January 2008
Volume 49, Issue 1
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Retinal Cell Biology  |   January 2008
Identification of Photoreceptor Precursors in the Pars Plana during Ocular Development and after Retinal Injury
Author Affiliations
  • Koji M. Nishiguchi
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Hiroki Kaneko
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Makoto Nakamura
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Shu Kachi
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Hiroko Terasaki
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Investigative Ophthalmology & Visual Science January 2008, Vol.49, 422-428. doi:10.1167/iovs.07-1008
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      Koji M. Nishiguchi, Hiroki Kaneko, Makoto Nakamura, Shu Kachi, Hiroko Terasaki; Identification of Photoreceptor Precursors in the Pars Plana during Ocular Development and after Retinal Injury. Invest. Ophthalmol. Vis. Sci. 2008;49(1):422-428. doi: 10.1167/iovs.07-1008.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To study the distribution and differentiation of photoreceptor precursors in the ciliary epithelium in mice.

methods. Proliferating cells in flat-mount specimens of the ciliary body and retina were studied by bromodeoxyuridine (BRDU; 150 mg/kg) labeling in young C57Bl mice. Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically to assess the distribution and differentiation of photoreceptor progenitors or precursors. Mice injected intraperitoneally with N-methyl-N-nitrosourea (MNU; 60 mg/kg) were also examined.

results. Part of the neuroblast layer composed of BrdU-positive retinal progenitor cells was identified within the ciliary epithelium of the pars plana in continuation of the layer of the peripheral retina during ocular development. In both the ciliary epithelium and the retina, the layer size decreased rapidly and disappeared mostly by postnatal day (P)9. Within the ciliary epithelium of the pars plana, numerous postmitotic rod and cone photoreceptor precursors were identified that rapidly differentiated morphologically and decreased in number with ocular development. Rod precursors were no longer seen in the pars plana at P12, whereas rare presumptive cone precursors persisted even at P120. An increase in the number of presumptive cone precursors (approximately 16-fold) was identified in the pars plana of adult mice with MNU-induced photoreceptor degeneration.

conclusions. Rod and cone precursors were identified in the ciliary epithelium of the murine pars plana during ocular development but nearly disappeared after the completion of histogenesis. However, in response to retinal injury, an increased number of presumptive cone precursors was found even in the adult pars plana.

