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J Neurosci. Author manuscript; available in PMC 2009 June 10.
Published in final edited form as:
J Neurosci. 2008 December 10; 28(50): 13684–13695.
doi: 10.1523/JNEUROSCI.4333-08.2008.
PMCID: PMC2637769
NIHMSID: NIHMS82523
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Intact RPE maintained by Nok is essential for retinal epithelial polarity and cellular patterning in zebrafish
Jian Zou,1 Kira L. Lathrop,1 Ming Sun,2 and Xiangyun Weicorresponding author1,3
1Department of Ophthalmology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15213
2Center for Biologic Imaging, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine
3Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine
corresponding authorCorresponding author.
Corresponding author: Xiangyun Wei, E-mail: weix/at/upmc.edu Tel: 412-383-5845; Fax: 412-647-5880
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Abstract
Within the vertebrate eye, the retinal pigment epithelium (RPE) juxtaposes with the retina, but how the RPE plays a role in retinal morphogenesis remains elusive. It has been shown that the loss of function of the polarity proteins, such as Nagie oko (Nok), disrupts RPE integrity and retinal lamination. However, it is unclear whether or not such defects are caused in a tissue-autonomous fashion. Here, by taking advantage of the nok mutation, we have generated a transgenic model to restore the Nok function in the RPE, but not in the retina. With this model, we show that Nok is required for RPE integrity in a tissue-autonomous manner. However, proper retinal epithelial polarity does not require retinal expression of Nok prior to embryonic photoreceptor genesis; rather, it requires a Nok-mediated intact RPE. Interestingly, sporadic wildtype RPE donor cells are not sufficient to maintain proper retinal polarity. We further show that RPE-mediated retinal epithelial polarity underlies proper patterning of retinal ganglion cells and the cells of the inner nuclear layer. Nevertheless, during embryonic photoreceptor genesis, an intact RPE is not sufficient to maintain retinal epithelial polarity and retinal cellular pattern formation. Our results show that the subcellular architecture and cellular pattern formation of a tissue may be regulated by neighboring tissues through tissue-tissue interactions.
Keywords: RPE, retina, cellular pattern formation, Nok, polarity, transgenesis
In vertebrates, the optic cup originates from invagination of the optic vesicle. The outer layer of the cup becomes the RPE, and the inner layer becomes the retina. During retinal neurogenesis, the retinal cells stratify into a layered structure. Each retinal layer is occupied by distinct types of cells that are positioned in specific geometric patterns (Dowling, 1970), but how such a retinal cytoarchitecture is formed during development is still not fully understood.
Because of the juxtaposition of the RPE with the retina, whether and how the RPE regulates retinal development has been an important research subject. Despite previous studies on this subject, the cellular and molecular mechanisms by which the RPE regulates retinal cellular pattern formation remain elusive. RPE ablation in transgenic mice showed that the RPE is needed to maintain the survival of the retina (Raymond and Jackson, 1995). In vitro culture of dissociated chicken retinal cells suggested that the RPE may secrete unknown factor(s) to regulate retinal lamination (Vollmer et al., 1984; Rothermel et al., 1997; Nakagawa et al., 2003). In addition, blastomere transplantation experiments revealed that the mosaic eyes (moe) and nagie oko (nok) genes function in a non-cell-autonomous manner in patterning retinal cells (Jensen et al., 2001; Wei and Malicki, 2002; Zolessi et al., 2006). While these experiments indicated that retinal development requires extrinsic regulations, each of these studies has particular limitations on revealing the mechanisms involved: the absence of the RPE in the transgenic mouse models made it difficult to analyze direct physical interactions between the RPE and the retina, and it was hard to distinguish the RPE's trophic function from its other functions; the in vitro systems may not fully represent the in vivo conditions; and blastomere transplantations in zebrafish generated uncontrollable distribution of donor cells in the host RPE and retina, making it difficult to determine unequivocally whether the RPE-retina or the retina-retina interactions are essential for the proper patterning of retinal cells. Thus, a different in vivo experimental approach is needed to provide further insights into the RPE-retina interactions.
To achieve this goal, we took advantage of the zebrafish nok mutation and generated a transgenic zebrafish model (pt106) to restore the Nok functions in the RPE but not in the retina. The nok gene encodes a member of the membrane associated guanylate kinase protein family (Wei and Malicki, 2002; Funke et al., 2005). Loss of Nok function causes patchy RPE and retinal lamination defects (Wei and Malicki, 2002). Because the loss of Nok function affects the development of both the RPE and the retina, the tissue-specific restoration of the RPE by transgenic Nok expression in pt106 will provide a unique in vivo system to answer certain questions about RPE-retina interactions: Does the loss of nok function cause the retinal and RPE defects in a tissue-autonomous fashion? How does an intact RPE regulate retinal development at the cellular and subcellular levels? We demonstrate for the first time that the maintenance of retinal epithelial polarity requires an intact wildtype RPE but not a few sporadic wildtype donor RPE cells. This RPE-mediated retinal epithelial polarity is essential for cellular pattern formation during retinal neurogenesis.
