Gravagna NG , Knoeckel CS , Taylor AD , Hultgren BA , Ribera AB .

P30 NS048154 NINDS NIH HHS , P30 NS048154-05 NINDS NIH HHS , R01 NS025217-19 NINDS NIH HHS , R01 NS25217 NINDS NIH HHS , T32 HD041697 NICHD NIH HHS , T32 HD041697-08 NICHD NIH HHS , R01 NS025217 NINDS NIH HHS

	Figure 1. Anti-Xenopus Kv2.2 specifically recognized both heterologously expressed and native Kv2.2. Protein extracts from Xenopus laevis adult brain tissue (brain), untransfected HEK cells (–), HEK cells transfected with Kv2.2 (2.2), and HEK cells transfected with Kv2.1 (2.1) were electrophoretically separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with either Kv2.2 antibody (left lanes, Kv2.2) or preblocked Kv2.2 antibody (right lanes, +peptide). Numbers on the left indicate the molecular weights of corun standards.
	Figure 2. In the spinal cord, Kv2.2 immunoreactivity was detected during early stages of postmitotic neuronal differentiation. Transverse cryostat (18 μm) sections of St 20–50 Xenopus embryos and tadpoles were incubated with the Kv2.2 antibody or preblocked Kv2.2 antibody (see, e.g., I). Sections were counterstained with Hoechst (blue). In all panels, dorsal is upward. cc, Central canal; rp, roof plate; fp, floor plate. A: At St 20, Kv2.2 immunolabeling was comparable to or barely above background. B–D: Kv2.2 immunoreactivity in the St 32 (B) and 37 (D) resembled a camera lucida image of a retrogradely labeled motor neuron (C, arrow) in the spinal cord of a St 35/36 Xenopus embryo (Roberts and Clarke,1982; adapted with permission). Comparison of the motor neuron cell body in C with the immunoreactive signals in B and D suggested that Kv2.2-immunoreactive protein localized to both the somata and the dendrites of primary spinal motor neurons. E–H: Between St 41 and St 50, the Kv2.2 immunopositive region of ventrolateral spinal cord progressively expanded, consistent with growth of neuronal processes. I: Preincubation of the Kv2.2 antibody with Kv2.2 peptide (see Materials and Methods) blocked the immunoreactive signal. J: The percentage of spinal cord cross-sectional area positive for Kv2.2 immunoreactivity steadily increased between St 20 and St 50. For each stage, 2–10 sections were examined. *P < 0.05 vs. St. 50, **P < 0.001 vs. St. 50. Scale bars = 50 μm.
	Figure 3. In the spinal cord, acetylated α-tubulin and syntaxin colocalized with the majority of Kv2.2 immunolabeling. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody and the acetylated α-tubulin (aat) or syntaxin (syn) monoclonal antibodies. In the merged images of C,F,I,L, the Hoechst counterstain is shown (blue). A–C: At St 41, Kv2.2 (A, green) and aat (B, red) were both present in the lateral regions of the spinal cord, as well as in peripheral motor axons in the ventral roots (C, arrow). A merged image (C) highlights the close apposition of Kv2.2 immunolabeling to that of aat. D–F: At St 41, Kv2.2 (D, green) and syn (E, red) predominated in lateral regions of the spinal cord. Syn labeling (E) was more restricted to the lateral margins, whereas Kv2.2 immunolabeling was detected dorsally near the roof plate (rp) as well as processes surrounding medial portions of the cord (F). G–I: At St 44, Kv2.2 (G, green) and aat (H, red) persisted in lateral regions of the spinal cord. However, in comparison with St 41 (A–C) Kv2.2 (G, green) and aat (H, red) were now present in more medial regions and continued to show extensive colocalization. J–L: At St 44, Kv2.2 (J, green) and syn (K, red) persisted together in lateral spinal cord, but Kv2.2 protein was also detected without syn in more medial regions (L). cc, Central canal; fp, floor plate; nc, notocord; rp, roof plate. Scale bar = 20 μm.