In fish and amphibians, a circumferential zone in the retinal margin, named the ciliary marginal zone (CMZ), produces all types of retinal neurons and thereby continuously regenerates the retina throughout life. 1 2 3 However, in mammals, an analogous zone for retinal neurogenesis or regeneration in the peripheral retina or the surrounding ciliary body has not been identified. Evidence suggests that the ciliary epithelium contains stem cells that have the potential to proliferate and express markers specific to retinal neurons in mammals, 4 5 6 7 including primates. 8 However, the exact potential of these cells, including whether they can produce morphologically differentiated retinal neurons, remains unknown. 
In mice, the ciliary body is composed of two distinct anatomic substructures. 9 The anterior aspect of the ciliary body with ciliary processes is known as the pars plicata. The pars plana, a flat circumferential zone not more than 12 to 16 cells wide, forms the posterior part of the ciliary body and connects the retina and the pars plicata. 9 Developmentally, both the ciliary epithelium and the retina share a common origin and arise from the inner layer of the optic cup. However, these two anatomic structures are thought to eventually follow different developmental routes because each has a distinct role in the eye; the retina translates light stimuli into neural signals and conveys them to the brain, whereas the ciliary epithelium secretes aqueous humor to maintain the intraocular pressure and integrity of the eye. In young mice, when gross structures of the eye, including the ciliary body, are already formed, a layer composed of proliferating retinal progenitor cells—called the neuroblast layer—plays an important role in the development of retinal neurons that migrate to form distinct retinal layers. As the maturation of the retina begins around the optic nerve and extends gradually toward the retinal margin, the neuroblast layer in the peripheral retina, adjacent to the pars plana, persists at the late stages of retinal development. 
Here, we report that part of the neuroblast layer resides within the ciliary epithelium of the pars plana during postnatal ocular development in mice. Moreover, cone and rod photoreceptor precursors in various stages of morphologic development are identified in the pars plana during this period and after retinal injury, even in adult animals. 
Methods
Animals
All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and to the guidelines for the use of animals at Nagoya University School of Medicine. C57BL/6J mice used were kept under a 12-hour light/12-hour dark cycle. 
BrdU Labeling
To label cells in the S-phase of the cell cycle, mice were injected intraperitoneally with bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) at a dose of 150 mg/kg and were humanely killed after 2 hours so that eyes could be collected. 
Immunohistochemistry
Immunohistochemical analyses were conducted as previously described, with slight modification. 10 For eye sections, the eyecups were fixed in 4% paraformaldehyde for 2 hours, followed by cryoprotection in 30% sucrose overnight at 4°C. The eyes were embedded in OCT compound (Tissue-Tek; Sakura Finetek Japan Co. Ltd., Tokyo, Japan), and frozen sections 12-μm thick were cut through the dorsal to ventral meridian at −21°C. After sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 15 minutes, they were incubated in 4 N HCl for 15 minutes, neutralized with 0.1 M Na2B4O7 for 5 minutes, blocked in 5% goat serum in PBS for 30 minutes, and incubated with primary antibodies for 1 hour and with secondary antibodies for 1 hour. 
For flat-mount specimens, after radial incisions were made to flatten the eyecups, the samples were fixed in 4% paraformaldehyde for 2 hours, and the vitreous was carefully removed. The eyecups were permeabilized in 0.5% PBST for 4 hours, incubated in 2 N HCl for 45 minutes, and neutralized with 0.1 M Na2B4O7 for 15 minutes. After blocking with 5% goat serum in PBS for 1 hour, the samples were incubated with primary antibodies for 12 hours and with secondary antibody for 9 hour. All procedures were conducted at room temperature unless indicated otherwise. 
Sections and flat-mounts were stained with primary antibodies for BrdU (1:1000; Oxford Biotechnology, Oxford, UK), rhodopsin (1:1000; Chemicon, Temecula, CA), recoverin (1:1000; Chemicon), Pax6 (1:200; Developmental Studies Hybridoma Bank), and proliferating cell nuclear antigen (PCNA; 1:200; BD Biosciences, San Jose, CA) followed by combinations of fluorescent dye-conjugated secondary antibodies (Alexa 405, 488, and 568; all at 1:1000; Molecular Probes, Eugene, OR), peanut agglutinin (PNA; 1:100; Molecular Probes), and diamidino-2-phenylindole (DAPI; 1:1000; Molecular Probes). 
Identification of the Retinal Margin and Pars Plana with Confocal Microscope
The pars plana was distinguished clearly from the retina in flat-mounts because the following features were observed in immunohistochemical analyses. First, the pars plana had features of both the retina and the pars plicata of the ciliary body. The inner surface of the pars plana was flat, showing a contour similar to that of the retina, whereas in the deeper layers it had discernible folds that appeared to be the continuum of the pars plicata. Second, with rhodopsin or recoverin staining, an intervening space was frequently detected between immunopositive cells at the retinal margin and pars plana, particularly in adult mice with toxin-induced retinal degeneration. Third, the retina showed dense staining, forming a plane of rhodopsin- or recoverin-positive cells in contrast to the pars plana, which had much less densely distributed cells aligned circumferentially. Fourth, photoreceptor precursors in the pars plana resided only in the surface layer parallel to the retina, consistent with their location in the nonpigmented ciliary epithelium. Meanwhile, retinal photoreceptors located deep in the outermost nuclear layer were oriented perpendicularly to the vitreoretinal interface and comprised multiple layers of neurons of the same cell type. Therefore, cells in the pars plana could be selectively visualized by scanning the flat-mounts at the level of the ciliary epithelium with a confocal microscope. Although most of these features were confirmed before obtaining images, not all are presented in every figure to highlight the cells of interest. 
N-methyl-N-nitrosourea Injection
P60 C57BL/6J mice were intraperitoneally injected with N-methyl-N-nitrosourea (MNU; 60 mg/kg; Sigma) and were humanely killed at P75 for histologic analysis. 
Results
Neuroblast Layer Encompasses Peripheral Retina and Ciliary Epithelium of Pars Plana in Postnatal Mouse
To test the hypothesis that retinal progenitors or precursors are present in the ciliary epithelium in the developing mammalian eye, mitotic cells were labeled by intraperitoneal injection of BrdU into young mice of various ages 2 hours before enucleation. To improve the accuracy of the interpretations of the experimental results, we defined retinal progenitors in this study as mitotic cells with the potential to generate retinal neurons and retinal precursors as postmitotic cells with morphologic evidence of neuronal differentiation not yet integrated into the retina. We first studied the distribution and quantity of BrdU-positive cells in the ciliary epithelium and peripheral retina during (P6 and P9) and after (P12 and P18) histogenesis of the retina using eye sections. At P6, many BrdU-positive cells were observed in the ciliary epithelium and the retina (Figs. 1a 1b) . These cells formed the presumptive neuroblast layer in the retina and were most frequently identified around the pars plana, close to the retinal margin in the ciliary epithelium. The BrdU-positive cells decreased rapidly by P9 and continued to decrease further thereafter (Figs. 1c 1d) . Consequently, when BrdU was administered at P18, cells in the ciliary epithelium and retinal margin were only rarely labeled in histologic sections. 
To confirm the findings in eye sections, we repeated the experiment using flat-mounts of the ciliary body and retina from mice ranging in age from P6 to P60. The anatomies of the retina and ciliary body are compared between histologic sections and a flat-mount specimen in Figure 2 . Immunohistochemical evaluation of BrdU incorporation in P6 flat-mounts revealed a discrete zone of dense BrdU-positive cells consistent with the neuroblast layer (Fig. 3a) , as supported by the presence of cells positive for Pax6, a marker for retinal progenitors, and BrdU immunoreactivity (Supplementary Fig. S1). The BrdU-positive cells were also immunoreactive for PCNA, a marker for the G1 to S phase of cell cycle, which confirms that they are proliferating (data not shown). However, concomitant staining of the flat-mounts with anti–rhodopsin antibody revealed that a part of the discrete zone of dense BrdU-positive cells roughly corresponded to the pars plana of the ciliary body, in which numbers of photoreceptor precursors were identified (Fig. 3b) . These cells—reactive to anti–rhodopsin and recoverin antibodies—never stained with anti–Pax6 antibodies. We failed to identify the presence of other classes of retinal neurons, namely bipolar or ganglion cells, using anti–PKC and Brn3 antibodies, respectively (data not shown). 
The BrdU-positive cells occupied a significant portion of the ciliary body up to P6 (Fig. 3b) , but their numbers decreased rapidly by P9, when the neuroblast layer was no longer identified in the retina and pars plana in many eyes (Fig. 3c) . Minor proportions of BrdU-positive cells at P6 and most of those after P9 were also rhodopsin positive. Their morphology 11 12 (Supplementary Fig. S2) and distinct location—they were present not within the retina or the ciliary epithelium but on the surfaces of these structures, they were associated with the presence of residual vitreous (Supplementary Fig. S2), and they were scattered in the pars plana and pars plicata—indicate that these cells were probably hyalocytes. Meanwhile, BrdU-negative rod photoreceptor precursors had a characteristic morphology and rhodopsin expression pattern (described here in detail), resided within the retina or ciliary epithelium, and were never identified in the pars plicata. Taken together, these results indicate that the rhodopsin staining in BrdU-positive cells was probably false positive. At P12, there were only sporadic BrdU-positive cells in the ciliary body (Fig. 3d) , some of which were also immunoreactive for Pax6 (Figs. 3e 3f) , suggesting that they maintained the potential to develop into retinal neurons. The BrdU-positive cells continued to decrease in number. 
