A model illustrates the steps by which the Nok-mediated intact RPE plays its essential role in retinal development (See the first paragraph in the discussion section for a detailed explanation.). RC stands for postmitotic migrating retinal cells.

Retinal expression of wildtype Nok is not sufficient to rescue the retinal neuroepithelial polarity defect in nok^m520. A. Nok and ZO-1 expressed in the wildtype pt104 donor cells localize apically in the pt106 host retinas at 36 hpf (arrows). Arrowheads indicate the intact RPE as revealed by DIC imaging. B. Nok and ZO-1 expressed in the wildtype pt104 donor cells localize ectopically to the interior of the nok^m520 host retinas at 36 hpf (arrows). Arrowheads indicate the fragmented patchy RPE as revealed by DIC imaging. The tissue sections were oriented with the RPE on top and the lens at bottom.

The retinal epithelial polarity is recovered in pt106 at early stages of retinal development. A, B, C. As in wildtype (A), transgenic expression of Nok (red, arrowheads) in the RPE restores the apical localization of apical markers ZO-1(blue, arrows) and adherens junction associated actin bundles (green, arrows) in pt106 retina at 36 hpf (C). These apical markers localize ectopically to the interior of the retina in nok^m520 (arrows, B). Embryos were treated with PTU so that transgenic Nok expressed in the RPE would not be obscured by melanin pigmentation.

The nok gene is expressed in both the neural retina and the RPE. A, B. At 35 hpf, Nok (red, arrows) localizes to the vicinity of ectopic adherens junction clusters marked by ZO-1 (blue) and actin bundles (green) in the interior of the nok^m520 mutant retinal neuroepithelium. The lens is indicated by an asterisk. The dashed line in A demarks the boundary of the retina. B is shown at a higher magnification than that in A. C. A cell, dissociated from 72 hpf eyes, displays signals for both Nok (red) and RPE specific marker zpr2 (blue).

Sporadic wildtype RPE cells were not sufficient to rescue the retinal epithelial polarity. Wildtype pt104 donor cells (visualized by GFP, green) were transplanted into nok^m520 mutants. The localization of retinal M-phase nuclei (white arrows, blue TO-PRO staining for the nuclei) and the apical marker ZO-1 (red) were analyzed at 36 hpf. Near the regions that contained wildtype RPE cells (arrowheads), both wildtype retinal donor cells (red arrows) and host retinal cells displayed an identical polarity defect of interior localized M-phase nuclei and apical marker ZO-1 (red), as did the retinal cells that were distant from the wildtype RPE donor cells or in pure nok^m520 mutants (for statistical analysis, see text).

At early stages of embryonic retinal neurogenesis, cell divisions occur predominantly at the apical region of the retina in pt106 as in wildtype. A, B, C. The localization of M-phase nuclei in wildtype, nok^m520, and pt106 at 33 hpf, 42 hpf, and 53 hpf were revealed by phospho-histone 3 staining (red) or YO-PRO nuclear staining (green, for chromatin condensation). Arrows indicate that some nuclei in late cell division stages (anaphase and telophase) display weak or no PH3 staining. D. A chart to show the percentage of cell division at the apical region of the retina at different developmental stages in pt106, wildtype, and nok^m520. The numbers of nuclei in metaphase, anaphase, or telophase at each stage are presented in the table on the right, as determined by PH3 staining or by the morphological condensation of chromatin as revealed by YO-PRO staining.

The retinal lamination is largely restored in pt106. A. A JB4 histological analysis revealed a laminar cellular structure in the 5-dpf pt106 retinas. PCL, photoreceptor cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer; ON, optic nerve. B. Unlike in nok^m520 mutants, ganglion cells (zn8 staining in blue) and Lin7 positive cells (red) are positioned properly in pt106 as in wildtype at 4.5 dpf. C. Muller cells (red, CA staining for carbonic anhydrase) are also positioned in the inner nuclear layer in pt106 as in wildtype at 4.5 dpf. D. Double cones (red, zpr1 staining) localize to three cellular layers in pt106 at 4.5 dpf. Phalloidin staining for actin (green) highlights the nearly wildtype-like organization of the inner and outer interplexiform layers in pt106 (B, C, D).

The nok^m520 mutation causes aberrant retinal cell movements during retinal neurogenesis. In the retina, cellular stratification is reflected by the laminar position of the cell nuclei. To examine cellular movement in live embryos, we utilized the transgenic expression of Histone2A-GFP (Pauls et al., 2001) to visualize the cell nuclei. The positions of nuclei were examined and recorded under confocal microscopy every five minutes from 32 hpf to 33 hpf. To visualize the directions of nuclear movements, two images collected five minutes apart were displayed and superimposed in red and green (red for an earlier time point; green for a later time point). The five-minute interval was chosen because the majority of the nuclei did not move, so we could use them as reference points for precise superimposition of the red and green images. Thus, the nuclei that did not move over the five-minute period appeared yellow, whereas the nuclei that underwent apparent movements would display a color shift and the orientations of the color shift would illustrate the directions of the nuclear/cellular movements. The inset in A displays a magnified region showing a nucleus with an apparent color shift. The axes of the apparent red-green shifts (arrows) reveal a trend of radial nuclear/cellular movements in wildtype retina (A, C) and irregular movements in nok^m520 (B, D).

