Cell type-specific effects of p27KIP1 loss on retinal development

Abstract

Background: Cyclin-dependent kinase (CDK) inhibitors play an important role in regulating cell cycle progression, cell cycle exit and cell differentiation. p27^KIP1 (p27), one of the major CDK inhibitors in the retina, has been shown to control the timing of cell cycle exit of retinal progenitors. However, the precise role of this protein in retinal development remains largely unexplored. We thus analyzed p27-deficient mice to characterize the effects of p27 loss on proliferation, differentiation, and survival of retinal cells.

Methods: Expression of p27 in the developing and mature mouse retina was analyzed by immunohistochemistry using antibodies against p27 and cell type-specific markers. Cell proliferation and differentiation were examined in the wild-type and p27-deficient retinas by immunohistochemistry using various cell cycle and differentiation markers.

Results: All postmitotic retinal cell types expressed p27 in the mouse retinas. p27 loss caused extension of the period of proliferation in the developing retinas. This extra proliferation was mainly due to ectopic cell cycle reentry of differentiating cells including bipolar cells, Müller glial cells and cones, rather than persistent division of progenitors as previously suggested. Aberrant cell cycle activity of cones was followed by cone death resulting in a significant reduction in cone number in the mature p27-deficient retinas.

Conclusions: Although expressed in all retinal cell types, p27 is required to maintain the quiescence of specific cell types including bipolar cells, Müller glia, and cones while it is dispensable for preventing cell cycle reentry in other cell types.

Keywords: Cone death; Cyclin-dependent kinase inhibitor; Ectopic cell cycle reentry; Retinal development; p27KIP1.

Fig. 1

Cellular localization of p27 in the mouse retina during postnatal development. a Immunofluorescence for p27 in the wild-type (WT) and p27 knockout (KO) retinas at P0, P6 and P21. At P0, the ganglion cell layer (*), amacrine cell layer (**), and photoreceptor layer (***) are intensely immunoreactive. p27 is detected in all nuclear layers at P6 and P21. Arrowheads indicate Müller glia with intense immunoreactivity. No staining is observed in the KO retinas showing antibody specificity. b p27 expression in cones and horizontal cells in the P0 retinas. Double immunofluorescence for p27 in combination with the cone markers S-opsin and RXRγ and horizontal cell marker calbindin (Calb) showing colocalization (arrows). c P21 mouse retinas showing expression of p27 in Brn3+ ganglion cells, syntaxin (Syn) + amacrine cells, Sox9+ Müller glia, Chx10+ bipolar cells, calbindin (Calb) + horizontal cells, and RXRγ + cones. Arrows indicate colocalization. NBL, neuroblastic layer; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars = 20 μm

Fig. 2

Ectopic cell division in the absence of p27. a Double immunofluorescence for BrdU and phospho-histone H3 (pH3) in the wild-type (WT) and p27 knockout (KO) retinas at different postnatal ages. The central retinal regions are shown. BrdU+ and pH3+ cells are observed only in the KO retinas at P6 and thereafter. Note pH3+ cells are ectopically positioned in the inner nuclear layer (INL) at P9 (arrows). GCL, ganglion cell layer; NBL, neuroblastic layer; ONL, outer nuclear layer. Dotted lines indicate the border between the ONL and INL. Scale bar = 20 μm. b Quantification of BrdU+ and pH3+ cells in the KO retinas. Each bar represents the mean ± SEM (n = 3 per age)

Fig. 3

Progenitor characteristics of the proliferating cells in the p27 knockout retinas at P6. Double immunofluorescence for BrdU (a) or phospho-histone H3 (pH3) (b) with progenitor markers (Chx10, Sox9, Sox2, and Pax6) showing colocalization (arrows). Scale bars = 20 μm

Fig. 4

Ectopic cell cycle reentry of bipolar cells and Müller glia in the p27 knockout retinas at P9. a Triple immunofluorescence for BrdU, Chx10, and Sox9. Arrows indicate BrdU+ cells stained weakly for Chx10 and intensely for Sox9 (Müller glia) while arrowheads denote BrdU+ cells which are intensely Chx10+ and Sox9- (bipolar cells). b Triple immunofluorescence for BrdU, Chx10, and glutamine synthetase (GS). Arrows indicate BrdU+, weakly Chx10+ and GS+ cells (Müller glia). Arrowhead shows BrdU+, intensely Chx10+, and GS- cell (bipolar cell). c Triple immunofluorescence for BrdU, Chx10, and Otx2. Some BrdU+ cells are weakly Chx10+ and Otx2- (arrows, Müller glia) while others are intensely positive for both Chx10 and Otx2 (arrowheads, bipolar cells). d Triple immunofluorescence for phospho-histone H3 (pH3), Chx10, and Sox9. Arrowheads indicate ectopic M-phase cells strongly positive for Chx10, but negative for Sox9 (bipolar cells). e Triple immunofluorescence for pH3, Chx10, and Otx2. Arrows denote pH3+ cells stained weakly for Chx10 and negative for Otx2 (Müller glia). Arrowheads indicate pH3+/Chx10+/Otx2+ cells (bipolar cells). f Double immunofluorescence for pH3 and PKCα (PKC) showing colocalization (arrowheads, bipolar cells). Scale bar = 20 μm