Generation of Photoreceptor Precursors in the Pars Plana during Ocular Development
We studied the immunoreactivity for rhodopsin, a marker specific for rod photoreceptors, in the ciliary epithelium using flat-mounts. Within the pars plana, we found radially aligned postmitotic rhodopsin-positive cells with morphologic features of rod photoreceptor precursors (Figs. 3b 3c 4a 4b 4c 4f) . However, these cells were found only during retinal development. Unlike late rod photoreceptor precursors that express rhodopsin predominantly in the outer segment, rod photoreceptor precursors were immunoreactive for rhodopsin over the entire cell surface up to P6 (Figs. 4a 4b) . Many of these cells had one process with budding at the end, consistent with rod spherules, and another with appreciable inner and outer segment-like structures (Fig. 4c) . However, most of the rhodopsin-positive cells were not immunoreactive for recoverin (97.5% ± 2.7% [mean ± SD]; n = 6; Fig. 4b ), a marker for rod and cone photoreceptors. 13 14 This was in contrast to photoreceptors in the retinal margins of the same P6 mice, in which recoverin immunoreactivity was detected in nearly half the rhodopsin-positive cells (Fig. 4b) . By P9, when retinal histogenesis is almost complete, rod photoreceptor precursors were absent from a large portion of the ciliary epithelium, whereas the remaining cells resided within a narrow band along the pars plana (Figs. 3c 4f) . Most of these precursors were immunoreactive for rhodopsin predominantly in their outer segments (90.9% ± 4.4%; n = 5; Fig. 4f ), consistent with its expression pattern in mature rods. The visible outer segments were aligned radially, and some bridged the retina and pars plana. However, such rod photoreceptor precursors were no longer observed by P12 (n = 19) with the completion of retinal histogenesis. 15 16 17  
Meanwhile, at P6, most of the recoverin-positive cells in the ciliary epithelium had a uniform appearance; one or two processes were typically shorter than those of rod photoreceptor precursors in the retina (91.8% ± 7.7%; n = 5; Figs. 4d 4e ). These cells were probably cone photoreceptor precursors. As we confirmed in some of the cells, one of the processes was positive for peanut agglutinin (PNA; Fig. 4d ), a marker specific for the inner and outer segments and pedicles of cone photoreceptors. 18 19 This is consistent with previous reports indicating that in the developing eye, cone photoreceptor precursors express recoverin earlier than rod photoreceptor precursors. 13 14 Unfortunately, accurate evaluation of cone photoreceptor precursors was difficult because of the relatively weak signal and the high background of PNA staining, particularly during retinal development. 18 20 By P9, many recoverin-positive retinal precursors in the pars plana had developed a longer process (65.1% ± 17.1%; n = 4) and aligned radially (Figs. 4g 4h) . In addition to the appreciable inner and outer segment structures (Figs. 4h 4i) , some of these long processes showed PNA-positive terminal budding consistent with the pedicles of cone photoreceptors 18 19 20 (Supplementary Fig. S3). The observed morphologic features of recoverin-positive cells are in close agreement with immature cone precursors found in the developing retina, 20 which is summarized in Supplementary Figure S4. Some of these cells bridged the ciliary body and the retina. At P12, many recoverin-positive cells persisted in the pars plana, most of which had a detectable PNA-positive process (68.1% ± 4.4%; n = 6). Some areas of the pars plana still contained recoverin-positive cells at P18 (Fig. 5a) , but by P30 these cells were nearly absent from the ciliary epithelium (Figs. 5b 5c) . However, rare cells persisted in the pars plana up to at least P120 (data not shown). Most of these persistent recoverin-positive cells had morphology consistent with early cone photoreceptor precursors. 
Photoreceptors in the Pars Plana of Adult Mice with MNU-Induced Retinal Degeneration
Our findings suggest that the existence of rod and cone photoreceptor precursors in the ciliary epithelium of the pars plana is mostly restricted to a short period during retinal development in wild-type mice. In the brain, it is well known that neural stem cells proliferate in response to neural injury even in adult mammals. 21 22 23 The same is true for retinal progenitor/stem cells in mice with certain genetic conditions. 24 25 To examine whether photoreceptor precursors are identified in the adult ciliary epithelium, perhaps by neurogenesis and neural differentiation in mice with severe retinal injury, flat-mounts of the eyes from toxin-induced retinal degeneration were evaluated. We analyzed the pars plana of P75 C57Bl mice injected with MNU at P60. By 7 days after intraperitoneal injection of MNU, severe photoreceptor degeneration is observed in injected animals. 26 In addition to the reappearance of rare rhodopsin-positive rod photoreceptor precursors (data not shown), an increased number of recoverin-positive photoreceptor precursors in the pars plana was seen in MNU-treated mice compared with untreated control mice at P60 (10.7-fold; P = 2.1 × 10−5) or P75 (16.5-fold; P = 4.3 × 10−6; Fig. 6 ). Most of these cells had a prominent process with relatively large budding at the end, simulating cone pedicle. They were aligned randomly, in contrast to cells in the adjacent retina, which were organized radially (Fig. 6a) . Many of these cells also had a PNA-positive process. A few recoverin-positive cells had two relatively long processes with a small budding in one of its end resembling rod photoreceptors (Fig. 6a) . These cells were rarely found before MNU treatment and appeared to be consistent with newly generated photoreceptor precursors. However, we did not obtain direct evidence that these cells were generated from retinal progenitors after MNU injection, despite analyzing the eyes collected at variable times after BrdU labeling (data not shown). 
Discussion
Based on the analyses of flat-mounts and sections from the murine ciliary body and peripheral retina, it appears that at least part of the ciliary epithelium in the pars plana and neural retina are derived from the same neuroblast layer from which photoreceptor precursors are produced during postnatal ocular development in mice. We could not detect the presence of bipolar or ganglion precursors in the ciliary epithelium with the condition and antibodies used in this study. However, in the pars plana, presumptive early cone photoreceptor precursors were rarely found after P30. Interestingly, morphologically mature photoreceptor precursors were not found in the pars plana. The observations that some of the photoreceptor precursors bridged both the retina and the pars plana and that the precursors rapidly disappeared after retinal development raise the possibility that these cells migrate into the peripheral retina to participate in its histogenesis. It is also possible that these cells die in situ with no virtual significance. Meanwhile, our results suggest that the potential of ciliary epithelium to produce photoreceptor precursors may not be limited to ocular development under certain circumstances. In the pars plana of adult mice with MNU-induced retinal degeneration, increased numbers of photoreceptor precursors were found. The morphology of most of these cells was consistent with that of cone photoreceptor precursors with well-developed cone pedicles, but they lacked the prominent inner and outer segment structures seen frequently at approximately P9 in the pars plana and at P4 in the retina during ocular development 20 (Supplementary Fig. S3). However, we were able to find little evidence supporting their de novo neurogenesis from retinal progenitors or stem cells with pulse-chase study using BrdU (data not shown), possibly because of the adverse effect of BrdU incorporation on retinal progenitors, such as the inhibition of differentiation or death of these cells. 27 Meanwhile, it is possible that retinal precursors in the pars plana arose from cells difficult to detect with BrdU labeling, such as slowly cycling or quiescent retinal progenitors, postmitotic retinal precursors, a small number of rapidly dividing stem cells, or even ciliary epithelial cells by transdifferentiation. 
The role and distribution of reported multipotential retinal stem cells 4 5 remains unknown, but the relatively small number of photoreceptor precursors identified in the pars plana suggests that the physiological significance, if any, of these cells with regard to their ultimate contribution to retinal functional may be limited. This is unlike the adult CMZ in the retinal margin of the lower vertebrates, which contains numerous dividing retinal progenitors capable of replacing large number of various classes of neurons throughout the retina. 1 2 3  
In the pars plana, we found postmitotic photoreceptor precursors of late ontogenetic stages that have recently been shown to be an ideal source for photoreceptor transplantation. 28 The only other known sources of such cells are the immature retina. 28 29 30 Although fetal retinal tissue transplants yielded photoreceptors consistent with rod and cone photoreceptors, 29 30 cell transplants derived from the immature retina developed almost exclusively into rod photoreceptors 28 responsible for vision under dim light only. On the other hand, we found that large proportions of photoreceptor precursors generated in the pars plana were presumably of the cone photoreceptor lineage, which is by far the most important class of photoreceptors mediating daylight vision. Therefore, the use of retinal progenitors/precursors from the pars plana may have a significant advantage when considering cone photoreceptor cell replacement therapy for treating most types of photoreceptor degeneration. However, its simple clinical application may be hampered by the relatively small amount of collectable cells. 
In conclusion, at least a part of the ciliary epithelium in the pars plana is derived from the neuroblast layer. Rod and cone photoreceptor precursors are identified in the pars plana during murine retinal development and may participate in the histogenesis of the peripheral retina. In adult mice with severe retinal injury, increased numbers of photoreceptor precursors are also observed in the pars plana. These results raise the possibility that the pars plana may serve as a potential source for photoreceptor replacement therapy. 
 
