March 2016
Volume 57, Issue 3
Open Access
Retinal Cell Biology  |   March 2016
DNA Damage Response in Proliferating Müller Glia in the Mammalian Retina
Author Affiliations & Notes
  • Kaori Nomura-Komoike
    Department of Anatomy School of Medicine, Tokyo Women's Medical University, Tokyo, Japan
  • Fuminori Saitoh
    Department of Anatomy School of Medicine, Tokyo Women's Medical University, Tokyo, Japan
  • Yuta Komoike
    Department of Hygiene and Public Health I, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan
  • Hiroki Fujieda
    Department of Anatomy School of Medicine, Tokyo Women's Medical University, Tokyo, Japan
  • Correspondence: Hiroki Fujieda, Department of Anatomy, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan; hfujieda@research.twmu.ac.jp
Investigative Ophthalmology & Visual Science March 2016, Vol.57, 1169-1182. doi:10.1167/iovs.15-18101
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      Kaori Nomura-Komoike, Fuminori Saitoh, Yuta Komoike, Hiroki Fujieda; DNA Damage Response in Proliferating Müller Glia in the Mammalian Retina. Invest. Ophthalmol. Vis. Sci. 2016;57(3):1169-1182. doi: 10.1167/iovs.15-18101.

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

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Abstract

Purpose: Müller glia, the principal glial cell type in the retina, have the potential to proliferate and regenerate neurons after retinal damage. However, unlike the situation in fish and birds, this capacity of Müller glia is extremely limited in mammals. To gain new insights into the mechanisms that hamper retinal regeneration in mammals, we examined the cell cycle progression and DNA damage response in Müller glia after retinal damage.

Methods: Expression of cell cycle-related proteins and DNA damage response were analyzed in adult rat and mouse retinas after N-methyl-N-nitrosourea (MNU)- or N-methyl-D-aspartate (NMDA)-induced retinal damage. Zebrafish and postnatal rat retinas were also investigated for comparison. Analysis was conducted by using immunofluorescence, Western blotting, and quantitative real-time polymerase chain reaction.

Results: In the rat retina, most Müller glia reentered the cell cycle after MNU-induced photoreceptor damage while no proliferative response was observed in the mouse model. Cell cycle reentry of rat Müller glia was accompanied by DNA damage response including the phosphorylation of the histone variant H2AX and upregulation of p53 and p21. The DNA damage response was also observed in rat Müller glia after NMDA-induced loss of inner retinal neurons, but not in zebrafish Müller glia or rat retinal progenitor cells.

Conclusions: Our findings suggest that the DNA damage response induced by unscheduled cell cycle reentry may be one of the mechanisms that limit the proliferative and regenerative capacity of Müller glia in the mammalian retina.