Generation of the pt106 and pt104 embryos
We used the Fugu tyrosinase promoter to drive a transgenic expression of a wildtype Nok gene in the RPE. The Fugu tyrosinase promoter was amplified from pF3xho5' (a gift from Dr. Friedrich Beermann) by PCR and used to replace the EF1α promoter between the Apa I and BamH I sites of the Tol2 transgenesis construct pT2KXIGΔin (Zou et al., 2006; Camacho-Hubner, 2000). The wildtype Nok ORF, including a TGA stop codon, was inserted between the Fugu tyrosinase promoter and GFP ORF by an Age I and Fse I restriction ligation. The resulting construct (pTol2-ftyp-Nok-GFP, Fig. 2AFigure 2) was used for transgenesis according to the established Tol2 methodology (Zou et al., 2006). To make transgenic founder fish, about 10-20 pg of pTol2-ftyp-Nok-GFP along with 25-50 pg of Tol2 transposase mRNA were co-injected at 1-4-cell-stages into embryos obtained from crosses between nokm520 carriers. Seven transgenic positive founder fish were outcrossed with nokm520 heterozygous fish. Transgenic positive F1 fish were identified by a PCR genotyping analysis using primers 5'-aacaaaaatgatgactttg-3' and 5'-tcagcgcagccaggatgaag-3'. To screen for F1 individuals that carry a genome-integrated transgene with a good RPE rescuing capability, individual F1 transgenic fish that are also heterozygous for nokm520 were crossed with regular nokm520 heterozygous fish for examination of their F2 progeny. Out of 74 transgenic and nokm520 F1 carriers, we identified two F1s that rescued the patchy RPE defect in 50% of the mutant embryos, indicating that these two fish carried a single copy of the transgene. One line was further characterized in this study and is designated pt106bing-33. The transgenic mutant embryos produced from the crosses between pt106bing-33 and regular nokm520 carries were named pt106 embryos.
Figure 2
Figure 2
Figure 2
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 (more ...)
To generate a stable transgenic line that express GFP in an ubiquitous fashion, we injected Tol2 transposase mRNA along with pT2KXIGΔin, which contains a GFP open reading frame downstream of the EF1α promoter, into AB wildtype embryos. This line was named pt104. Care of experimental animals was in accordance with University Pittsburgh guidelines.
In situ hybridization
In situ hybridization analyses of transgenic Nok expression and zebrafish tyrosinase related protein 1 (TYRP1) were performed according to a previous publication (Zou et al., 2006).
The generation of rabbit-anti-Nok C terminal polyclonal antibody
The C-terminal region of Nok505-703 (from amino acid 505 to amino acid 703) was PCR amplified and cloned into the His tag expression vector pET32a+ (Novagen) between EcoR I and Hind III. The construct was transformed into BL21 competent cells (Invitrogen) and used to express the Nok505-703-His fusion protein. Two milligrams of Nok505-703-His was purified with a His-trap column (Amersham) and used to immunize rabbits using the service provided by Proteintech Group, Inc. Antibodies that only recognize Nok down-stream of the m520 mutation site were affinity purified using a Aminolink Plus immobilization affinity column (Pierce) that was conjugated with 1 mg of GST-Nok547-703 fusion protein (expressed in the pGex-5x-1 system (Amersham)).
Immunohistochemistry
Immunohistological analyses were performed using the procedure and reagents as described previously (Wei et al., 2006b), except that the embryos were fixed at RT for 30 minutes for anti-Nok547-703 immunostaining.
Blastomere transplantation
Wildtype pt104 donor blastomeres were transplanted into either regular nokm520 mutant or pt106 host embryos at 3-4 hpf using the standard transplantation technique (Wei and Malicki et al., 2002). The resulting mosaic embryos were raised in egg water at 28.5 °C till 36 hpf before fixation with 4% paraformaldehyde at room temperature for 2 hours. Fixed embryos were subjected to standard immunohistochemical analyses for exanimation of retinal polarity phenotypes.