Figure 1.
 
Distribution of BrdU-positive cells in the ciliary epithelium of the pars plana during ocular development in histologic sections. Double arrows indicate the pars plana. (a) At P6, the neuroblast layer composed of BrdU-positive retinal progenitors (red) is present in the eye section. BrdU was injected 2 hours before enucleation (applies to all the data presented in Fig. 1 ). (b) Enlarged image of (a) overlaid with DAPI staining (blue). (c) By P9, the numbers of BrdU-positive cells (red) in both the ciliary epithelium and the retina had decreased markedly. (d) The number of BrdU-positive cells in the entire ciliary epithelium and retina 200 μm from the cilioretinal margin during and after retinal histogenesis counted from eye sections (mean ± SEM). The number of BrdU-positive cells in the P6 retina was 106 ± 5, which is indicated by an off-the-scale bar (asterisk). (d) 8, 7, 7, and 7 animals were used at P6, P9, P12, and P18, respectively. Scale bar, 100 μm. NBL, neuroblast layer; CB, ciliary body; Cor, cornea; Scl, sclera; I, iris; CPE, ciliary body pigmented epithelium; RPE, retinal pigmented epithelium; Rho, rhodopsin.
Figure 1.
 
Distribution of BrdU-positive cells in the ciliary epithelium of the pars plana during ocular development in histologic sections. Double arrows indicate the pars plana. (a) At P6, the neuroblast layer composed of BrdU-positive retinal progenitors (red) is present in the eye section. BrdU was injected 2 hours before enucleation (applies to all the data presented in Fig. 1 ). (b) Enlarged image of (a) overlaid with DAPI staining (blue). (c) By P9, the numbers of BrdU-positive cells (red) in both the ciliary epithelium and the retina had decreased markedly. (d) The number of BrdU-positive cells in the entire ciliary epithelium and retina 200 μm from the cilioretinal margin during and after retinal histogenesis counted from eye sections (mean ± SEM). The number of BrdU-positive cells in the P6 retina was 106 ± 5, which is indicated by an off-the-scale bar (asterisk). (d) 8, 7, 7, and 7 animals were used at P6, P9, P12, and P18, respectively. Scale bar, 100 μm. NBL, neuroblast layer; CB, ciliary body; Cor, cornea; Scl, sclera; I, iris; CPE, ciliary body pigmented epithelium; RPE, retinal pigmented epithelium; Rho, rhodopsin.
Figure 2.
 
Comparison of ocular structures between sections and a flat-mount. (a) Normal histologic section containing the ciliary body and the retina. (ac) Upper and lower dashed lines correspond to the anterior and posterior border of the pars plana, respectively. (b) Histologic section of flat-mount specimen. Note that the ciliary processes of the pars plicata are displaced toward the iris. (c) Flat-mount of the ciliary body and the retina stained with anti-recoverin antibody at P6. Note that the retinal margin is demarcated by dense recoverin-immunopositive cells. Figures 3 4 5 6and S1–S3 are all images of flat-mounts and are presented in the same orientation as this but at different magnifications. Scale bar, 100 μm.
Figure 2.
 