Müller glia, the principal glial cell type in the retina, have a range of functions to support retinal neurons, comparable to those of astrocytes in the brain.1 Recent evidence has further indicated that Müller glia have the potential to regenerate neurons in certain pathologic conditions. In the fish retina, damage stimulates Müller glia to dedifferentiate, proliferate, and regenerate retinal neurons to completely restore the lost tissue.2,3 Although less robust, retinal regeneration by Müller glia is also reported in the postnatal chick retina.4 Unlike the situation in fish and birds, the regenerative capacity of Müller glia is extremely limited in mammals.5 Treatment with mitogenic factors such as Wnt, Sonic hedgehog, and EGF has been shown to promote the regenerative response of mammalian Müller glia69; however, the regenerative response of mammalian retina is insufficient, even after mitogenic stimulation, to restore the structure and function of the damaged retina. 
One of the key barriers to regeneration in the mammalian retina is the extremely limited proliferative potential of Müller glia in vivo.5,10,11 When Müller glia proliferate in response to damage, most of them form glial scars, contributing to the pathogenesis of proliferative vitreoretinopathy (PVR).12 Moreover, only a small fraction of the progeny of Müller glia divisions survives.5,8,13 The mechanism responsible for the limited proliferation and survival of mammalian Müller glia is currently unknown. A better understanding of the molecular events occurring in Müller glia after retinal damage may provide important insights into how to improve the poor regenerative response of the mammalian retina. 
In the present study, we exploited the rodent model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU) treatment and carefully monitored the expression of various cell cycle markers in Müller glia as they reenter and exit the cell cycle. In the rat retina, most Müller glia reentered the cell cycle after photoreceptor damage, while in the mouse retina, no proliferative response was observed. Interestingly, cell cycle reentry of rat Müller glia was accompanied by DNA damage response such as the phosphorylation of the histone variant H2AX and p53 activation. These findings suggest that the DNA damage response may be one of the mechanisms that limit the proliferation and survival of Müller glia in the mammalian retina. 
Materials and Methods
Animals and Tissue Preparation
Male C57BL/6J mice (5 weeks old) and Wistar rats (5 weeks old) obtained from Charles River Laboratories Japan (Yokohama, Japan) were maintained under a 12:12-hour light:dark cycle with food and water ad libitum. Animals were killed by decapitation or cervical dislocation under anesthesia with isoflurane. Adult zebrafish (Danio rerio) approximately 20 months of age were maintained in a circulating water tank system at 28.5°C under a 14:10-hour light:dark cycle. Fish were killed by decapitation under anesthesia with ice-cold water. 
For immunohistochemistry, the eyecups with the cornea and lens removed were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour, rinsed in 15% and 30% sucrose in phosphate buffer, and frozen with dry ice–isopentane. Cryostat sections were cut at 10 μm through the optic disc along the dorsoventral axis and collected on MAS-coated glass slides (Matsunami Glass, Osaka, Japan). For Western blotting and RT-PCR, the retinas were dissected and kept frozen at −80°C until use. All experimental procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the research protocols approved by the institutional animal care committee. 
Induction of Retinal Damage
To induce photoreceptor damage, mice and rats received a single intraperitoneal injection of MNU (70 mg/kg body weight; Sigma-Aldrich Corp., St. Louis, MO, USA) as reported previously.14 Zebrafish were exposed to 150 mg/L MNU dissolved in tank water for 1 hour, as reported previously.15 
To induce cell death of inner retinal neurons, rats were intravitreally injected with 5 μL 40 mM N-methyl-D-aspartate (NMDA; Nacalai tesque, Kyoto, Japan) as reported previously,5 under anesthesia with sodium pentobarbital (35 mg/kg, intraperitoneal) supplemented with topical application of a few drops of 0.4% oxybuprocaine. 
BrdU Incorporation Assay
To label S-phase cells, mice and rats received a single intraperitoneal injection of bromodeoxyuridine (BrdU; 100 mg/kg body weight; Sigma-Aldrich Corp.) 2 hours before euthanasia. To track the fate of BrdU-labeled cells, some animals injected with BrdU were allowed to survive for various periods of time before euthanasia. Zebrafish were treated with 10 mM BrdU dissolved in tank water for 1 hour before euthanasia. 
Immunofluorescence
Immunofluorescence was conducted as described previously.16 For BrdU labeling, sections were treated with 2 M HCl at 37°C for 30 minutes prior to incubation with primary antibodies. Primary antibodies are listed in Supplementary Table S1. Secondary antibodies included donkey anti-mouse IgG (Alexa Fluor 488 and 555), donkey anti-rabbit IgG (Alexa Fluor 488 and 555), donkey anti-rat IgG (Alexa Fluor 594), and donkey anti-goat IgG (Alexa Fluor 647), all of which were purchased from Invitrogen (Eugene, OR, USA). Nuclear counterstaining was conducted with 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich Corp.). Fluorescence images were obtained using a fluorescence microscope (DM IL LED; Leica, Tokyo, Japan) and confocal laser scanning microscope (LSM710; Carl Zeiss, Jena, Germany). 
TUNEL Assay
TUNEL assay was performed using the in situ cell death detection kit, TMR red (Roche, Mannheim, Germany), according to the manufacturer's instructions. 
Cell Counting
Cells immunoreactive for specific cell markers were counted on vertically sliced retinal sections. Three sections per animal containing the optic nerve head were selected for analysis (three animals per stage). Two images per section (each measuring 650 by 650 μm) were captured from the central region adjacent to the optic nerve head under the Leica fluorescence microscope using a ×20 objective lens. Immunoreactive cells were counted and the density was calculated per millimeter retina. Due to their paucity, phosphorylated histone H3 (pH3)-positive cells were counted in the whole section. 
Western Blotting
Western blotting was conducted as described previously.17 Briefly, tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% Deoxycholate, and 0.1% SDS. The lysate was subject to SDS-PAGE and immunoblot analysis. Primary antibodies are listed in Supplementary Table S1. Secondary antibodies included horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG, HRP-donkey anti-mouse IgG, and HRP-donkey anti-goat IgG, all of which were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Proteins were visualized by enhanced chemiluminescence reagent Immunostar (WAKO, Tokyo, Japan) and visualized with a charge-coupled device video camera system (LumiVision PRO 400EX; AISIN, Aichi, Japan). 
Quantitative (Real-Time) RT-PCR
Retinas from both eyes of individual animals were pooled, from which total RNA was isolated using the RNeasy kit (Qiagen, Tokyo, Japan). RNA was reverse transcribed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover Kit (Toyobo, Osaka, Japan). Quantitative PCR was carried out by StepOnePlus (Applied Biosystems, Tokyo, Japan) using THUNDERBIRD SYBR qPCR Mix (Toyobo). The list of primers is shown in Supplementary Table S2. Data were normalized to Gapdh expression and shown relative to control levels. 
Statistical Analysis
Statistical analysis was conducted by 1-way analysis of variance (ANOVA) followed by Tukey's post hoc comparisons using the SPSS software (IBM, Armonk, NY, USA). Significance was evaluated at P < 0.05. 
Results
Cell Cycle Reentry of Müller Glia After MNU-Induced Photoreceptor Damage in the Rat Retina
We first performed TUNEL assays to examine the time course of photoreceptor damage in the mouse and rat retinas after MNU treatment. In both species, most, if not all, photoreceptors became TUNEL+ by day 1 after MNU treatment, and the labeling in the outer nuclear layer (ONL) was even more intense at day 2 (Fig. 1A, Supplementary Fig. S1A). Most TUNEL+ photoreceptor cells were eliminated by day 3 in the rat retinas (Fig. 1A). We also analyzed the expression of photoreceptor genes by quantitative RT-PCR (qRT-PCR) and confirmed that, in both species, the expression of rod (Rho and Gnat1) and cone genes (Opn1sw and Opn1mw) substantially decreased by day 1, followed by an almost complete loss of their expression by day 2 (Fig. 1B, Supplementary Fig. S1B). On the other hand, the expression of Calb1 (horizontal and amacrine cells18), Calb2 (amacrine cells18), and Brn3a (ganglion cells19) did not differ significantly between the control and MNU-treated rat retinas (day 3) while the Vsx2 (bipolar cells and Müller glia20,21) levels were significantly elevated after MNU treatment, most likely reflecting the progenitor-like changes of Müller glia after retinal damage22 (Supplementary Fig. S2). 
Figure 1
 
Induction of photoreceptor-specific cell death by MNU treatment. (A) TUNEL assay in the rat retina. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Real-time PCR analysis of photoreceptor genes in the rat retinas after MNU treatment. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01.
Figure 1
 
Induction of photoreceptor-specific cell death by MNU treatment. (A) TUNEL assay in the rat retina. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Real-time PCR analysis of photoreceptor genes in the rat retinas after MNU treatment. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01.
We next conducted immunofluorescence for cell cycle markers to assess whether MNU-induced photoreceptor damage causes cell cycle reentry of Müller glia. Phosphorylated histone H3 and BrdU were used as M- and S-phase markers, respectively. Geminin was also used to label S/G2/M-phase cells.23 These cell cycle markers were colabeled with the Müller glia markers S100β,24 Sox9,25 or Lhx2.26 We confirmed that these Müller glia markers were almost completely colocalized in the control and MNU-treated rat retinas (Supplementary Fig. S3). In the mouse retinas, no labeling for pH3 or BrdU was observed at any stage after MNU treatment (Supplementary Fig. S1C). In the rat retinas, in comparison, pH3+ Müller glia were first detected at day 2.5, peaked in number at day 3, and decreased drastically by day 3.5 (Figs. 2A, 2B). Incorporation of BrdU was also detected first at day 2.5, when more than 60% of Müller glia were labeled for BrdU (Figs. 2A, 2B). The proportion of BrdU+ Müller glia was subsequently decreased to approximately 30% at day 3 and 5% at day 3.5 (Figs. 2A, 2B). Geminin+ Müller glia were observed at days 2.5 and 3, confirming the results with pH3 and BrdU (Fig. 2A). Notably, the nuclei of many Müller glia were displaced in the ONL by day 3.5 (Fig. 2A). 
Figure 2
 
Cell cycle reentry of Müller glia after MNU-induced photoreceptor damage in the rat retina. (A) Double immunofluorescence for cell cycle markers (phospho-histone H3 [pH3], BrdU, and geminin) and Müller glia markers (S100β [S100], Sox9, and Lhx2). DAPI images are shown only for pH3/S100 staining. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer. (B) The number of pH3+ Müller glia per retinal section and the proportion of BrdU+ Müller glia. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 2
 