Immunohistochemical analysis of dissociated RPE cells
Wildtype embryos were raised in E3 egg water with 0.003% of pigmentation-blocking chemical 1-phenyl-2-thiourea (PTU, Sigma) till 72 hpf. 40 eyes were removed from these embryos and digested with 200 μl TrypLE Express (Gibco; containing amphotericin B and Penicillin-Streptomycin) at RT for 30 minutes. The digestion was stopped by adding 20 μl (10%) FBS and chilled to 4 °C. The supernatant of the digest was further microcentrifuged at 2000 rpm for 5 minutes to collect the dissociated cells. The cell pellet was re-suspended with 100 μl 1x PBS at 4 °C and spread on a poly-L-lysine-coated slide (Fisher), followed by an incubation of one hour at 4 °C. The cells adhered to the slide were then fixed with 2% PFA for 1 hour. To determine whether or not RPE cells express Nok, the slide was immunostained with the rabbit polyclonal anti-Nok28-208 antibodies (1:200, Wei and Malicki, 2002) and the mouse monoclonal zpr2 antibody (1:200, ZFIN), which recognizes an RPE-specific antigen. Expression of Nok in zpr2-positive cells were examined under confocal microscopy.
The nok gene is expressed in both the RPE and the retina
A display of a heritable developmental defect in a tissue does not necessarily mean that the tissue expresses a particular mutant gene. In order to restore the RPE integrity in nokm520 by expressing a wildtype nok transgene, we first need to confirm that the nok gene is indeed expressed in the RPE. Previously, we revealed that the Nok protein localizes to the interface between the retinal neuroepithelium and the RPE (Wei and Malicki, 2002). However, because the apical surfaces of the two tissues are in close contact at early epithelial stages, the limited resolution of conventional light microscopy does not allow definitive assertion about Nok's tissue expression pattern. In fact, another study has since suggested that Nok was expressed in the RPE but not in the retina (Jensen and Westerfield, 2004). To unambiguously confirm that both the RPE and the retina express Nok, we need to spatially separate the apical surfaces of the two tissues when examining Nok's expression patterns. We thus first analyzed the distribution of the Nok proteins in the 35-hpf N-cadm117 mutant retinas, where the apical surface of the retinal neuroepithelial cells localizes ectopically to the interior of the retina, 3 to 7 cells away from the RPE (Erdmann et al., 2003). Indeed, we found that Nok localizes to the interior of the N-cadm117 retina, demonstrating that the retina expresses Nok (Fig. 1A, BFigure 1). To determine if Nok is also expressed in the RPE, we chose to analyze dissociated individual RPE cells but not the RPE tissue in N-cadm117 to avoid the potential signal interference from the adjacent choroidal cells. Indeed, Nok is expressed in the RPE (Fig. 1CFigure 1). The expression of Nok in both the RPE and the retina makes it legitimate to examine the effects of RPE restoration on retinal development in nokm520 by expressing a wildtype transgenic nok gene in the RPE.
Figure 1
Figure 1
Figure 1
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 nokm520 mutant retinal (more ...)
Transgenic expression of Nok in the RPE rescues the patchy RPE defect caused by the nokm520 mutation in a tissue-autonomous manner
To achieve an RPE-specific transgenic expression of Nok in the eye, we used the Fugu tyrosinase gene promoter to express a wildtype transgenic nok gene in the nokm520 mutant background (Fig. 2AFigure 2) (Zou et al., 2006). Out of 74 distinct F1 transgenic fish lines, we established one stable transgenic model (pt106) that is homozygous for the nokm520 mutant gene and heterozygous for the wildtype nok transgene. In pt106, the expression of the transgenic nok gene restores RPE integrity, but the developmental defects of a curled body axis and paracardiac edema remain the same as that of regular nokm520 mutant embryos (Fig. 3A-FFigure 3). To verify the specificity of the transgenic expression, we generated polyclonal antibodies that recognize the full-length Nok protein but not the truncated endogenous m520 mutant Nok protein (Fig. 2Figure 2). Using the antibodies, we determined that the transgenic Nok protein is expressed in the RPE (Fig. 3G-KFigure 3). An in situ hybridization analysis further confirmed that the transgene is expressed in the RPE but not in the retina (Fig. 2AFigure 2; Fig. 3L, MFigure 3). Thus, Nok maintains RPE integrity in a tissue autonomous manner.
Figure 3
Figure 3
Figure 3
Transgenic expression of Nok in the RPE of pt106 is sufficient to rescue the patchy RPE defect caused by the nokm520 mutation. A-F. The expression of transgenic Nok in pt106 (E, F) restores the RPE integrity to a level indistinguishable from that of wildtype (more ...)