Comparison of ocular structures between sections and a flat-mount. (a) Normal histologic section containing the ciliary body and the retina. (ac) Upper and lower dashed lines correspond to the anterior and posterior border of the pars plana, respectively. (b) Histologic section of flat-mount specimen. Note that the ciliary processes of the pars plicata are displaced toward the iris. (c) Flat-mount of the ciliary body and the retina stained with anti-recoverin antibody at P6. Note that the retinal margin is demarcated by dense recoverin-immunopositive cells. Figures 3 4 5 6and S1–S3 are all images of flat-mounts and are presented in the same orientation as this but at different magnifications. Scale bar, 100 μm.
Figure 3.
 
Distribution of BrdU-positive cells and rod photoreceptor precursors in the pars plana in flat-mounts. (a) At P6, a distinct layer composed of BrdU-positive cells (red), the neuroblast layer, was identified. (b) Merged image with (a) showing BrdU (red) and rhodopsin (green) labeling. Numerous rhodopsin-positive rod photoreceptor precursors (filled arrowheads) are identified in the pars plana. Note that the retinal margin (open arrowheads) is clearly distinguished by an alteration in the density of rhodopsin-positive cells relative to the adjacent ciliary epithelium. (c) By P9, the pars plana contained sporadic BrdU-positive cells (red). Note that only the inner/outer segments of the rod photoreceptor precursors stain with anti-rhodopsin antibodies (green; open arrowheads) at this age. (d) Rod photoreceptor precursors were no longer seen in the pars plana at P12. Round to oval cells double-positive for rhodopsin and BrdU (c, d; filled arrowheads) are likely hyalocytes based on their location and morphology (Supplementary Fig. S2). 11 12 Rod photoreceptor precursors with evidence of morphologic differentiation were never labeled with BrdU. (e, f) Some BrdU-positive cells (red) in the pars plana were also immunoreactive for Pax6 (green; double-positive cells are indicated with filled arrowheads). Densely and evenly distributed postmitotic (BrdU-negative) Pax6-positive cells in the retinal surface are consistent with ganglion/amacrine cells. 31 32 Note that retinal vessels showed false-positive staining (open arrowheads). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; PP, pars plana.
Figure 3.
 
Distribution of BrdU-positive cells and rod photoreceptor precursors in the pars plana in flat-mounts. (a) At P6, a distinct layer composed of BrdU-positive cells (red), the neuroblast layer, was identified. (b) Merged image with (a) showing BrdU (red) and rhodopsin (green) labeling. Numerous rhodopsin-positive rod photoreceptor precursors (filled arrowheads) are identified in the pars plana. Note that the retinal margin (open arrowheads) is clearly distinguished by an alteration in the density of rhodopsin-positive cells relative to the adjacent ciliary epithelium. (c) By P9, the pars plana contained sporadic BrdU-positive cells (red). Note that only the inner/outer segments of the rod photoreceptor precursors stain with anti-rhodopsin antibodies (green; open arrowheads) at this age. (d) Rod photoreceptor precursors were no longer seen in the pars plana at P12. Round to oval cells double-positive for rhodopsin and BrdU (c, d; filled arrowheads) are likely hyalocytes based on their location and morphology (Supplementary Fig. S2). 11 12 Rod photoreceptor precursors with evidence of morphologic differentiation were never labeled with BrdU. (e, f) Some BrdU-positive cells (red) in the pars plana were also immunoreactive for Pax6 (green; double-positive cells are indicated with filled arrowheads). Densely and evenly distributed postmitotic (BrdU-negative) Pax6-positive cells in the retinal surface are consistent with ganglion/amacrine cells. 31 32 Note that retinal vessels showed false-positive staining (open arrowheads). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; PP, pars plana.
Figure 4.
 
Postmitotic rod and cone photoreceptor precursors in the pars plana in flat-mounts. Double arrows: pars plana. (a) At P6, within the pars plana, rhodopsin-positive (green) rod photoreceptor precursors (filled arrowhead) were found between BrdU-positive cells (red). BrdU was injected 2 hours before enucleation, which applies also to (d). (b) Rod photoreceptor precursors (green; filled arrowhead) in the pars plana were negative for recoverin immunoreactivity (blue), whereas many of those in the retina were positive (open arrowhead). (c) Schematic illustration of a rod photoreceptor precursor labeled with a filled arrowhead in (a, b). (d) At P6, many recoverin-positive cells (blue) with a short PNA-positive process (green; filled arrowhead) were found between BrdU-positive cells (red). (e) A selected area with recoverin-positive cells (green) in both the pars plana (open arrowhead) and the pars plicata (filled arrow) at P6. (f) At P9, rhodopsin immunoreactivity (green) was localized to the outer segments (filled arrowhead) of rod photoreceptor precursors. (g) At P9, recoverin-positive cells (green) developed a long process in addition to the short process positive for PNA (red). (h) Many recoverin-positive cells (green) with a long process were aligned perpendicularly to the retinal margin. (i) Schematic illustration of a recoverin-positive cell labeled with a filled arrowhead in (h). Scale bar, 50 μm. CB, ciliary body; PP, pars plana; Rho, rhodopsin; Rcv, recoverin; IS, inner segment; OS, outer segment.
Figure 4.
 