Cell cycle reentry of Müller glia after MNU-induced photoreceptor damage in the rat retina. (A) Double immunofluorescence for cell cycle markers (phospho-histone H3 [pH3], BrdU, and geminin) and Müller glia markers (S100β [S100], Sox9, and Lhx2). DAPI images are shown only for pH3/S100 staining. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer. (B) The number of pH3+ Müller glia per retinal section and the proportion of BrdU+ Müller glia. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
The above cell cycle markers label S, G2, and M phase, but not G1 phase. To visualize G1 phase, we conducted triple immunofluorescence for cyclin-dependent kinase 4 (CDK4) and cyclin D1, the core components of early G1-phase progression,27 in combination with the Müller glia marker Lhx2. Müller glia in the control rat retinas exhibited faint labeling for cyclin D1 but lacked CDK4 (Fig. 3A). Intense labeling for CDK4 was detected in the outer plexiform layer of the control retinas, which we considered nonspecific because of the lack of immunoreactivity by Western blotting (see below). Strikingly, intense CDK4 staining was found in most Müller glia as early as day 2 (Fig. 3A, Supplementary Fig. S4). Also, cyclin D1 expression reached the maximum levels by day 2 (Fig. 3A). The majority of Müller glia lost CDK4 labeling by day 3.5, when intense labeling for cyclin D1 was still detected (Fig. 3A, Supplementary Fig. S4). 
Figure 3
 
Expression of G1-phase regulators in rat Müller glia. (A) Triple immunofluorescence for CDK4, cyclin D1 (D1), and Lhx2 in the rat retina after MNU treatment. Arrowheads indicate CDK4−/D1+ Müller glia while arrows denote CDK4+/D1+ cells. Asterisk shows CDK4 staining in the outer plexiform layer in the control (Cont) retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for phosphorylated pRb (p-pRb), p27KIP1 (p27), and Lhx2 in the rat retina after MNU treatment. Arrowheads depict p-pRb-/p27+ Müller glia while arrows show p-pRb+/p27− Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm.
Figure 3
 
Expression of G1-phase regulators in rat Müller glia. (A) Triple immunofluorescence for CDK4, cyclin D1 (D1), and Lhx2 in the rat retina after MNU treatment. Arrowheads indicate CDK4−/D1+ Müller glia while arrows denote CDK4+/D1+ cells. Asterisk shows CDK4 staining in the outer plexiform layer in the control (Cont) retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for phosphorylated pRb (p-pRb), p27KIP1 (p27), and Lhx2 in the rat retina after MNU treatment. Arrowheads depict p-pRb-/p27+ Müller glia while arrows show p-pRb+/p27− Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm.
The robust increase in the expression of cyclin D1 and CDK4 at day 2 suggests that Müller glia may be in G1 phase of the cell cycle at this stage. To confirm this notion, we further examined the phosphorylation of retinoblastoma protein pRb, an event essential for G1-S progression.28 The expression of p27KIP1, a CDK inhibitor that maintains the quiescence of Müller glia29 and is downregulated upon their cell cycle reentry,30 was also examined. In the control retinas, Müller glia were intensely labeled for p27KIP1 but lacked phosphorylated pRb, indicating the quiescence of Müller glia (Fig. 3B). At day 2, however, a large fraction of Müller glia were labeled for phosphorylated pRb and lost p27KIP1 expression (Fig. 3B, Supplementary Fig. S4). Phosphorylation of pRb and loss of p27KIP1 were most prominent at day 2.5 (approximately 80%). The majority of Müller glia lost phosphorylated pRb and re-expressed p27KIP1 by day 3.5 (Fig. 3B, Supplementary Fig. S4). 
Pan-cell cycle markers, such as Ki67 and MCM6, have been used to visualize retinal cell proliferation during development and under pathologic conditions3133; however, these markers have been shown to label not only proliferating progenitor cells but also postmitotic differentiating neurons.31,32 We thus assessed the expression of Ki67 and MCM6 to test the reliability of these proteins as markers of proliferating Müller glia. No labeling for MCM6 and Ki67 was observed in the control rat retinas. At day 2, however, faint labeling for MCM6 was detected in many Müller glia despite the absence of Ki67 labeling, and the majority of Müller glia became intensely positive for MCM6 and Ki67 by day 2.5 (Figs. 4A, 4B). Notably, most Müller glia retained both MCM6 and Ki67 until day 4, when other cell cycle markers were mostly extinguished (Figs. 4A, 4B). Double staining for Ki67 and p27KIP1 revealed that virtually all Ki67+ cells were p27− at day 2.5 while most Ki67+ cells were p27+ at day 3.5, indicating that Ki67+ Müller glia were mostly quiescent at day 3.5 (Fig. 4C). 
Figure 4
 
Expression of MCM6 and Ki67 in rat Müller glia after MNU treatment. (A) Triple immunofluorescence for MCM6, Ki67, and Sox9 reveals MCM6+/Ki67− (arrowheads) and MCM6+/Ki67+ (arrows) Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer; Cont, control. Scale bar: 20 μm. (B) The proportions of Ki67+ Müller glia. Each bar represents the mean ± SEM (n = 3). (C) Double immunofluorescence for p27KIP1 (p27) and Ki67. Arrows point to Ki67+/p27− cells while arrowheads depict Ki67+/p27+ cells. Scale bar: 20 μm.
Figure 4
 
Expression of MCM6 and Ki67 in rat Müller glia after MNU treatment. (A) Triple immunofluorescence for MCM6, Ki67, and Sox9 reveals MCM6+/Ki67− (arrowheads) and MCM6+/Ki67+ (arrows) Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer; Cont, control. Scale bar: 20 μm. (B) The proportions of Ki67+ Müller glia. Each bar represents the mean ± SEM (n = 3). (C) Double immunofluorescence for p27KIP1 (p27) and Ki67. Arrows point to Ki67+/p27− cells while arrowheads depict Ki67+/p27+ cells. Scale bar: 20 μm.
To confirm the immunohistochemical findings, we also conducted Western blotting for selected cell cycle proteins in the rat retinas after MNU treatment. Phosphorylated histone H3 was detected only at day 3 (Fig. 5A). Expression of geminin, MCM6, CDK4, and cyclin D1 was detected as early as day 2 (Fig. 5A). Notably, high levels of MCM6 were still detected at day 4, when all other cell cycle markers were drastically diminished or extinguished. Overall, the Western blot results were consistent with the immunohistochemical findings. 
Figure 5
 