Fig. 5

The fate of dividing cells in the p27 knockout retinas. a Experimental scheme of BrdU incorporation assays indicating the postnatal stages of BrdU injection and sacrifice. b Single staining for BrdU in the P21 retinas treated with BrdU at P6 and P9. Arrows indicate positive cells in the outer nuclear layer (ONL). c P21 retina (BrdU injected at P6) double-stained for BrdU and the rod marker NR2E3 showing colocalization (arrows). d P21 retina (BrdU injected at P6) double-stained for BrdU and the cone marker M-opsin. No colabeling is observed. e Triple immunofluorescence for BrdU, Chx10 and Sox9 in the P21 retinas injected with BrdU at P6. Arrows indicate BrdU+ Müller glia labeled weakly for Chx10 and intensely for Sox9. Arrowheads denote BrdU+ bipolar cells expressing Chx10 but not Sox9. f P21 retina (BrdU injected at P6) double-labeled for BrdU and the bipolar marker PKCα (PKC) showing colocalization (arrows). g P21 retina (BrdU injected at P6) double-labeled for BrdU and the Müller marker glutamine synthetase (GS) showing colocalization (arrows). h Quantification of the cell types which were in the last S-phase at the time of BrdU injection. Rods were identified by NR2E3 labeling, and bipolar and Müller cells were determined based on the expression of Chx10 and Sox9. Each bar represents the mean ± SEM (n = 3 per stage). GCL, ganglion cell layer; INL, inner nuclear layer. Scale bars = 20 μm

Fig. 6

Aberrant cell cycle reentry of cones in the p27 knockout (KO) retinas. a Immunofluorescence for Ki67 in the WT and KO retinas at P6 and later stages showing proliferating cells only in the KO retinas. Arrowheads indicate Ki67+ cells in the outer nuclear layer (ONL) of the KO retina at P12. INL, inner nuclear layer. b The ONL of the KO retina at P12 immunolabeled for Ki67 in combination with the photoreceptor marker recoverin, rod marker NR2E3, and cone marker S-opsin. Note colocalization of Ki67 with recoverin and S-opsin, but not with NR2E3 (arrowheads). c Double immunofluorescence for Ki67 and S-opsin in the KO retinas at P6 and later stages. Ki67+/S-opsin + cones are shown by arrowheads. d Quantification of Ki67+ cones in the KO retinas. Graph data represent the means ± SEM (n = 3 per stage). e Cones identified by S-opsin or M-opsin immunoreactivity express proliferation markers PCNA, phospho-Rb, and MCM6 in the KO retinas at P12. Scale bars = 20 μm

Fig. 7

Cones are not generated de novo during the period of extra proliferation in the p27 knockout (KO) retinas. KO mice were treated with BrdU at P6 or daily from P7 to P11, and the fate of proliferating cells analyzed at P12. Double labeling for BrdU and S-opsin shows no colocalization. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar = 20 μm

Fig. 8

Impaired differentiation and survival of cones in the p27 knockout (KO) retinas. a Double immunofluorescence for Ki67 and S-opsin in the KO retina at P15. Note relatively weak S-opsin labeling in the Ki67+ cones (arrowheads). b Quantitative RT-PCR analyses of cone gene expression in the WT and KO retinas during postnatal development. The transcript levels are expressed relative to WT at P21 after normalized to Gapdh levels. Each value represents the mean ± SEM (n = 3 per stage and genotype). *P < 0.05, **P < 0.01, Student’s t test. c Double immunofluorescence for phospho-H2AX (pH2AX) and S-opsin in the WT and KO retinas at P15. Note pH2AX+ cones in the KO retina. d Quantification of retinal cell types in the WT and p27KO retinas at P56. Retinal sections or whole mounts were immunolabeled for cell-specific markers. Note a significant reduction in cone number in the KO retina. The number of rods and bipolar cells are also mildly reduced. Bars represent the mean ± SEM (n= 3 per genotype). *p < 0.05, **p < 0.01, Student’s t test. Scale bars are 20 μm