	Figure 4. The intensity of Kv2.2 immunofluorescence in the spinal cord correlated with that of some but not all markers examined. A–G: For individual confocal sections, we constructed scatterplots that showed, for each pixel, the intensity of Kv2.2 immunofluorescence (y-axis) and that of the marker being compared (x-axis): aat (A, St 41; B, St 50), syn (C, St 41; D, St 50), HNK-1 (E, St 44), 3A10 (F, St 44), and actin (G, St 50). The frequency of each combination of Kv2.2 and marker fluoresence intensities is indicated in pseudocolor (H). I: Pearson correlation coefficients indicated colocalization between Kv2.2 and aat, syn and HNK-1 but not 3A10 or actin.
	Figure 5. In the developing spinal cord, 3A10 and HNK-1 colocalize, to an extent, with the Kv2.2 immunolabeling. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody and 3A10 or HNK-1 mouse monoclonal antibodies. Antibody labelings were detected using goat anti-rabbit Alexa 488 (green; Kv2.2) and goat anti-mouse Alexa 568 (red) secondary antibodies. Sections were counterstained with Hoechst (blue). A–C: At St 44, Kv2.2 (A, green) was detected in lateral portions of the spinal cord that contain developing neuronal processes. 3A10 (B, red), a marker of commissural interneurons, showed a more restricted expression in lateral spinal cord in comparison with Kv2.2 (C). Moreover, crossing axons (asterisks, inset) that were 3A10 positive did not also show Kv2.2 immunoreactivity. D–F: At St 44, Kv2.2 (D, green) and HNK-1, (E, red) a marker for glycosylated neural cell adhesion molecules, predominated in lateral regions of the spinal cord and also decorated the plasma membranes of neuronal cell bodies. HNK-1 labeling appeared by itself (F, arrowheads) as well as colocalized with Kv2.2 immunoreactivity (F, arrows) in the medial cell body region. cc, Central canal; fp, floor plate; nc, notocord; rp, roof plate. Scale bar = 20 μm.
	Figure 6. Kv2.2 protein was present early in the developing retina. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody or preblocked Kv2.2 antibody (inset). Sections were counterstained with Hoechst (blue). A,B: In the retina of St 41 embryos, Kv2.2 immunoreactivity localized to the inner and outer plexiform layers (ipl, opl), axons in the ganglion cell layer (gcl), and somata of the photoreceptor layer (prl). Kv2.2 was also present in the optic nerve (on) and occasional cell bodies and processes within the inner nuclear layer (inl). C: In the St 44 retina, Kv2.2 protein was present in the ganglion cell layer, inner and outer plexiform layers, photoreceptor layer somata, and a subset of somata and processes within the inner nuclear layer. D,E: In the St 47 retina, Kv2.2 protein showed a distribution similar to that found at St 44. Inset: Preincubation of the Kv2.2 antibody with Kv2.2 peptide blocked the immunoreactive signal. F: In the stage 50 retina, the Kv2.2 protein displayed less intense immunoreactivity in the inner plexiform and ganglion cell layers in comparison with previous stages. In other regions, the pattern of immunolabeling was similar to that of earlier stages. Scale bars = 20 μm in A–C,E,F; 200 μm in D..