Expression of cell cycle regulators in the rat retina after MNU treatment. (A) Western blots for cell cycle markers used for immunohistochemical analyses. Shown are representative blots from at least three independent experiments. Actin serves as loading control. (B) qRT-PCR analysis of cyclin genes. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 5
 
Expression of cell cycle regulators in the rat retina after MNU treatment. (A) Western blots for cell cycle markers used for immunohistochemical analyses. Shown are representative blots from at least three independent experiments. Actin serves as loading control. (B) qRT-PCR analysis of cyclin genes. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
The expression of cyclins known to regulate G1 and S phases of the cell cycle was also monitored in the rat retina by qRT-PCR. The expression of cyclin D1 (Ccnd1), which regulates early G1 phase,27 and cyclin E (Ccne1, Ccne2), the regulator of G1/S transition,27 was upregulated as soon as day 1 and reached the maximum levels by day 2 (Fig. 5B). The levels of cyclin A2 (Ccna2), which promotes S-phase progression,27 was upregulated by day 2 and further increased by day 3. We also assessed cyclin gene expression in the mouse retina after MNU treatment. In contrast to the rat retina, upregulation of cyclin genes was not evident except for a moderate increase of cyclin D1 at day 1 (Supplementary Fig. S1D). 
DNA Damage Response in Rat Müller Glia After Photoreceptor Damage
Expression of cell cycle markers does not necessarily mean cell division. To confirm that Müller glia actually underwent cell division, we counted the number of Müller glia marked by Sox9 immunostaining at various time points after MNU treatment. As expected, the number of Müller glia in the central retina was increased after day 2.5 and approximately doubled by day 3.5, when most Müller glia had exited the cell cycle (Fig. 6A), suggesting that most Müller glia underwent a single round of division by day 3.5. However, the number of Müller glia significantly decreased between day 4 and day 7 (P < 0.01), and a further significant decline was observed by day 21 (P < 0.01) (Fig. 6A). The observed decrease in the number of Müller glia indicates that the progeny of Müller glia divisions may have died after their cell cycle exit. To test this possibility, we pulse-labeled proliferating Müller glia with BrdU at day 3 and counted the number of BrdU+ cells over the next few weeks. Similar to the changes in Sox9+ cell number, there was a dramatic decline of BrdU+ cells between day 4 and day 21 (Fig. 6B). 
Figure 6
 
Quantification of Müller glia number after MNU treatment. (A) Changes in the density of Sox9+ Müller glia in the central retina after MNU treatment. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (compared to control). (B) Changes in the density of BrdU+ cells in the central retina after BrdU pulse labeling at day 3. Each bar represents the mean ± SEM (n = 3). *P < 0.05.
Figure 6
 
Quantification of Müller glia number after MNU treatment. (A) Changes in the density of Sox9+ Müller glia in the central retina after MNU treatment. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (compared to control). (B) Changes in the density of BrdU+ cells in the central retina after BrdU pulse labeling at day 3. Each bar represents the mean ± SEM (n = 3). *P < 0.05.
Previous studies have indicated that forced cell cycle reentry induced by oncogene activation causes DNA damage, which triggers pathways leading to cell cycle arrest, senescence, or apoptosis.34 We thus hypothesized that the unscheduled cell cycle reentry of rat Müller glia after photoreceptor loss triggered a DNA damage response leading to cell cycle arrest or apoptosis. To address this hypothesis, we first conducted immunofluorescence for the phosphorylation of the histone variant H2AX at serine 139 (γ-H2AX), one of the earliest signs of the DNA damage response induced by various genotoxic stresses.3537 In the control rat retinas, no γ-H2AX labeling was observed (Fig. 7A). After MNU treatment, the cells in the ONL, most likely dying photoreceptors, became γ-H2AX+, indicating that DNA damage is involved in MNU-induced photoreceptor death (Fig. 7A, asterisk). At day 1 and 2, no γ-H2AX+ Müller glia were observed (Fig. 7A). At day 2.5, however, most Müller glia (82.4% ± 7.6%, n = 3) became intensely labeled for γ-H2AX (Fig. 7A). Immunoreactivity for γ-H2AX decreased thereafter, and only a few γ-H2AX+ cells were observed at day 7. Immunolabeling for γ-H2AX was also examined in the mouse retinas, in which Müller glia remained quiescent after MNU treatment. Virtually no γ-H2AX+ Müller glia were observed in the control and MNU-treated mouse retinas (Fig. 7B). 
Figure 7
 
Expression of the DNA damage marker γH2AX in the rat (A) and mouse retina (B) after MNU treatment. γH2AX colocalizes with the Müller marker Lhx2 in the rat retina after day 2.5 of MNU treatment (arrows). Arrowheads indicate γH2AX+/Lhx2− cells, most likely neurons, in the inner nuclear layer (INL). Asterisk shows γH2AX+ cells in the outer nuclear layer (ONL), most likely dying photoreceptors. Scale bar: 20 μm.
Figure 7
 
Expression of the DNA damage marker γH2AX in the rat (A) and mouse retina (B) after MNU treatment. γH2AX colocalizes with the Müller marker Lhx2 in the rat retina after day 2.5 of MNU treatment (arrows). Arrowheads indicate γH2AX+/Lhx2− cells, most likely neurons, in the inner nuclear layer (INL). Asterisk shows γH2AX+ cells in the outer nuclear layer (ONL), most likely dying photoreceptors. Scale bar: 20 μm.
We next performed immunofluorescence for the tumor suppressor protein p53, a key effector of DNA damage response that induces cell cycle arrest or apoptosis.38,39 In response to DNA damage, p53 is phosphorylated by ATM (ataxia-telangiectasia mutated) and its related kinase ATR (ATM and Rad3 related), particularly at serine 15, which leads to the stabilization and accumulation of p53 protein.40 Immunofluorescence for p53 and phosphorylated p53 (Ser15) revealed accumulation of p53 exclusively in Müller glia at day 2.5 but not before (Fig. 8A), indicating that p53 was rapidly activated in Müller glia following their S-phase reentry as a result of the DNA damage response. Staining of p53 was subsequently decreased in parallel with the decrease in γ-H2AX levels. 
Figure 8
 
Upregulation of p53 and its target p21CIP1 in the rat retina after MNU treatment. (A) Triple staining for p53, phosphorylated p53, and Lhx2 (Müller glia marker). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Double staining for p21CIP1 (p21) and Lhx2. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Real-time PCR analysis of the Cdkn1a gene. The transcript levels are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01. (D) Western blots for the DNA damage response markers. Two antibodies against p53 (from Santa Cruz and CST) were used. Shown are representative blots from at least three independent experiments. Actin serves as loading control.
Figure 8
 
