The islet-1 antibody stains all RGCs (red), but the Kv4.2 antibody (green) labels only a subset of these islet-1–positive RGCs. C: Double labelling (yellow) of a stage 41 retina with a Muller glial marker (3CB2) and the anti- Kv4.2 antibody shows that radial cell processes in the central third of the retina are immunopositive (arrows) for both markers, whereas processes in the peripheral retina are immunopositive for the Kv4.2- like subunit only (arrowheads). By this stage the optic nerve head (onh), but not RGC somata, are Kv4.2-like immunopositive. D: Merged image of E and F shows colocalization of the Kv3.4 subunit and actin filaments in an RGC growth cone. E: Actin labelled with rhodamine phalloidin. F: The Kv3.4 antibody labels the axon and growth cone. IPL, inner plexiform layer; L, lens; OPL, outer plexiform layer; RGCL, RGC layer. Scale bar 100 m for A–C and 10 m for D–F.
	Fig. 4. Voltage-dependent potassium 1.5 (Kv1.5)–like subunits are expressed in the Xenopus retina. A: The stage 33/34 retina shows immunolabelling of retinal somata and the ciliary marginal zone (CMZ) by the Kv1.5 subunit antibody. B: In the stage 37/38 retina, the antibody immunolabels the CMZ and cells in the central retina, most notably cells in the retinal ganglion cell layer (RGCL). C,D: Fluorescent micrographs of a stage 40 retina at low and high power. C: Immunoreactivity is observed in somata in all three layers of the central region of the retina. D: RGCs are particularly well labeled, and immunostaining is no longer observed in the proliferating CMZ and is reduced in the lens (L). D, dorsal; Hb, hindbrain; INL, inner nuclear layer; ONL, outer nuclear layer; PE, pigmented epithelium; V, ventral. Scale bar 50 m for A, B, and D and 100 m for C.
	Fig. 5. Voltage-dependent potassium 3.4 (Kv3.4)–like subunits are expressed in the Xenopus retina. A: Membrane-associated labelling with an anti-Kv3.4 antibody of all retinal and lens (L) cells in a stage 32 Xenopus retina. Arrow points to retinal ganglion cell (RGC) axon labelling at the vitreal surface. B,C: A stage 33/34 retina at low (B) and high (C) power shows abundant labelling of RGC processes at the vitreal surface (vs). D: In a stage 37/38 retina, membrane-associated labelling of most retinal and lens cells is maintained, and staining in the synaptic layers appears (IPL and OPL, inner and out plexiform layers). E: Low magnification micrograph demonstrates Kv3.4 immunolabelling in the brain neuropil, the optic nerve, and retina at stage 40. CMZ, ciliary marginal zone; D, dorsal; Mb, midbrain; NR, neural retina; onh, optic nerve head; V, ventral. Scale bar 50 m for A, B, and D, 100 m for E, and 20 M for C.
	Fig. 6. Voltage-dependent potassium 4.2 (Kv4.2)–like subunits are expressed in the Xenopus retina. Transverse sections through Xenopus retinas at different developmental stages. A: Kv4.2-like immunoreactivity is first detectable at stage 24 in the retina with labelling in the basal lamina surrounding the eye primordium (arrowhead). B: At stage 28, additional labelling is observed in radially oriented fibres (arrowhead), processes along the vitreal surface of the retina (arrow), and cell bodies. C: Higher magnification picture demonstrates staining of processes along the vitreal surface (vs) in a stage 30 retina. Arrows point to immunolabelling of the basal lamina. D: At stage 37/38, and earlier at stage 35/36, radial cell processes and a subset of retinal ganglion cell and cells in the outer nerve layer (ONL) are immunopositive for the Kv4.2-like protein. D, dorsal; Di, diencephalon; Ep, eye primordium; IPL, inner plexiform layer; L, lens; RGCL, retinal ganglion cell layer; V, ventral. Scale bar 200 m for A and B and 100 m for C and D.
	Fig. 7. Voltage-dependent potassium (Kv) subunits are differentially expressed in developing Xenopus retinal ganglion cell (RGC) growth cones. Stage 24 eye primordia were grown in culture for 24–48 hours and labelled with various Kv subunit antibodies. A: Phase micrograph of the same RGC axons shown in B. Arrowhead points to a filopodia. B: The Kv1.3 channel antibody labels the axon abundantly, but not the body of the growth cone, nor the filopodia or lamellipodia. C: The Kv1.5 antibody labels the RGC axon and the entire growth cone, including the filopodia (arrowhead). D: The Kv4.2 antibody labels the RGC axon and growth cone in a punctate manner. Scale bar 16 m for A–D.
	Fig. 8. Voltage-dependent potassium (Kv) subunit antibodies recognize Xenopus proteins of the expected molecular weights. Western blots of Xenopus head tissue used different rabbit polyclonal antibodies against mammalian Kv subunits. Molecular weights are based on standards that were run at the same time as the sample tissue. All bands observed correspond to previously reported values for their respective Kv channels. Lane 1: The Kv1.3 antibody detects a single band with an approximate molecular weight of 60 kDa. Lane 2: Anti-Kv1.5 detects a prominent band of approximately 55 kDa. Lane 3: The Kv3.4 antibody reacts with a protein of approximately 115 kDa. Lane 4: The Kv4.2 antibody recognizes a doublet at approximately 71 kDa.