Postmitotic rod and cone photoreceptor precursors in the pars plana in flat-mounts. Double arrows: pars plana. (a) At P6, within the pars plana, rhodopsin-positive (green) rod photoreceptor precursors (filled arrowhead) were found between BrdU-positive cells (red). BrdU was injected 2 hours before enucleation, which applies also to (d). (b) Rod photoreceptor precursors (green; filled arrowhead) in the pars plana were negative for recoverin immunoreactivity (blue), whereas many of those in the retina were positive (open arrowhead). (c) Schematic illustration of a rod photoreceptor precursor labeled with a filled arrowhead in (a, b). (d) At P6, many recoverin-positive cells (blue) with a short PNA-positive process (green; filled arrowhead) were found between BrdU-positive cells (red). (e) A selected area with recoverin-positive cells (green) in both the pars plana (open arrowhead) and the pars plicata (filled arrow) at P6. (f) At P9, rhodopsin immunoreactivity (green) was localized to the outer segments (filled arrowhead) of rod photoreceptor precursors. (g) At P9, recoverin-positive cells (green) developed a long process in addition to the short process positive for PNA (red). (h) Many recoverin-positive cells (green) with a long process were aligned perpendicularly to the retinal margin. (i) Schematic illustration of a recoverin-positive cell labeled with a filled arrowhead in (h). Scale bar, 50 μm. CB, ciliary body; PP, pars plana; Rho, rhodopsin; Rcv, recoverin; IS, inner segment; OS, outer segment.
Figure 5.
 
Rare recoverin-positive photoreceptor precursors in the pars plana after retinal histogenesis. Double arrows: pars plana. (a) Recoverin-positive cells with a short process (arrowhead) persisted in the pars plana and the pars plicata at P18. (b) At P30, there were only rare recoverin-positive retinal precursors in the retina. Most of these cells had irregular to oval cell bodies with a short single process, with a small budding at its end morphologically consistent with early cone photoreceptor precursors first seen in the embryonic retina. 20 (c) There were no recoverin-positive cells in most parts of the pars plana in P30 mice. Scale bar, 50 μm. CB, ciliary body; Rcv, recoverin.
Figure 5.
 
Rare recoverin-positive photoreceptor precursors in the pars plana after retinal histogenesis. Double arrows: pars plana. (a) Recoverin-positive cells with a short process (arrowhead) persisted in the pars plana and the pars plicata at P18. (b) At P30, there were only rare recoverin-positive retinal precursors in the retina. Most of these cells had irregular to oval cell bodies with a short single process, with a small budding at its end morphologically consistent with early cone photoreceptor precursors first seen in the embryonic retina. 20 (c) There were no recoverin-positive cells in most parts of the pars plana in P30 mice. Scale bar, 50 μm. CB, ciliary body; Rcv, recoverin.
Figure 6.
 
Photoreceptor precursors in the pars plana of mice with MNU-induced retinal degeneration. (a) At P75, an increased number of recoverin-positive photoreceptor precursors were identified in the pars plana (double arrow) of mice treated with MNU. Most of these cells had a single prominent process with relatively large budding at its end resembling cone pedicle (filled arrowhead). Rare cells had two processes, with small budding at one end simulating rod photoreceptor precursors (open arrowhead). Recoverin-positive cells in the pars plana were aligned randomly, whereas most of those in the peripheral retina were organized radially. Note that recoverin-positive cells at the retinal margin had prominent inner/outer segment structures and were morphologically consistent with mature cone photoreceptors (filled arrow). (b) An increased number of recoverin-positive photoreceptor precursors (mean ± SEM) was observed in the pars plana of P75 wild-type mice treated with MNU at P60 (n = 7) compared with untreated litters at P60 (10.7-fold; n = 6) or P75 (16.5-fold; n = 7). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; Rcv, recoverin; WT, wild-type.
Figure 6.
 
Photoreceptor precursors in the pars plana of mice with MNU-induced retinal degeneration. (a) At P75, an increased number of recoverin-positive photoreceptor precursors were identified in the pars plana (double arrow) of mice treated with MNU. Most of these cells had a single prominent process with relatively large budding at its end resembling cone pedicle (filled arrowhead). Rare cells had two processes, with small budding at one end simulating rod photoreceptor precursors (open arrowhead). Recoverin-positive cells in the pars plana were aligned randomly, whereas most of those in the peripheral retina were organized radially. Note that recoverin-positive cells at the retinal margin had prominent inner/outer segment structures and were morphologically consistent with mature cone photoreceptors (filled arrow). (b) An increased number of recoverin-positive photoreceptor precursors (mean ± SEM) was observed in the pars plana of P75 wild-type mice treated with MNU at P60 (n = 7) compared with untreated litters at P60 (10.7-fold; n = 6) or P75 (16.5-fold; n = 7). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; Rcv, recoverin; WT, wild-type.
Supplementary Materials
The authors thank Thaddeus P. Dryja and Motokazu Tsujikawa for helpful discussions and advice regarding this manuscript. Anti–Pax6 monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences at the University of Iowa (Iowa City, IA). 
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Figure 1.
 
Distribution of BrdU-positive cells in the ciliary epithelium of the pars plana during ocular development in histologic sections. Double arrows indicate the pars plana. (a) At P6, the neuroblast layer composed of BrdU-positive retinal progenitors (red) is present in the eye section. BrdU was injected 2 hours before enucleation (applies to all the data presented in Fig. 1 ). (b) Enlarged image of (a) overlaid with DAPI staining (blue). (c) By P9, the numbers of BrdU-positive cells (red) in both the ciliary epithelium and the retina had decreased markedly. (d) The number of BrdU-positive cells in the entire ciliary epithelium and retina 200 μm from the cilioretinal margin during and after retinal histogenesis counted from eye sections (mean ± SEM). The number of BrdU-positive cells in the P6 retina was 106 ± 5, which is indicated by an off-the-scale bar (asterisk). (d) 8, 7, 7, and 7 animals were used at P6, P9, P12, and P18, respectively. Scale bar, 100 μm. NBL, neuroblast layer; CB, ciliary body; Cor, cornea; Scl, sclera; I, iris; CPE, ciliary body pigmented epithelium; RPE, retinal pigmented epithelium; Rho, rhodopsin.
Figure 1.
 