Upregulation of p53 and its target p21CIP1 in the rat retina after MNU treatment. (A) Triple staining for p53, phosphorylated p53, and Lhx2 (Müller glia marker). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Double staining for p21CIP1 (p21) and Lhx2. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Real-time PCR analysis of the Cdkn1a gene. The transcript levels are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01. (D) Western blots for the DNA damage response markers. Two antibodies against p53 (from Santa Cruz and CST) were used. Shown are representative blots from at least three independent experiments. Actin serves as loading control.
We further assessed the expression of the CDK inhibitor p21CIP1, the principal mediator of p53-dependent cell cycle arrest in response to DNA damage.38,39 No p21CIP1+ cells were detected in the control retinas and MNU-treated retinas between day 1 and day 2.5 (Fig. 8B and data not shown). However, some p21CIP1+ Müller glia were identified at day 3, with the highest number observed at day 3.5 (Fig. 8B). The p21CIP1+ cells then decreased with time, and few positive cells were found at day 7 (Fig. 8B and data not shown). The upregulation of p21CIP1 was also analyzed by qRT-PCR. Significantly higher expression of Cdkn1a, which encodes p21CIP1, was observed at day 2 compared to the controls, and there was further significant increase between day 2 and day 4 (Fig. 8C). We further performed Western blotting to confirm immunohistochemical findings. Intense immunoreactivity for γ-H2AX and p53 was detected as early as day 1 (Fig. 8D), which likely reflects the DNA damage response in dying photoreceptor cells (Fig. 7A). We tested two different p53 antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (CST; Beverly, MA, USA) with similar results (Fig. 8D). Immunoreactivity for γ-H2AX, p53, and p21CIP1 between days 3 and 4 was consistent with the DNA damage response in Müller glia detected by immunofluorescence (Fig. 8D). 
To test the possibility that surviving photoreceptors express cell cycle or DNA damage-related proteins after MNU treatment, we conducted immunofluorescence for various cell cycle markers or DNA damage markers in combination with the photoreceptor marker recoverin and the Müller glia marker. A few recoverin+ photoreceptors were observed in the ONL at day 3 after MNU treatment (Supplementary Fig. S5); however, the cell cycle markers or DNA damage markers were expressed exclusively in Müller glia but not in surviving photoreceptors (Supplementary Fig. S5). 
DNA Damage Response in Rat Müller Glia After NMDA-Induced Retinal Damage
The DNA damage response in rat Müller glia upon cell cycle reentry may occur regardless of the type of retinal damage or only after MNU-induced photoreceptor loss. To address this question, we examined the response of Müller glia after retinal inner neuron damage induced by NMDA treatment. Intravitreous injection of NMDA in the rat retina induced TUNEL+ cell death in the inner nuclear layer (INL) and ganglion cell layer (Supplementary Fig. S6) as previously reported.41 Triple immunofluorescence for γ-H2AX, BrdU, and Lhx2 demonstrated that Müller glia in S phase were γ-H2AX+ in the NMDA-treated retinas while no labeling for BrdU and γ-H2AX was observed in the control retinas treated with PBS (Fig. 9A). Triple immunofluorescence for γ-H2AX, MCM6, and S100β also revealed that a subset of Müller glia that entered the cell cycle after NMDA treatment exhibited γ-H2AX (Fig. 9B). Müller glia also expressed p53 and p21 after NMDA treatment, both of which were absent in the control retinas (Figs. 9C, 9D). 
Figure 9
 
DNA damage response in rat Müller glia after NMDA-induced retinal damage. (A) Triple immunofluorescence for γH2AX, BrdU, and Lhx2 in the rat retinas 2 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for γH2AX, MCM6, and S100β (S100) in the rat retinas 3 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Triple immunofluorescence for p53, MCM6, and Sox9. Arrows indicate triple-positive cells in the NMDA-treated retina (day 3). (D) Triple immunofluorescence for p21, MCM6, and Sox9, indicating triple-positive cells (arrows).
Figure 9
 
DNA damage response in rat Müller glia after NMDA-induced retinal damage. (A) Triple immunofluorescence for γH2AX, BrdU, and Lhx2 in the rat retinas 2 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for γH2AX, MCM6, and S100β (S100) in the rat retinas 3 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Triple immunofluorescence for p53, MCM6, and Sox9. Arrows indicate triple-positive cells in the NMDA-treated retina (day 3). (D) Triple immunofluorescence for p21, MCM6, and Sox9, indicating triple-positive cells (arrows).
Absence of DNA Damage Response in Rat Retinal Progenitors and Proliferating Müller Glia in the Zebrafish Retina
Given that DNA damage response is a general mechanism that limits the proliferative potential of mammalian Müller glia, it is assumed that DNA damage response should be absent in Müller glia with high proliferative potential, such as those in the zebrafish retina.2,3 To verify this assumption, we performed immunofluorescence for γ-H2AX and p53 in the MNU-treated zebrafish retinas. A recent study has reported that MNU induces rod-specific cell loss followed by massive proliferation of Müller glia in the zebrafish retina.15 At days 3 and 8 after MNU treatment, BrdU+ cell clusters were detected in the INL of the zebrafish retina (Fig. 10A, Supplementary Fig. S7C), consistent with neurogenic clusters of Müller glia-derived progenitors reported previously.2,15,42 The zebrafish-specific antibody to γ-H2AX failed to label proliferating Müller glia or Müller glia-derived progenitors in the zebrafish retina (Fig. 10A, Supplementary Fig. S7C) while UV-irradiated zebrafish embryos were strongly immunoreactive (Supplementary Figs. S7A, S7B). This antibody also reacted with zebrafish photoreceptors at day 1 after MNU treatment, consistent with the observations in the MNU-treated rat retinas (Fig. 7A). Moreover, we used an antibody that recognizes both zebrafish and mammalian p53. While this antibody labeled Müller glia in the MNU-treated rat retina (data not shown), no labeling was observed in the MNU-treated zebrafish retina (Fig. 10A). 
Figure 10
 
DNA damage response is absent in zebrafish Müller glia and rat retinal progenitors. (A) Double immunofluorescence for γH2AX/BrdU or p53/BrdU in the zebrafish retina at day 8 after MNU treatment. BrdU+ neurogenic clusters are negative for both γH2AX and p53 (arrows). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 20 μm. (B) Double immunofluorescence for γH2AX/MCM6 or p53/MCM6 in the rat retina at postnatal day 3. MCM6+ retinal progenitors are negative for both γH2AX and p53. NBL, neuroblastic layer; GCL, ganglion cell layer. Scale bar: 20 μm.
Figure 10
 