Retinal lamination is largely restored in pt106
To analyze the effect of the restoration of RPE integrity on retinal cellular pattern formation in pt106, we performed histological analyses of retinal structure at 4.5 or 5 dpf. We found that retinal lamination is dramatically recovered in pt106 (Fig. 4AFigure 4). Specifically, we found that retinal ganglion cells, Muller cells, and Lin7-positive cells (mainly bipolar cells, amacrine cells, and some ganglion cells that are adjacent to the inner plexiform layer; Wei et al., 2006a) localize to proper cellular layers in pt106 (Fig. 4B, CFigure 4). The processes of bipolar cells and Muller cells are also oriented radially in pt106 as in wildtype, except that apical processes from Muller cells did not appear to extend to the outer nuclear layer (Fig. 4B, CFigure 4). The integrity of the inner and outer plexiform layers, as revealed by actin staining, is also greatly improved (Fig. 4B-DFigure 4). However, green/red double cones were found in all three layers, with only about 50% of them in the photoreceptor layer. In pt106, these double cones are not as elongated as their counterparts in wildtype, indicating their morphological defects at the cellular level. In addition, rods and blue and UV cones are also aberrantly positioned, similar to double cones in pt106 (data not shown). Interestingly, we rarely saw photoreceptor cells in the outer half of the inner nuclear layer where the cell bodies of bipolar cells localize (Fig. 4DFigure 4). We also found no apparent outer limiting membrane-like structure in pt106 at 4.5 dpf (Fig. 4B-DFigure 4). Thus, the rescue of RPE defects by RPE expression of Nok in pt106 has a dramatic positive effect on retinal cytoarchitecture, leading to the proper positioning of several major retinal cell types (except for photoreceptors) and the overall structural recovery of inner and outer plexiform layers.
Figure 4
Figure 4
Figure 4
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 (more ...)
Positive correlation between apical cell division and the patterning of retinal cells
While complex, the process of cellular patterning for a given postmitotic cell can be divided into two general steps. First, the cell needs to migrate to a proper place if its birth place is not its final destination. Second, stabilizing mechanisms are utilized to ensure that the cell makes proper contacts with its neighbor cells and does not move away from its destination. Here, the differential effects of pt106 transgenic Nok expression on the positioning of ganglion cells, inner nuclear layer cells, and photoreceptors may provide an opportunity to analyze how the cellular patterning process goes wrong in nokm520 retinas. The results will provide insights into the mechanisms by which retinal cells are normally patterned in wildtype.
To dissect the cellular basis of the retinal pattern formation, we first analyzed the distribution of M-phase nuclei at distinct developmental stages when ganglion cells, inner nuclear layer cells, or photoreceptors are each predominantly generated (Hu and Easter, 1999). The rationale for this analysis is that during neurogenesis, some cell divisions are final cell divisions, after which those cells become postmitotic; therefore, the locations of M-phase nuclei will provide information about the start sites of postmitotic cell migrations (although we recognize that this analysis does not distinguish final cell divisions from other cell divisions). To more accurately define the start sites of post-division cell migrations, we calculated the percentage of apical cell division events by counting the nuclei in late M-phase, namely, in metaphase, anaphase, or telophase (some cells in prophase are still moving towards the apical surface and therefore, their locations do not accurately represent the start sites of post-division migration, so these were not included). We found that, as in wildtype, the majority of retinal cell divisions occur apically in pt106 when ganglion cells and inner nuclear cells are being generated (Fig. 5A, B, DFigure 5). During embryonic photoreceptor genesis, the percentage of apical cell divisions declines in pt106 (though still higher than that in regular nokm520 mutant retinas) (Fig. 5C, DFigure 5). Because retinal ganglion cells and inner nuclear cells are patterned more properly than photoreceptors in pt106 (Fig. 4Figure 4), the above observations reveal a positive correlation between the frequency of apical localization of cell divisions and the degree of proper cellular patterning. This correlation suggests that apical localization of final cell divisions may play an essential role in proper postmitotic cellular patterning.
Figure 5
Figure 5
Figure 5
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, nokm520, and pt106 at 33 hpf, 42 hpf, and 53 hpf (more ...)
Besides the starting sites of cell migration, the migratory directions of postmitotic cells also influence the final destinations of cells. To analyze the features of cell movements during retinal neurogenesis in wildtype and in nokm520, we examined nuclear movements in living embryos. Consistent with previous findings (Hinds and Hinds, 1974; Das et al. 2003; Baye and Link, 2007), we found that the majority of retinal interphase nuclei move radially along the apicobasal axis in wildtype (Fig. 6A, CFigure 6; Supplementary movie 1). However, in mutant retinas many nuclei did not move radially; many round nuclei moved perpendicularly to the apicobasal axis of the retinal neuroepithelium (Fig. 6B, DFigure 6). In addition, many cells in the mutant retinas move towards the interior of the retina prior to cell division: Cells in the inner half of the retina move apically to the interior to divide, whereas cells in the outer half of the retina move basally to the interior to divide (Supplementary movie 2). Thus, the retinal epithelium is split in two in nokm520, with each half displaying reversed directions of cellular movements.