	Figure 7. In the retina, Kv2.2 immunolabeling colocalized with α-tubulin but not F-actin. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody or preblocked Kv2.2 antibody (inset) and aat mouse monoclonal or fluorescently labeled phalloidin to detect F-actin. In some sections, nuclei were counterstained with Hoechst (A–F, blue). A–C: At St 47, Kv2.2 (A, green) was abundant in synaptic layers (opl, ipl) and the cell bodies of the photoreceptor layer (prl). Less abundant labeling was also detected within the inner nuclear layer (inl) and ganglion cell layers (gcl). Aat (B, red) localized to the ipl and opl, the inl, the gcl, and optic nerve (on). A merged image (C) illustrates the close apposition of aat and Kv2.2 immunolabeling, specifically in the ipl, opl, inl, and gcl. D–F: At St 47, Kv2.2 (D, green) was present in the prl and both the ipl and the opl. F-actin (E, red) was present in the prl, ipl, opl, and lens (le). F: In contrast to aat (C), F-actin did not colocalize extensively with Kv2.2. Although Kv2.2 and F-actin were both present in the ipl and opl, their labeling patterns showed only occasional overlap in the merged image (F). G–I: A magnified view of the St 47 prl indicated that Kv2.2 (G, green) was detected in the cell bodies of photoreceptor cells and the opl. Inset: Preincubation of the Kv2.2 antibody with the Kv2.2 peptide block effectively eliminated Kv2.2 immunolabeling. F-actin (H, red) was present in both the inner and the outer segments of the photoreceptor cell as well as in the opl. A merged image (I) demonstrated that in some regions, notably the opl, Kv2.2 and actin were adjacent to each other. In photoreceptor cells, the majority of Kv2.2 protein and F-actin were detected in different locations. Scale bars = 50 μm in C (applies to A–F); 50 μm in I (applies to G–I).
	Figure 8. Kv2.2 protein was detected in the developing otic vesicle and the hindbrain. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody or preblocked Kv2.2 antibody (inset). Sections were counterstained with Hoechst (blue). A: At St 41, Kv2.2 immunoreactivity was present in the ventral lateral hindbrain and the medial epithelium of the otic vesicle (asterisk), where the sacculus forms. B: A magnified image of the boxed area in A illustrates the localization of Kv2.2 protein to the medial side of the otic epithelium. Kv2.2 protein was also detected in the innervating nerve (arrowhead) and the spiral ganglion cells (sg). C: At St 44, the Kv2.2 immunolabeling in the hindbrain had expanded both dorsally and medially. Kv2.2 protein was again detected in the saccular portion of the otic epithelium. D: A magnified view of the boxed area in C shows Kv2.2 protein again at the medial side of the otic epithelium. Also, Kv2.2 immunolabeling persisted in the innervating nerve and the spiral ganglion cells. Inset: Preincubation of the Kv2.2 antibody with the Kv2.2 peptide block effectively eliminated Kv2.2 immunolabeling. E: At St 50, Kv2.2 protein expression had continued both dorsal and medial expansion in the hindbrain. The Kv2.2 immunolabeling in the otic epithelium was again present. F: A magnified view of the boxed area in D illustrates a strong Kv2.2 presence in the otic epithelium as well as the spiral ganglion cells and innervating nerve. In the inset, Kv2.2 immunolabeling is detected around the membrane of the developing hair cell, with large cytosolic portions of the protein evident toward the apical portion of the soma. nc, Notochord; ov, otic vesicle; rp, roof plate; sg, spiral ganglion. Scale bars = 200 μm in A,C,E; 20 μm in B; 20 μm in F (applies to D,F), 5 μm for inset.
	Figure 9. In the otic vesicle, Kv2.2 was present at the apical end of hair cells. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody and aat monoclonal or fluorescently labeled phalloidin. Sections were counterstained with Hoechst (blue). A–C: At St 47, Kv2.2 (A, green) was abundant in the otic epithelium, at the apical side (ap) of developing hair cells. Kv2.2 was also present along the long axis of the hair cells flanking the soma and along the innervating nerve (arrowheads). aat (B, red) marked the kinocilia of the hair cells as well as the innervating nerve and portions of the soma. A merged image (C) illustrates the localization of the Kv2.2 protein at the base of the kinocilia at the apical end of the hair cells. Kv2.2 and aat colocalized in the innervating nerve. D–F: At St 47, Kv2.2 (D, green) continued to be present apically, along the long axis of hair cells, and in the innervating nerve. F-actin (E, red) was present in the hair cell stereocilia. A merged image (F) demonstrates the localization of the Kv2.2 immunolabeling at the base of the actin-containing stereocilia. G–I: At St 50, Kv2.2 immunolabeling (G, green) persisted at the apical side of the hair cells and along the long axis flanking the soma. aat (H, red) marked the hair cell kinocilia and the innervating nerve. A merged image (I) shows strong Kv2.2 immunolabeling at the base of kinocilia. J–L: At St 50, Kv2.2 immunolabeling (J, green) was detected in the apical portion of hair cells. F-actin (K, red) marked the hair cell stereocilia. A merged image (L) displays the localization of Kv2.2 protein at the base of the stereocilia. ap, Apical membrane; bl, basolateral membrane; kc, kinocilium; stc, stereocilia. Scale bars = 20 μm.