DNA damage response is absent in zebrafish Müller glia and rat retinal progenitors. (A) Double immunofluorescence for γH2AX/BrdU or p53/BrdU in the zebrafish retina at day 8 after MNU treatment. BrdU+ neurogenic clusters are negative for both γH2AX and p53 (arrows). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 20 μm. (B) Double immunofluorescence for γH2AX/MCM6 or p53/MCM6 in the rat retina at postnatal day 3. MCM6+ retinal progenitors are negative for both γH2AX and p53. NBL, neuroblastic layer; GCL, ganglion cell layer. Scale bar: 20 μm.
We also assumed that DNA damage response should be absent in highly proliferative retinal progenitors in the developing retina. We thus performed immunofluorescence for γ-H2AX and p53 in the rat retina at postnatal day 3. Many MCM6+ retinal progenitors were present, but none of them were positive for γ-H2AX and p53 (Fig. 10B). 
Discussion
In the present study, we attempted to define the exact timing of cell cycle reentry and exit of rat Müller glia using various cell cycle markers. The expression patterns of cell cycle markers in rat Müller glia after MNU treatment are summarized in Figure 11. Bromodeoxyuridine incorporation assays revealed that the majority of Müller glia entered S phase between day 2.5 and 3. Intriguingly, expression of CDK4, a regulator of G1 phase, and phosphorylation of Rb, an event essential for G1/S transition, were identified as early as day 2 prior to S-phase entry. Also, p27KIP1, known to be downregulated upon cell cycle reentry, was lost in many Müller glia by day 2. Furthermore, mRNA expression of cyclin E1, E2, and A2, essential regulators of G1/S transition, were upregulated by day 2. All these findings indicate that most Müller glia enter G1 phase by day 2, progressing into S phase by day 2.5. The dramatic decline of BrdU incorporation and cell cycle marker expression by day 3.5 indicates that the majority of Müller glia exit the cell cycle by this time point. The recovery of p27KIP1 expression in most Müller glia at day 3.5 also supports this notion. 
Figure 11
 
Summary of the timing of expression of cell cycle markers and DNA damage response (DDR) markers in rat Müller glia after MNU-induced photoreceptor damage. Black represents expression of each marker. Time scale indicates days after MNU treatment.
Figure 11
 
Summary of the timing of expression of cell cycle markers and DNA damage response (DDR) markers in rat Müller glia after MNU-induced photoreceptor damage. Black represents expression of each marker. Time scale indicates days after MNU treatment.
Ki67 is a commonly used cell cycle marker expressed in all phases of the cell cycle.43 However, previous reports suggest that cells entering G1 phase for the first time are negative for Ki67.44,45 Consistent with these reports, our data showed that rat Müller glia did not express Ki67 in the initial G1 phase at day 2. Furthermore, Müller glia retained Ki67 expression until day 4, when other cell cycle markers were mostly extinguished. Likewise, MCM6 was expressed in Müller glia for a certain period of time after they exited the cell cycle. Recent studies have indicated that both Ki67 and MCM6 are expressed not only in proliferating cells but also in postmitotic differentiating cells in the developing retina.31,32 It has also been reported that cells continue to express Ki67 even after arrested following DNA damage.46 Thus, caution is in order when assessing Müller glia proliferation using Ki67 or MCM6. These cell cycle markers may not be suitable to assess current division, but should be used to mark cells with recent cell cycle activity. Cyclin D1 is also considered a pan-cell cycle marker in the developing retina.31,32 However, the present work and that of others11,21,47 showed that cyclin D1 is expressed in quiescent Müller glia in the healthy retina and that photoreceptor damage induces cyclin D1 expression in mouse Müller glia despite failure to enter the cell cycle. Thus, although established as a retinal progenitor marker,31,32 cyclin D1 may not be a reliable marker to monitor cell cycle reentry of Müller glia after retinal damage. 
The present study has revealed considerable variation in the proliferative response of Müller glia between two rodent species, rats and mice. The majority of rat Müller glia incorporated BrdU and expressed a variety of cell cycle markers indicating induction of cell cycle reentry. Cell counting also suggested that many, if not all, Müller glia completed a full mitotic cycle in the rat retina. In striking contrast, BrdU incorporation and cell cycle marker expression were not observed in mouse Müller glia. The mechanism underlying the different responses of Müller glia in the two species is currently unknown. Suga et al.47 have recently reported that the proliferative potential and gene expression pattern of Müller glia after retinal damage varies between mouse strains. Further investigations on the proliferative response of Müller glia and underlying signal transduction mechanisms48 in different mammalian species and strains may give important insights into key molecules involved in the decision whether Müller glia reenter the cell cycle or remain quiescent following retinal damage. 
It is of note that the nuclei of many Müller glia migrated to the ONL during their cell cycle activity. Previous reports indicated that Müller glia nuclei migrate to the ONL to undergo mitotic division following retinal detachment in rabbits12 or light-induced photoreceptor damage in zebrafish.49 In our model, however, pH3+ Müller glia nuclei were found in both the INL and ONL, suggesting that not all Müller glia migrate to the ONL to divide but that some Müller glia progress into M phase before or without nuclear migration. This discrepancy may be explained by the difference in animal species or the type of damage induced. It would be interesting to determine whether the location of mitotic division is related to the fate of Müller glia after division. 
The present findings that rat Müller glia underwent DNA damage response upon cell cycle reentry afford new insights into the molecular basis underlying the limited proliferative capacity of mammalian Müller glia. In zebrafish, retinal damage induces generation of Müller glia-derived progenitors, which divide multiple times and form neurogenic cell clusters,2,42 while rat Müller glia dedifferentiate only partially and undergo only a single round of division.5 DNA damage is known to activate p53, which induces cell cycle arrest through transcriptional activation of the CDK inhibitor p21CIP1.50,51 The peak expression of p21CIP1 in rat Müller glia at day 3.5 after MNU treatment is consistent with the timing of their cell cycle exit, suggesting that p21CIP1 induction by p53 may be one of the mechanisms that prompt Müller glia to exit the cell cycle. This notion is supported by recent reports that the p53-p21CIP1 pathway plays a role in inhibiting Müller glia proliferation in vitro.52,53 The late occurrence of p21CIP1 compared to p53 is likely due to the time lag required for the transcriptional activation of p21CIP1 by p53. Moreover, p53 is known to induce cell death when DNA damage is unrepairable.50,51 Thus, the substantial decrease in the progeny of Müller glia divisions observed in the present study and others5,8,13 may also be accounted for by DNA damage-induced activation of the p53 pathway. Further functional studies will be required to define the exact role of the p53 pathway in the control of the damage-induced response of mammalian Müller glia. 
The phosphorylation of H2AX (γ-H2AX), one of the earliest cellular responses to DNA double-strand breaks,3537 was detected in rat Müller glia at day 2.5 after MNU treatment, consistent with the timing of their S-phase entry (Fig. 11), indicating that DNA replication may trigger DNA damage in rat Müller glia. This possibility is supported by the lack of DNA damage response in mouse Müller glia, which failed to enter S phase in the present retinal damage model. Importantly, the DNA damage response was observed not only after MNU-induced photoreceptor damage, but also after NMDA-induced cell death of inner retinal neurons, indicating that Müller glia entering S phase undergo DNA damage regardless of the type of retinal damage. Furthermore, we found no evidence for the presence of DNA damage in proliferating retinal progenitors in the developing rat retina and zebrafish Müller glia, which robustly proliferate after MNU-induced photoreceptor loss. Thus, the DNA damage response associated with cell cycle reentry may be a unique feature of mammalian Müller glia. The mechanism by which Müller glia activate the DNA damage response pathway remains unknown. Replication stress induced by aberrant growth signals, such as overexpression of oncogenes, is known to activate DNA damage response, leading to p53-dependent cell cycle arrest, senescence, or apoptosis, thus providing a barrier to tumor progression.5456 Increased DNA damage and apoptosis are also observed in highly regenerative cells, such as those derived from the MRL mouse.57 Similarly, reprogramming of somatic cells to induced pluripotent stem (iPS) cells has been shown to stimulate DNA damage checkpoints including p53 activation.58,59 Thus, in the mammalian species, the DNA damage response triggered by replication stress may be an intrinsic barrier to oncogenic transformation or reprogramming of differentiated cells to a highly proliferative dedifferentiated state. The DNA damage response in rat Müller glia observed in the present study strongly indicates the presence of replication stress due to aberrant growth signals associated with severe retinal damage. Müller glia in mammals, although capable of proliferation under certain pathologic conditions, may be in a terminally differentiated state, such that unscheduled cell cycle reentry causes a conflict between proliferation and differentiation, giving rise to replication stress and DNA damage. We propose that the DNA damage response in proliferating Müller glia might be one of the mechanisms that hamper retinal regeneration in mammals. If so, attempts to force these cells to proliferate by growth factor stimulation may simply cause the DNA damage response, leading to cell cycle arrest and cell death. Strategies to minimize replication stress may be required to promote proliferation and survival of Müller glia, the prerequisites for successful retinal regeneration. 
Acknowledgments
Supported in part by Grant-in-Aid for Scientific Research 22591968 (HF) and 25861656 (FS) from Japan Society for the Promotion of Science. The authors alone are responsible for the content and writing of the paper. 
Disclosure: K. Nomura-Komoike, None; F. Saitoh, None; Y. Komoike, None; H. Fujieda, None 
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Figure 1
 