Figure 6
Figure 6
Figure 6
The nokm520 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 (more ...)
Taken together, we infer that the ectopic interior localization of cell divisions and aberrant directions of cell migrations may directly contribute to the eventual cellular patterning defect in nokm520 mutant retinas.
Proper early retinal epithelial polarity requires Nok expression in the RPE but not necessarily in the retina
Because the localization of M-phase nuclei reflects how the epithelium polarizes (Hinds and Hinds, 1974), the above results suggest that the rescue of RPE defects by RPE expression of Nok restores the polarity of the retinal epithelium in pt106 prior to embryonic photoreceptor genesis. To confirm this, we next examined additional epithelial polarity markers ZO-1 and adherens junction-associated actin bundles. Indeed, unlike in nokm520 (Fig. 7BFigure 7), these two markers are largely positioned at the apical surface of the retina in pt106 and wildtype retinal epithelia (Fig. 7A, CFigure 7). Thus, transgenic expression of Nok in the RPE can sufficiently restore the proper polarity of retinal neuroepithelium at early stages of retinal development in pt106.
Figure 7
Figure 7
Figure 7
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) (more ...)
We next investigated whether or not retinal expression of Nok plays an active role in the polarization of the retinal epithelium. We transplanted wildtype retinal donor cells to either regular nokm520 or pt106 hosts. To visualize donor cells, we first generated a stable transgenic fish line (pt104) that expresses GFP under the ubiquitous EF1α promoter in a wildtype genetic background. The transgenic GFP expression allows a visualization of live donor cells, avoiding the signal interference from dead donor cells when labeled with conventional non-degradable dextran conjugates (Catalano et al., 2007). We found the apical markers Nok and ZO-1 of the wildtype pt104 donor cells localized apically in pt106 host retinas but internally in nokm520 host retinas (Fig. 8A, BFigure 8). Because the only genetic difference between the regular nokm520 and pt106 host eyes is that pt106 RPE expresses the wildtype transgenic nok gene, the above results suggest that the retinal expression of Nok is not sufficient to maintain proper polarity of the donor retinal epithelial cells at early developmental stages; instead, Nok expression in the RPE, which restores the RPE integrity, is required and sufficient to maintain proper retinal epithelial polarity.
Figure 8
Figure 8
Figure 8
Retinal expression of wildtype Nok is not sufficient to rescue the retinal neuroepithelial polarity defect in nokm520. A. Nok and ZO-1 expressed in the wildtype pt104 donor cells localize apically in the pt106 host retinas at 36 hpf (arrows). Arrowheads (more ...)
An intact RPE but not sporadic wildtype RPE cells is required to restore the mutant retinal epithelial polarity defect in nokm520
Restoration of RPE integrity and retinal epithelial polarity in pt106 raises interesting questions: Is RPE integrity important for maintaining retinal epithelial polarity? Or, can sporadic wildtype RPE cells be sufficient to maintain retinal epithelial polarity? To analyze the correlation of the RPE integrity and retinal polarity defects in nokm520, we first examined the temporal course of the disintegration of nokm520 RPE with a zebrafish TYRP1 riboprobe as an early RPE integrity indicator (Zou et al., 2006). We found that at 24 hpf, the RPE in nokm520 is as intact as in wildtype (Fig. 9AFigure 9). At 26 hpf in nokm520, the retinal epithelial polarity is as normal as in wildtype (Fig. 9BFigure 9). At around 28 hpf when the polarity of retinal epithelium starts to deteriorate (data not shown), the RPE in nokm520 begins to lose its intactness, as indicated by the presence of occasional gaps in the epithelial sheet (Fig. 9AFigure 9). The disruption of RPE integrity becomes more severe and apparent around 32 hpf, when retinal polarity is severely perturbed (Fig. 9AFigure 9). Thus, the loss of retinal epithelial polarity is linked to the disruption of the RPE integrity in a tight temporal manner.
Figure 9
Figure 9
Figure 9
The disruption of retinal epithelial polarity in nokm520 is correlated with the loss of RPE integrity in a tight temporal manner. A. An in situ hybridization analysis of wildtype and nokm520 embryos with an antisense RNA probe against the zebrafish TYRP1 (more ...)
In the nok mutant retinas at 48 hpf, retinal ganglion cells prefer to accumulate at regions adjacent to the basement membrane of the retina or to apical retinal regions that lack RPE cells (Zolessi et al., 2006; and Fig. 9CFigure 9). Zolessi et al. proposed that the RPE might play a role in blocking the attracting influence from the Bruch's membrane, which is normally constituted from the basement membranes of the RPE and choroidal cells. This led us to wonder whether or not the local coverage of RPE patches influences the polarity of retinal epithelial cell in their vicinity at early developmental stages in nokm520. We thus examined the spatial relationship between the patches of the RPE and M-phase nuclei and apical marker ZO-1 in nokm520 retinas at 36 hpf. Interestingly, we found that the coverage of retina by RPE patches did not relieve the retinal polarity defects locally (Fig. 9DFigure 9), suggesting RPE patches in nokm520 are not equivalent to an intact RPE in pt106 in mediating retinal epithelial polarity.