Distribution of BrdU-positive cells in the ciliary epithelium of the pars plana during ocular development in histologic sections. Double arrows indicate the pars plana. (a) At P6, the neuroblast layer composed of BrdU-positive retinal progenitors (red) is present in the eye section. BrdU was injected 2 hours before enucleation (applies to all the data presented in Fig. 1 ). (b) Enlarged image of (a) overlaid with DAPI staining (blue). (c) By P9, the numbers of BrdU-positive cells (red) in both the ciliary epithelium and the retina had decreased markedly. (d) The number of BrdU-positive cells in the entire ciliary epithelium and retina 200 μm from the cilioretinal margin during and after retinal histogenesis counted from eye sections (mean ± SEM). The number of BrdU-positive cells in the P6 retina was 106 ± 5, which is indicated by an off-the-scale bar (asterisk). (d) 8, 7, 7, and 7 animals were used at P6, P9, P12, and P18, respectively. Scale bar, 100 μm. NBL, neuroblast layer; CB, ciliary body; Cor, cornea; Scl, sclera; I, iris; CPE, ciliary body pigmented epithelium; RPE, retinal pigmented epithelium; Rho, rhodopsin.
Figure 2.
 
Comparison of ocular structures between sections and a flat-mount. (a) Normal histologic section containing the ciliary body and the retina. (ac) Upper and lower dashed lines correspond to the anterior and posterior border of the pars plana, respectively. (b) Histologic section of flat-mount specimen. Note that the ciliary processes of the pars plicata are displaced toward the iris. (c) Flat-mount of the ciliary body and the retina stained with anti-recoverin antibody at P6. Note that the retinal margin is demarcated by dense recoverin-immunopositive cells. Figures 3 4 5 6and S1–S3 are all images of flat-mounts and are presented in the same orientation as this but at different magnifications. Scale bar, 100 μm.
Figure 2.
 
Comparison of ocular structures between sections and a flat-mount. (a) Normal histologic section containing the ciliary body and the retina. (ac) Upper and lower dashed lines correspond to the anterior and posterior border of the pars plana, respectively. (b) Histologic section of flat-mount specimen. Note that the ciliary processes of the pars plicata are displaced toward the iris. (c) Flat-mount of the ciliary body and the retina stained with anti-recoverin antibody at P6. Note that the retinal margin is demarcated by dense recoverin-immunopositive cells. Figures 3 4 5 6and S1–S3 are all images of flat-mounts and are presented in the same orientation as this but at different magnifications. Scale bar, 100 μm.
Figure 3.
 
Distribution of BrdU-positive cells and rod photoreceptor precursors in the pars plana in flat-mounts. (a) At P6, a distinct layer composed of BrdU-positive cells (red), the neuroblast layer, was identified. (b) Merged image with (a) showing BrdU (red) and rhodopsin (green) labeling. Numerous rhodopsin-positive rod photoreceptor precursors (filled arrowheads) are identified in the pars plana. Note that the retinal margin (open arrowheads) is clearly distinguished by an alteration in the density of rhodopsin-positive cells relative to the adjacent ciliary epithelium. (c) By P9, the pars plana contained sporadic BrdU-positive cells (red). Note that only the inner/outer segments of the rod photoreceptor precursors stain with anti-rhodopsin antibodies (green; open arrowheads) at this age. (d) Rod photoreceptor precursors were no longer seen in the pars plana at P12. Round to oval cells double-positive for rhodopsin and BrdU (c, d; filled arrowheads) are likely hyalocytes based on their location and morphology (Supplementary Fig. S2). 11 12 Rod photoreceptor precursors with evidence of morphologic differentiation were never labeled with BrdU. (e, f) Some BrdU-positive cells (red) in the pars plana were also immunoreactive for Pax6 (green; double-positive cells are indicated with filled arrowheads). Densely and evenly distributed postmitotic (BrdU-negative) Pax6-positive cells in the retinal surface are consistent with ganglion/amacrine cells. 31 32 Note that retinal vessels showed false-positive staining (open arrowheads). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; PP, pars plana.
Figure 3.
 
Distribution of BrdU-positive cells and rod photoreceptor precursors in the pars plana in flat-mounts. (a) At P6, a distinct layer composed of BrdU-positive cells (red), the neuroblast layer, was identified. (b) Merged image with (a) showing BrdU (red) and rhodopsin (green) labeling. Numerous rhodopsin-positive rod photoreceptor precursors (filled arrowheads) are identified in the pars plana. Note that the retinal margin (open arrowheads) is clearly distinguished by an alteration in the density of rhodopsin-positive cells relative to the adjacent ciliary epithelium. (c) By P9, the pars plana contained sporadic BrdU-positive cells (red). Note that only the inner/outer segments of the rod photoreceptor precursors stain with anti-rhodopsin antibodies (green; open arrowheads) at this age. (d) Rod photoreceptor precursors were no longer seen in the pars plana at P12. Round to oval cells double-positive for rhodopsin and BrdU (c, d; filled arrowheads) are likely hyalocytes based on their location and morphology (Supplementary Fig. S2). 11 12 Rod photoreceptor precursors with evidence of morphologic differentiation were never labeled with BrdU. (e, f) Some BrdU-positive cells (red) in the pars plana were also immunoreactive for Pax6 (green; double-positive cells are indicated with filled arrowheads). Densely and evenly distributed postmitotic (BrdU-negative) Pax6-positive cells in the retinal surface are consistent with ganglion/amacrine cells. 31 32 Note that retinal vessels showed false-positive staining (open arrowheads). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; PP, pars plana.
Figure 4.
 