Induction of photoreceptor-specific cell death by MNU treatment. (A) TUNEL assay in the rat retina. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Real-time PCR analysis of photoreceptor genes in the rat retinas after MNU treatment. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01.
Figure 1
 
Induction of photoreceptor-specific cell death by MNU treatment. (A) TUNEL assay in the rat retina. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Real-time PCR analysis of photoreceptor genes in the rat retinas after MNU treatment. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01.
Figure 2
 
Cell cycle reentry of Müller glia after MNU-induced photoreceptor damage in the rat retina. (A) Double immunofluorescence for cell cycle markers (phospho-histone H3 [pH3], BrdU, and geminin) and Müller glia markers (S100β [S100], Sox9, and Lhx2). DAPI images are shown only for pH3/S100 staining. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer. (B) The number of pH3+ Müller glia per retinal section and the proportion of BrdU+ Müller glia. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 2
 
Cell cycle reentry of Müller glia after MNU-induced photoreceptor damage in the rat retina. (A) Double immunofluorescence for cell cycle markers (phospho-histone H3 [pH3], BrdU, and geminin) and Müller glia markers (S100β [S100], Sox9, and Lhx2). DAPI images are shown only for pH3/S100 staining. Scale bar: 20 μm. Cont, control; ONL, outer nuclear layer; INL, inner nuclear layer. (B) The number of pH3+ Müller glia per retinal section and the proportion of BrdU+ Müller glia. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 3
 
Expression of G1-phase regulators in rat Müller glia. (A) Triple immunofluorescence for CDK4, cyclin D1 (D1), and Lhx2 in the rat retina after MNU treatment. Arrowheads indicate CDK4−/D1+ Müller glia while arrows denote CDK4+/D1+ cells. Asterisk shows CDK4 staining in the outer plexiform layer in the control (Cont) retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for phosphorylated pRb (p-pRb), p27KIP1 (p27), and Lhx2 in the rat retina after MNU treatment. Arrowheads depict p-pRb-/p27+ Müller glia while arrows show p-pRb+/p27− Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm.
Figure 3
 
Expression of G1-phase regulators in rat Müller glia. (A) Triple immunofluorescence for CDK4, cyclin D1 (D1), and Lhx2 in the rat retina after MNU treatment. Arrowheads indicate CDK4−/D1+ Müller glia while arrows denote CDK4+/D1+ cells. Asterisk shows CDK4 staining in the outer plexiform layer in the control (Cont) retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for phosphorylated pRb (p-pRb), p27KIP1 (p27), and Lhx2 in the rat retina after MNU treatment. Arrowheads depict p-pRb-/p27+ Müller glia while arrows show p-pRb+/p27− Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm.
Figure 4
 
Expression of MCM6 and Ki67 in rat Müller glia after MNU treatment. (A) Triple immunofluorescence for MCM6, Ki67, and Sox9 reveals MCM6+/Ki67− (arrowheads) and MCM6+/Ki67+ (arrows) Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer; Cont, control. Scale bar: 20 μm. (B) The proportions of Ki67+ Müller glia. Each bar represents the mean ± SEM (n = 3). (C) Double immunofluorescence for p27KIP1 (p27) and Ki67. Arrows point to Ki67+/p27− cells while arrowheads depict Ki67+/p27+ cells. Scale bar: 20 μm.
Figure 4
 
Expression of MCM6 and Ki67 in rat Müller glia after MNU treatment. (A) Triple immunofluorescence for MCM6, Ki67, and Sox9 reveals MCM6+/Ki67− (arrowheads) and MCM6+/Ki67+ (arrows) Müller glia. ONL, outer nuclear layer; INL, inner nuclear layer; Cont, control. Scale bar: 20 μm. (B) The proportions of Ki67+ Müller glia. Each bar represents the mean ± SEM (n = 3). (C) Double immunofluorescence for p27KIP1 (p27) and Ki67. Arrows point to Ki67+/p27− cells while arrowheads depict Ki67+/p27+ cells. Scale bar: 20 μm.
Figure 5
 