The above results promoted us to ask whether the RPE integrity or the expression of wildtype Nok in the RPE cells is important to maintain retinal epithelial polarity. Previously, it was described that sporadic wildtype donor RPE cells might be able to restore the apical localization of moe mutant M-phase retinal nuclei by presumably secreting some signaling factors, as suggested by the over four-fold reduction of ectopic localization of moe M-phase nuclei in the presence of sporadic wildtype RPE donor cells (Jensen et al., 2001). To investigate if sporadic wildtype RPE cells can restore retinal epithelial polarity defects in nokm520 mutants, we transplanted wildtype pt104 blastomeres into nokm520 mutants and examined the retinal regions that were adjacent to wildtype RPE donor cells. Surprisingly, in contrast to the results of Jensen et al. (2001), we found that the percentages of interiorly localized M-phase nuclei showed no apparent difference, whether or not there were wildtype RPE donor cells in the vicinity (Fig. 10Figure 10) (When adjacent to wildtype RPE donor cells, about 87.8% of the M-phase nuclei localized interiorly (n=41); when distant from wildtype RPE donor cells, about 89.3% of the M-phase nuclei localized interiorly (n=47)). These percentages of ectopic nuclei were similar to that in the regular nokm520 mutants (Fig. 5DFigure 5). In addition, apical marker ZO-1 also localized interiorly in the host retina (Fig. 10Figure 10, right panel, red). The inability of sporadic wildtype RPE cells to rescue retinal polarity defects is not likely due to the nokm520 mutation, because the wildtype donor retinal cells also displayed the polarity defects whether or not they were adjacent to wildtype RPE donor cells (Fig. 8BFigure 8; Fig. 10Figure 10, red arrow). Considering the restoration of retinal epithelial polarity in pt106 (Fig. 7Figure 7; Fig. 8AFigure 8), these results indicate that an intact wildtype RPE is required to maintain the proper retinal epithelial polarity, but sporadic wildtype RPE cells are not sufficient to do so.
Figure 10
Figure 10
Figure 10
Sporadic wildtype RPE cells were not sufficient to rescue the retinal epithelial polarity. Wildtype pt104 donor cells (visualized by GFP, green) were transplanted into nokm520 mutants. The localization of retinal M-phase nuclei (white arrows, blue TO-PRO (more ...)
In summary, our study revealed a series of novel developmental steps by which the nok gene regulates retinal development through RPE-retina interactions (Fig. 11Figure 11): The expression of Nok in the RPE is required and sufficient to maintain RPE integrity. An intact RPE ensures proper polarity of retinal neuroepithelium prior to photoreceptor genesis, and a properly polarized retinal epithelium ensures apical localization of cell divisions and provides a proper environment for postmitotic cell migration and patterning. Finally, even though retinal expression of Nok is not required for retinal cell polarity and patterning during the genesis of retinal ganglion cells and inner nuclear cells, it is required during embryonic photoreceptor genesis.
Figure 11
Figure 11
Figure 11
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.
The autonomy and non-autonomy of Nok function in the RPE and the retina
In the RPE, Nok maintains its integrity in a tissue autonomous manner. This autonomy resembles the way in which Nok homologs play a role in maintaining the polarity and integrity of the fly embryonic epithelium and MDCK cell monolayer (Tepass and Knurt, 1993; Kamberov et al., 2000; Hong et al., 2001; Bachmann et al.., 2001; Straight et al., 2004). By contrast, Nok cannot maintain proper retinal epithelial polarity in an autonomous manner (Fig. 8 A, BFigure 8). Such differential requirements of Nok suggest a mechanical variation in maintaining epithelial polarity and integrity of the RPE and the retina.
RPE's role in maintaining retinal epithelial polarity
Sporadic wildtype donor RPE cells were suggested to be able to rescue the polarity defect of retinal cells in the moe mutant hosts (Jensen et al., 2001). However, our similar blastomere transplantation analysis in the nok background did not support such a capability (Fig. 10Figure 10). The reasons for this discrepancy are unclear. We cannot rule out the possibility that different mutation background made the difference. In fact, unlike the apical localization of Nok, the distribution of Moe to the entire cell membrane suggests that the molecular mechanisms by which Moe regulates retinal epithelial polarity are different from that for Nok (Hsu et al., 2006). Alternatively, because moe mutation does not cause a severe retinal polarity defect at early developmental stages (see Fig. 3FFigure 3 of the Jensen et al., 2001 paper; and our data not shown), it might be challenging to assess to what extent sporadic wildtype RPE cells really restored the apical localization of M-phase nuclei in moe. Thus, to our knowledge, our study is the first to demonstrate with a stable genetic system that retinal epithelial polarity is dependent on an intact RPE, and sporadic wildtype RPE cells are not sufficient to regulate retinal epithelial polarity.