	Figure 10. Kv2.2 immunoreactivity was present in the developing olfactory system. Transverse cryostat (18 μm) sections were incubated with the Kv2.2 antibody or peptide-blocked Kv2.2 antibody (inset) and aat or fluorescently labeled phalloidin to detect F-actin. Sections were counterstained with Hoechst (blue in A,C). A: At St 47, Kv2.2 (green) was present in the developing nares, throughout the dense cellular layer, and distally near the principal cavity (asterisk). F-actin (red) was present in the microvilli, proximal to the Kv2.2 imunolabeling near the principal cavity (asterisk). B: A peptide block (inset) effectively eliminated Kv2.2 immunolabeling present in the St 47 retina. C: At St 50, Kv2.2 (green) immunolabeling in the developing nares was similar to but stronger than that observed at St 47. D: At St 50, aat (red) localized to the cilia and ventral portions of the cellular area of the nares. As was true in other tissues, Kv2.2 (green) colocalized with aat (yellow) in the cilia and cellular region. Scale bar = 50 μm.
	Figure 11. In culture, Kv2.2 protein colocalized with α-tubulin and was present in long processes. In cultured cells, Kv2.2 immunolabeling was detected in neuronal cell bodies and axons, as well as in myocytes and undifferentiated cells. Cells were cultured from dissociated neural plates of St 15–17 Xenopus embryos. Cultures were incubated with the Kv2.2 and aat antibodies. A–C: Kv2.2 (A, green) was present in a myocyte (myo), an undifferentiated cell (un), and a neuron (white outline). The Kv2.2 protein was found in the cell body, process, and terminal of the differentiated neuron (white outline). aat (B, red) localized to the cell body, process, and terminal of the neuron; the terminal contacted the myocyte. A merged image (C) supported colocalization of Kv2.2 and aat immunolabeling in the neuronal cell body, process, and terminal. D–F: Kv2.2 (D, green) was present in the cell body and long process of a differentiated neuron. aat (E, red) was also detected in the cell body and long process of the neuron. A merged image (F) demonstrated strong colocalization of Kv2.2 and aat immunolabeling. Scale bar = 20 μm.
	Figure 12. In culture, a majority of Kv2.2 protein colocalized with α-tubulin. Neurons in culture were fixed, immunolabeled for Kv2.2 (green) and aat (red), and imaged by confocal microscopy. A: Kv2.2 (green) and aat (red) immunolabeling were present in the long process and cell body of a neuron in culture. B: For the image in A, a scatterplot showed the intensity of Kv2.2 immunofluorescence (y-axis) and that of aat immunofluorescence (x-axis) for each pixel. The lower left corner (region 0) contains pixels that were excluded from the analysis, because they were below threshold (background) levels. On the basis of the Kv2.2 and aat immunofluorescence intensities, the remainder of the pixels are grouped into one of three regions: 1, aat immunofluorescent with little to no Kv2.2 immunofluorescence; 2, Kv2.2 immunofluorescent with little to no aat immunofluorescence; 3, immunofluorescent for both Kv2.2 and aat. C: The neuron shown in A is presented as a masked image according to regions in B: region 1 (green), region 2 (red), region 3 (yellow). D: For the individual lines drawn through the neuron in A, profiles of the Kv2.2 and aat immunofluorescence intensities as a function of distance are plotted. In each case, the aat signal either followed the Kv2.2 signal closely or was not present. Scale bar = 10 μm.

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