The disruption of retinal epithelial polarity in nok^m520 is correlated with the loss of RPE integrity in a tight temporal manner. A. An in situ hybridization analysis of wildtype and nok^m520 embryos with an antisense RNA probe against the zebrafish TYRP1 revealed the presence of an intact RPE structure in nok^m520 embryos at 24 hpf. The disruption of RPE integrity progressively worsens as retinal neurogenesis unfolds; arrows indicate the disrupted RPE regions in nok^m520 at 28 and 32 hpf. B. At 26 hpf, the polarity of the retinal epithelium is still largely unaffected by the nok^m520 mutation. Apical markers ZO-1 (blue, arrowheads) and M-phase nuclei (green, arrows) localize to the apical surface in both wildtype and nok^m520 mutant retinas. C. At 48 hpf in nok^m520, ectopic retinal ganglion cells (red, Zn8 staining, arrows) prefer to accumulate at the apical retinal regions adjacent to disrupted RPE areas (DIC imaging). Actin staining (green) is for the background morphology. D. At 36 hpf, the ectopic localization of M-phase nuclei (green, YO-PRO, arrows) and ZO-1 maker (red, arrows) in nok^m520 is not influenced by local presence or absence of the RPE patches (right two panels). The left two panels are controls for wildtype.

Transgenic expression of Nok in the RPE of pt106 is sufficient to rescue the patchy RPE defect caused by the nok^m520 mutation. A-F. The expression of transgenic Nok in pt106 (E, F) restores the RPE integrity to a level indistinguishable from that of wildtype RPE (A, B). The phenotypes of a curled body axis and a paracardiac edema are still present in pt106 (E) as in nok^m520 mutants (C) at 36 hpf. Panels B, D, and F show magnified eye regions in A, C, and E, respectively. G, H. Anti-Nok^547-703 antibody visualized peripheral localization of transgenic Nok (red) in the eyes of pt106 at 33 hpf (arrows). G and H show images of the same embryo at different confocal optical sections. The inset shows a magnified RPE area that expresses transgenic Nok (red). I-K. Transgenic Nok (red, arrows) localizes to the RPE that was visualized with RPE specific marker zpr2 (green) at 72 hpf. The arrowhead indicates the non specific staining of the anti-Nok^547-703 antibody (also see Figure 2). I shows the overview of an entire eye. L, M. Using a GFP riboprobe that recognizes the mRNA of the transgenic nok gene but not the endogenous nok gene (also see Figure 2), an in situ hybridization analysis of 33-hpf PTU-treated wildtype pt106^bing-33 embryos confirmed that the transgene is expressed in the RPE (arrows) but not in the neural retina (K). A control regular wildtype embryo showed no detectable signal (L).

Strategies for transgenic expression of Nok in the RPE and for selection of desired transgenic zebrafish lines. A. The promoter of Fugu tyrosinase gene was used to direct pigment-cell-specific expression of the full-length wildtype nok gene. Individual protein domains are indicated with grey boxes. A GFP coding region was inserted between the stop codon of the nok gene and the SV40 polyA signal-containing 3'UTR. This GFP sequence serves as the target of a GFP antisense probe for in situ analyses. The Nok C-terminal region downstream of the m520 non-sense mutation site was used to make an affinity column to purify the anit-Nok^547-703 polyclonal antibodies. Anti-Nok^28-208 antibodies (generated in a previous study) (Wei and Malicki, 2002) recognize both the full-length Nok and the truncated Nok^m520. B. Western blot analyses showed that anti-Nok^547-703 antibodies recognized the full-length Nok protein from wildtype fish samples (wt) and the recombinant Nok-His and GST-Nok^547-703 fusion proteins expressed in E oli.. Anti-Nok^547-703 antibodies do not recognize the truncated Nok^m520 mutant protein (m520 mutants). A few weaker bands indicate the existence of non-specific reactions of the antibodies to some fish proteins. The positions of full-length and m520 mutant Nok bands were indicated with arrows on the right. C. An immunohistochemical analysis demonstrated that the anit-Nok^547-703 antibodies recognize full-length Nok in wildtype at 72 hpf. Nok localizes to the apical regions of the RPE and the retina (arrows). The arrowheads indicate the non-specific staining of cells of an unknown class, which appear to be randomly distributed in the brain and retina. In mutant retinas, only non-specific staining was observed (arrowheads). Nuclear dye YO-PRO was used to visualize the overall shape of the eyes.