Expression of cell cycle regulators in the rat retina after MNU treatment. (A) Western blots for cell cycle markers used for immunohistochemical analyses. Shown are representative blots from at least three independent experiments. Actin serves as loading control. (B) qRT-PCR analysis of cyclin genes. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 5
 
Expression of cell cycle regulators in the rat retina after MNU treatment. (A) Western blots for cell cycle markers used for immunohistochemical analyses. Shown are representative blots from at least three independent experiments. Actin serves as loading control. (B) qRT-PCR analysis of cyclin genes. The transcript levels of each gene are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 6
 
Quantification of Müller glia number after MNU treatment. (A) Changes in the density of Sox9+ Müller glia in the central retina after MNU treatment. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (compared to control). (B) Changes in the density of BrdU+ cells in the central retina after BrdU pulse labeling at day 3. Each bar represents the mean ± SEM (n = 3). *P < 0.05.
Figure 6
 
Quantification of Müller glia number after MNU treatment. (A) Changes in the density of Sox9+ Müller glia in the central retina after MNU treatment. Each bar represents the mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (compared to control). (B) Changes in the density of BrdU+ cells in the central retina after BrdU pulse labeling at day 3. Each bar represents the mean ± SEM (n = 3). *P < 0.05.
Figure 7
 
Expression of the DNA damage marker γH2AX in the rat (A) and mouse retina (B) after MNU treatment. γH2AX colocalizes with the Müller marker Lhx2 in the rat retina after day 2.5 of MNU treatment (arrows). Arrowheads indicate γH2AX+/Lhx2− cells, most likely neurons, in the inner nuclear layer (INL). Asterisk shows γH2AX+ cells in the outer nuclear layer (ONL), most likely dying photoreceptors. Scale bar: 20 μm.
Figure 7
 
Expression of the DNA damage marker γH2AX in the rat (A) and mouse retina (B) after MNU treatment. γH2AX colocalizes with the Müller marker Lhx2 in the rat retina after day 2.5 of MNU treatment (arrows). Arrowheads indicate γH2AX+/Lhx2− cells, most likely neurons, in the inner nuclear layer (INL). Asterisk shows γH2AX+ cells in the outer nuclear layer (ONL), most likely dying photoreceptors. Scale bar: 20 μm.
Figure 8
 
Upregulation of p53 and its target p21CIP1 in the rat retina after MNU treatment. (A) Triple staining for p53, phosphorylated p53, and Lhx2 (Müller glia marker). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Double staining for p21CIP1 (p21) and Lhx2. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Real-time PCR analysis of the Cdkn1a gene. The transcript levels are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01. (D) Western blots for the DNA damage response markers. Two antibodies against p53 (from Santa Cruz and CST) were used. Shown are representative blots from at least three independent experiments. Actin serves as loading control.
Figure 8
 
Upregulation of p53 and its target p21CIP1 in the rat retina after MNU treatment. (A) Triple staining for p53, phosphorylated p53, and Lhx2 (Müller glia marker). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Double staining for p21CIP1 (p21) and Lhx2. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Real-time PCR analysis of the Cdkn1a gene. The transcript levels are expressed relative to controls (Cont) after normalization to Gapdh levels. Each bar represents the mean ± SEM (n = 3). **P < 0.01. (D) Western blots for the DNA damage response markers. Two antibodies against p53 (from Santa Cruz and CST) were used. Shown are representative blots from at least three independent experiments. Actin serves as loading control.
Figure 9
 
DNA damage response in rat Müller glia after NMDA-induced retinal damage. (A) Triple immunofluorescence for γH2AX, BrdU, and Lhx2 in the rat retinas 2 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for γH2AX, MCM6, and S100β (S100) in the rat retinas 3 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Triple immunofluorescence for p53, MCM6, and Sox9. Arrows indicate triple-positive cells in the NMDA-treated retina (day 3). (D) Triple immunofluorescence for p21, MCM6, and Sox9, indicating triple-positive cells (arrows).
Figure 9
 
DNA damage response in rat Müller glia after NMDA-induced retinal damage. (A) Triple immunofluorescence for γH2AX, BrdU, and Lhx2 in the rat retinas 2 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (B) Triple immunofluorescence for γH2AX, MCM6, and S100β (S100) in the rat retinas 3 days after intravitreous injection of PBS (cont) or NMDA. Arrows indicate triple-positive cells in the NMDA-treated retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar: 20 μm. (C) Triple immunofluorescence for p53, MCM6, and Sox9. Arrows indicate triple-positive cells in the NMDA-treated retina (day 3). (D) Triple immunofluorescence for p21, MCM6, and Sox9, indicating triple-positive cells (arrows).
Figure 10
 
DNA damage response is absent in zebrafish Müller glia and rat retinal progenitors. (A) Double immunofluorescence for γH2AX/BrdU or p53/BrdU in the zebrafish retina at day 8 after MNU treatment. BrdU+ neurogenic clusters are negative for both γH2AX and p53 (arrows). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 20 μm. (B) Double immunofluorescence for γH2AX/MCM6 or p53/MCM6 in the rat retina at postnatal day 3. MCM6+ retinal progenitors are negative for both γH2AX and p53. NBL, neuroblastic layer; GCL, ganglion cell layer. Scale bar: 20 μm.
Figure 10
 
DNA damage response is absent in zebrafish Müller glia and rat retinal progenitors. (A) Double immunofluorescence for γH2AX/BrdU or p53/BrdU in the zebrafish retina at day 8 after MNU treatment. BrdU+ neurogenic clusters are negative for both γH2AX and p53 (arrows). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 20 μm. (B) Double immunofluorescence for γH2AX/MCM6 or p53/MCM6 in the rat retina at postnatal day 3. MCM6+ retinal progenitors are negative for both γH2AX and p53. NBL, neuroblastic layer; GCL, ganglion cell layer. Scale bar: 20 μm.
Figure 11
 
Summary of the timing of expression of cell cycle markers and DNA damage response (DDR) markers in rat Müller glia after MNU-induced photoreceptor damage. Black represents expression of each marker. Time scale indicates days after MNU treatment.
Figure 11
 
Summary of the timing of expression of cell cycle markers and DNA damage response (DDR) markers in rat Müller glia after MNU-induced photoreceptor damage. Black represents expression of each marker. Time scale indicates days after MNU treatment.
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