Postmitotic rod and cone photoreceptor precursors in the pars plana in flat-mounts. Double arrows: pars plana. (a) At P6, within the pars plana, rhodopsin-positive (green) rod photoreceptor precursors (filled arrowhead) were found between BrdU-positive cells (red). BrdU was injected 2 hours before enucleation, which applies also to (d). (b) Rod photoreceptor precursors (green; filled arrowhead) in the pars plana were negative for recoverin immunoreactivity (blue), whereas many of those in the retina were positive (open arrowhead). (c) Schematic illustration of a rod photoreceptor precursor labeled with a filled arrowhead in (a, b). (d) At P6, many recoverin-positive cells (blue) with a short PNA-positive process (green; filled arrowhead) were found between BrdU-positive cells (red). (e) A selected area with recoverin-positive cells (green) in both the pars plana (open arrowhead) and the pars plicata (filled arrow) at P6. (f) At P9, rhodopsin immunoreactivity (green) was localized to the outer segments (filled arrowhead) of rod photoreceptor precursors. (g) At P9, recoverin-positive cells (green) developed a long process in addition to the short process positive for PNA (red). (h) Many recoverin-positive cells (green) with a long process were aligned perpendicularly to the retinal margin. (i) Schematic illustration of a recoverin-positive cell labeled with a filled arrowhead in (h). Scale bar, 50 μm. CB, ciliary body; PP, pars plana; Rho, rhodopsin; Rcv, recoverin; IS, inner segment; OS, outer segment.
Figure 4.
 
Postmitotic rod and cone photoreceptor precursors in the pars plana in flat-mounts. Double arrows: pars plana. (a) At P6, within the pars plana, rhodopsin-positive (green) rod photoreceptor precursors (filled arrowhead) were found between BrdU-positive cells (red). BrdU was injected 2 hours before enucleation, which applies also to (d). (b) Rod photoreceptor precursors (green; filled arrowhead) in the pars plana were negative for recoverin immunoreactivity (blue), whereas many of those in the retina were positive (open arrowhead). (c) Schematic illustration of a rod photoreceptor precursor labeled with a filled arrowhead in (a, b). (d) At P6, many recoverin-positive cells (blue) with a short PNA-positive process (green; filled arrowhead) were found between BrdU-positive cells (red). (e) A selected area with recoverin-positive cells (green) in both the pars plana (open arrowhead) and the pars plicata (filled arrow) at P6. (f) At P9, rhodopsin immunoreactivity (green) was localized to the outer segments (filled arrowhead) of rod photoreceptor precursors. (g) At P9, recoverin-positive cells (green) developed a long process in addition to the short process positive for PNA (red). (h) Many recoverin-positive cells (green) with a long process were aligned perpendicularly to the retinal margin. (i) Schematic illustration of a recoverin-positive cell labeled with a filled arrowhead in (h). Scale bar, 50 μm. CB, ciliary body; PP, pars plana; Rho, rhodopsin; Rcv, recoverin; IS, inner segment; OS, outer segment.
Figure 5.
 
Rare recoverin-positive photoreceptor precursors in the pars plana after retinal histogenesis. Double arrows: pars plana. (a) Recoverin-positive cells with a short process (arrowhead) persisted in the pars plana and the pars plicata at P18. (b) At P30, there were only rare recoverin-positive retinal precursors in the retina. Most of these cells had irregular to oval cell bodies with a short single process, with a small budding at its end morphologically consistent with early cone photoreceptor precursors first seen in the embryonic retina. 20 (c) There were no recoverin-positive cells in most parts of the pars plana in P30 mice. Scale bar, 50 μm. CB, ciliary body; Rcv, recoverin.
Figure 5.
 
Rare recoverin-positive photoreceptor precursors in the pars plana after retinal histogenesis. Double arrows: pars plana. (a) Recoverin-positive cells with a short process (arrowhead) persisted in the pars plana and the pars plicata at P18. (b) At P30, there were only rare recoverin-positive retinal precursors in the retina. Most of these cells had irregular to oval cell bodies with a short single process, with a small budding at its end morphologically consistent with early cone photoreceptor precursors first seen in the embryonic retina. 20 (c) There were no recoverin-positive cells in most parts of the pars plana in P30 mice. Scale bar, 50 μm. CB, ciliary body; Rcv, recoverin.
Figure 6.
 
Photoreceptor precursors in the pars plana of mice with MNU-induced retinal degeneration. (a) At P75, an increased number of recoverin-positive photoreceptor precursors were identified in the pars plana (double arrow) of mice treated with MNU. Most of these cells had a single prominent process with relatively large budding at its end resembling cone pedicle (filled arrowhead). Rare cells had two processes, with small budding at one end simulating rod photoreceptor precursors (open arrowhead). Recoverin-positive cells in the pars plana were aligned randomly, whereas most of those in the peripheral retina were organized radially. Note that recoverin-positive cells at the retinal margin had prominent inner/outer segment structures and were morphologically consistent with mature cone photoreceptors (filled arrow). (b) An increased number of recoverin-positive photoreceptor precursors (mean ± SEM) was observed in the pars plana of P75 wild-type mice treated with MNU at P60 (n = 7) compared with untreated litters at P60 (10.7-fold; n = 6) or P75 (16.5-fold; n = 7). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; Rcv, recoverin; WT, wild-type.
Figure 6.
 
Photoreceptor precursors in the pars plana of mice with MNU-induced retinal degeneration. (a) At P75, an increased number of recoverin-positive photoreceptor precursors were identified in the pars plana (double arrow) of mice treated with MNU. Most of these cells had a single prominent process with relatively large budding at its end resembling cone pedicle (filled arrowhead). Rare cells had two processes, with small budding at one end simulating rod photoreceptor precursors (open arrowhead). Recoverin-positive cells in the pars plana were aligned randomly, whereas most of those in the peripheral retina were organized radially. Note that recoverin-positive cells at the retinal margin had prominent inner/outer segment structures and were morphologically consistent with mature cone photoreceptors (filled arrow). (b) An increased number of recoverin-positive photoreceptor precursors (mean ± SEM) was observed in the pars plana of P75 wild-type mice treated with MNU at P60 (n = 7) compared with untreated litters at P60 (10.7-fold; n = 6) or P75 (16.5-fold; n = 7). Scale bar, 100 μm. CB, ciliary body; Rho, rhodopsin; Rcv, recoverin; WT, wild-type.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
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