While the RPE can rescue the retinal epithelial polarity defect in pt106 prior to embryonic photoreceptor genesis, the later loss of retinal epithelial polarity indicates that retinal expression of Nok is still required to sustain the polarity during the period that embryonic photoreceptors are generated (Fig. 5Figure 5). Thus, the requirement of the RPE for retinal epithelial polarity and retinal cellular pattern formation gradually decreases during development.
Mechanisms of RPE's maintenance of retinal epithelial polarity
There are several possible mechanisms by which the RPE maintains retinal epithelial polarity prior to embryonic photoreceptor genesis. During the formation of the optic cup in zebrafish, the apical surfaces faces of the RPE and the retina juxtapose tightly with each other (Schmitt and Dowling, 1994). It is likely that there is a physical adhesion between the apical surfaces of the two epithelia. This potential adhesion may serve as a pulling force to maintain the polarity of the retinal epithelium at early stages of development. The loss of RPE integrity, as caused in nokm520, may reduce the adhesion between the two tissues, leading to the loss of retinal epithelial polarity. This hypothesis is consistent with the tight temporal association between the patchy RPE defect and retinal polarity defect in nokm520 (Fig. 9Figure 9; data not shown). However, the molecular nature of this proposed physical adhesion is not known. A previous EM observation suggested that desmosomes may play a role in the adhesion of the two tissues in human embryos (Hollenberg and Spira, 1973). Our EM and immunohistochemical analyses, however, revealed no apparent subcellular structures that resemble conventional cell-cell junctional complexes, such as desmosomes, between the two tissues in zebrafish embryos (data not shown).
The close contact between the RPE and the retina is essential but not sufficient to prove that the RPE regulates retinal epithelial polarity through physical adhesion. Other alternative possibilities warrant further discussion here. First, the RPE may secrete diffusible factors (Vollmer et al., 1984; Rothermel et al., 1997; Layer et al., 1998; Nakagawa et al., 2003) to mediate retinal epithelial polarity. While the inability of the sporadic wildtype RPE cells to rescue the polarity defect of their neighbor nokm520 retinal cells (Fig. 10Figure 10) does not favor this secreted-factor theory, it cannot rule it out, either. This is because the sufficient secretion of any RPE factors may require a properly polarized intact RPE tissue. Sporadic wildtype RPE donor cells may not be able to make proper connections with neighboring host mutant RPE cells, leading to the loss of their secretion function. Or it could be that a few sporadic wildtype RPE donor cells cannot constitute the critical mass needed to secrete enough factors for proper functions. Second, the RPE may block some polarity-disrupting signals from the Bruch's membrane or the choroid, just as the RPE prevents retinal ganglion cells from migrating incorrectly to the apical retinal regions (Zolessi et al., 2006; Fig. 9CFigure 9). However, this possibility is not supported by the fact that the retinal polarity defect exists in all retinal regions in nokm520 mutants regardless of whether or not there are RPE cells nearby (Fig. 9 DFigure 9).
The functions of retinal Nok
Previously, we have shown that the adherens junctions in the retina are likely the precursor of the OLM, and Nok plays a role in the development and maintenance of the OLM (Wei et al., 2006b). These findings are further supported by the current observation that the OLM does not develop in pt106, which lacks retinal Nok function (Fig. 4Figure 4). It is likely that the disruption of Nok-mediated photoreceptor-photoreceptor adhesion/stabilization is one of the underlying causes of the improper positioning of photoreceptors in pt106. The Nok is required to target Crumbs to the inner segment region to potentially mediate physical adhesion via the extracellular domain of the Crumbs proteins (Wei et al., 2006b). Consistent with this notion, we found that Crumbs is internalized in photoreceptors in pt106 as in regular nokm520 mutants (Supplementary Figure 1; Wei et al., 2006b). Furthermore, the decline of apical cell division and the worsening of retinal polarity in pt106 retina occur immediately before embryonic photoreceptor genesis (Fig. 5DFigure 5; Hu and Easter, 1999). This suggests that Nok also plays an important role in the transition of the adherens junctions between undifferentiated retinal epithelial cells to the OLM in the ONL. Nevertheless, during the genesis of ganglion cells and inner nuclear cells, the maintenance of retinal epithelial polarity does not require retinal Nok (Fig. 5Figure 5).
