The RPE influences the ability of retinal ganglion cells to polarize in ectopic positions. (a) Head region of wild-type, morphant and mutant embryos, showing the distribution of pigment around the retina. Two examples of nok morpholino-injected embryos (0.17 pmoles per embryo) are shown. (b) Cryosection of the eye from a nok-/-, ath5:gap-gfp transgenic embryo, labeled with an anti-laminin 1 antibody (red). The bright-field image, inverted and pseudo-colored in blue, shows the distribution of the retinal pigment epithelium (RPE). Arrowheads indicate ath5:gap-gfp-positive retinal ganglion cells. D, dorsal; L, lateral; M, medial. Scale bar, 20 μm. (c) Transmission electron micrograph of the apical region of a nok mutant retina, showing the distribution of the RPE cells (dotted lines). Inset, a higher magnification of the boxed region, showing two apparent transverse-sectioned axons (Ax) at the RPE-free apical surface of the retina. Scale bars: low-magnification image, 5 μm; inset, 0.5 μm. (d) Transmission electron micrographs of the apical region of a wild-type (Wt) and a has-/- embryo. The RPE (blue arrows) is a very organized simple epithelium in the wild type, and it seems disrupted in the has mutant. Nevertheless, the picture does not show any actual gap between the RPE cells in the has-/-embryo. Red arrowheads indicate mitotic cells in a normal (apical) position in the wild-type, and in an ectopic position in the mutant. Scale bar, 5 μm. (e) Fluorescence intensity profiles made on confocal sections of 48 hpf ath5:gap-gfp transgenic embryos labeled with an anti-RPE antibody (Zpr-2). For the measurements a 20-pixel wide line was drawn along a radius of the retina as in (f). The intensity profile of the green channel (GFP) was plotted and the values were normalized (maximum intensity) and averaged for each embryo; the resulting plots were then normalized to each other (integrated intensity). To compare the profiles in relation to the distribution of the RPE, we positioned the line either in regions where zpr-2 immunoreactivity was detected (RPE) or not (NO RPE). For the nok mutants, only measurements of areas without detectable RPE were used. Measurements were made in three to ten different areas from one wild-type, five nok mutant, four nok morphant and three has mutant retinas. The inset picture shows a region of a wild-type retina, like those used for the measurements, which is aligned with the profile plot to show the correspondence with the retinal layers. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OFL, optic fiber layer; OPL, outer plexiform layer; PL, photoreceptor layer. (f) Optical sections of wild-type and mutant retinas of ath5:gap-gfp transgenic embryos labeled in red with the Zpr-2 antibody, which stains the RPE. The yellow rectangles show an example of the lines used for the measurements presented in (e). Scale bar, 25 μm.