Regulations of cell migration for retinal pattern formation
As for how cell migration underlies cellular patterning process of the retina, our study suggests several aspects, which might be mutually inclusive. First, when M-phase cells are ectopically positioned in the interior of the retina, as in nokm520, postmitotic cells are liable to migrate in abnormal directions, rather than the basal direction in wildtype (Supplementary movie 2; Fig. 6Figure 6). Second, the undifferentiated retinal neuroepithelial cells that span the entire thickness of the retina may serve as a guiding mechanism for differentiating retinal cells by providing a continuous surface for postmitotic cells to adhere to during migration, or by providing proper migration cues. In nokm520, the splitting of the mutant retinal epithelium into two halves (Figs 5Figure 5, ,7,7Figure 7, ,8),8Figure 8), with the outer half displaying a reversed apical-basal polarity compared to the adjacent inner half, will likely present confusing migration guidance cues to postmitotic cells, leading to irregular cellular movements (for comparison, see Supplementary movies 1 and 2). Consistent with this hypothesis, in pt106, the degree to which a given type of retinal cell is properly positioned is positively correlated with the extent of the restoration of retinal epithelial polarity during the developmental stage when those cells are predominantly being generated (Figs. 4Figure 4, ,5).5Figure 5). In addition, as shown by Zolessi et al, the RPE might block the Bruch's membrane from attracting retinal ganglion cells to migrate to the apical end (Zolessi et al., 2006; Fig. 9CFigure 9). Thus, the disruption of RPE integrity will further worsen the patterning process in nokm520. However, RPE's blockage function may not be sufficient/applicable for photoreceptors, because they localize improperly in pt106, even with an intact RPE during embryonic photoreceptor genesis (Fig. 4Figure 4).
Mosaic analyses by blastomere transplantation vs. transgenesis
Blastomere transplantation technology has been utilized by many researchers to study the autonomy of gene function. Although this technology is powerful and convenient, its strength can be limited if multiple types of cell-cell interactions exist simultaneously in the mosaic animals. Thus, uncontrollable distribution of donor cells can cause ambiguity when one tries to determine if a particular cell-cell interaction causes certain effects. This limitation manifests itself when determining whether the extrinsic regulation of the patterning of individual retinal cells is directed by the RPE or by the neighboring retinal cells (Jensen et al., 2001; Wei and Malicki, 2002; Zolessi et al., 2006). This limitation can be dramatically overcome by utilizing transgenic models, where gene expression/function in desired cells or tissues can be manipulated in a stable and reproducible fashion.
The transgenic approach has its own limitations as well: Proper promoters for cell- or tissue-specific expression of the transgenes might not be available. In addition, the position effects of transgenesis may alter the desired expression patterns in certain transgenic lines, so it requires vigorous selection and verification to obtain desired transgenic lines. To study the requirement of the RPE for retinal development, Raymond et al. ablated the RPE via transgenic expression of the toxic protein diphtheria toxin-A in two transgenic lines: line A (anophthalmic) and line M (microphthalmic) (Raymond and Jackson, 1995). The brain abnormality and the earlier-than-expected onset of retinal defect in line A at E11 raised concern of ectopic retinal expression of the toxic transgene (The study unfortunately did not verify the tissue specificity of the transgenic expression). The late onset of RPE ablation in line M, at E14.5, when the retina had already developed nearly perfect lamination of an inner and outer neuroblastic layers, made line M unsuitable for revealing RPE functions during early retinal development. By contrast, the restoration of the RPE in pt106 preserves the RPE's neurotrophic function for retinal cell survival (Ishida et al., 1997) and establishes pt106 as a unique system for characterizing RPE's roles in retinal epithelial polarity at early developmental stages and in cellular pattern formation during retinal neurogenesis.
Supplementary Material
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Acknowledgements
This study was supported by a NIH core grant (5P30EY008098-17) and the following funds to XW: NIH R01EY016099, University of Pittsburgh School of Medicine startup fund, and Research to Prevent Blindness Career Development Award. We are grateful to Dr. Pamela Raymond and Ms. Lynne Sunderman for critical editing of the manuscript, to Dr. Friedrich Beermann for providing Fugu tyrosinase gene promoter, to Drs. Jan Wijnholds and Penny Rashbass for anti-Crumbs antibodies, and to Dr. Paul Linser for providing the anti-Carbonic Anhydrase antibodies. We also thank Dr. Donna Stolz and Dr. Simon Watkins for providing the TEM facilities at the Center for Biologic Imaging at University of Pittsburgh School of Medicine. Dr. Richard Bilonick provided assistance in statistic analysis.
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