Cyclin D1 inactivation extends proliferation and alters histogenesis in the postnatal mouse retina

Abstract

Background: The cell-cycle regulator Cyclin D1 is expressed in embryonic retinal progenitor cells (RPCs) and regulates their cell-cycle rate and neurogenic output. We report here that Cyclin D1 also has important functions in postnatal retinal histogenesis.

Results: The initial production of Müller glia and bipolar cells was enhanced in Cyclin D1 knockout (Ccnd1(-/-) ) retinas. Despite a steeper than normal rate of depletion of the RPC population at embryonic ages, postnatal Ccnd1(-/-) retinas exhibited an extended window of proliferation, neurogenesis, and gliogenesis. Cyclin D3, normally confined to Müller glia, was prematurely expressed in Ccnd1(-/-) RPCs. However, Cyclin D3 did not compensate for Cyclin D1 in regulating cell-cycle kinetics or neurogenic output.

Conclusions: The data presented in this study along with our previous finding that Cyclin D2 was unable to completely compensate for the absence of Cyclin D1 indicate that Cyclin D1 regulates retinal histogenesis in ways not shared by the other D-cyclins.

Figure 1. Persistent proliferation in the postnatal Ccnd1^−/− retina

(A–D) CCND1 expression in wild type retinas from P0 to P14. CCND1 was expressed in a pattern consistent with RPCs and their temporal depletion, which is complete by P14. Arrows in C denote laminar position of CCND1⁺ cells. The staining at the edge of the GCL is due to secondary antibody cross-reactivity. (E–L) Proliferation in P6 wild type and Ccnd1^−/− retinas. PCNA is a general RPC marker and EdU identifies cells that were in S-phase during the labeling interval (2.5 hours). Arrows in E,I, and K denote PCNA⁺ mitotic figures; arrowheads in L denote EdU⁺ cells. Notable differences between the wild type and Ccnd1^−/− retinas are the presence of fewer EdU⁺ cells in the peripheral retina of the mutant (compare F and J) and the persistence of PCNA⁺ and EdU⁺ cells in the central retina of the mutant (compare G and H to K and L). (M,N) Wide field views of wild type (M) and Ccnd1^−/− retinas (N) reveal the extensive persistence of PCNA⁺ cells in the mutant at P6. Dashed lines mark the apico-basal boundaries of retina. Abbreviations: DCL, differentiated cell layer; NBL, neuroblast layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 100 μm

Figure 2. CCND3 expression in the postnatal retina

(A–E) CCND3 expression in wild type retinas from P0 to adult. CCND3 expression was not detected until P6. Once activated, CCND3 remains expressed into adulthood. The expression pattern was primarily restricted to the INL. Asterisks show examples of secondary antibody cross-reactivity. (F) CCND3 expression is not detected in Ccnd3^−/− retina. (G–I) CCND3 (G) and CCND1 (H) expression in the wild type retina at P6 (merged images in I). CCND1 expression is extinguished along the centro-peripheral axis of the retina and CCND3 expression is activated along the same axis in a complementary manner. Arrowheads show examples of cells expressing both proteins. Scale bars: 100 μm

Figure 3. Precocious activation and altered patterns of CCND3 expression in the Ccnd1^−/− retina

(A–D) CCND3 expression was detected by P0 and onward, mainly in the neuroblast layer (NBL). At P6 (C) and P14 (D), CCND3⁺ cells were also found in the ONL. Arrow in C points to an area of focal cell death in the mutant retina (Ma et al., 1998). Asterisks indicate secondary antibody cross-reactivity. Bright regions beyond the apical retinal boundary (asterisk in B) are due to pigmentation in the retinal pigmented epithelium and choroid. (E–G) CCND3 (E) and VSX2 (F) expression in the Ccnd1^−/− retina at P0 (merged images in G). Note the extensive overlap in expression patterns. Arrows point to examples of CCND3⁻, VSX2⁺ cells and arrowheads point to CCND3⁺, VSX2⁻ cells, both of which are rare compared to the double positive cells. (H–K) Triple-labeling with DAPI (H), CCND3 (I), and RLBP1 (J) reveals an unusual bilayer arrangement of CCND3⁺, RLBP1⁺ cells in the Ccnd1^−/− retina at P14 (merged images in K). Dashed line indicates the boundary between the ONL and INL and corresponds to the outer plexiform layer. Scale bars: 100 μm

Figure 4. Ccnd3 does not compensate for Ccnd1 in regulating proliferation

(A–F) P0 Ccnd1^−/− and Ccnd1^−/−; Ccnd3^−/− retinas were cultured successively in BrdU for 2 hours and EdU for 30 minutes. Sections were triple-labeled for PCNA (A,D), BrdU and EdU (B,E) and EdU only (C,F). Arrows point to RPCs that incorporated BrdU, but not EdU (PCNA⁺, BrdU⁺, EdU⁻ ). (G) The percentages of total cells expressing PCNA were unchanged between the two mutants (left graph), as was total cell cycle time (T_c) and S-phase time (T_s; middle graph). The percentage of RPCs in S-phase (right graph) was elevated in the double mutant, but the magnitude of the change was small. Numbers inside bars represent number of animals analyzed and error bars represent standard deviation from the mean. Scale bar: 100 μm.

Figure 5. Ccnd3 does not influence precursor cell type output from Ccnd1^−/− RPCs

P0 Ccnd1^−/− and Ccnd1^−/−, Ccnd3^−/− retinas were stained with antibodies against the RGC precursor marker POU4F2 (A,B), the horizontal and amacrine precursor marker PTF1A (C,D), the RGC and cone photoreceptor precursor marker RXRγ (E,F), and the rod photoreceptor precursor marker NR2E3 (G,H). Scale bar: 100 μm.

Figure 6. Precocious production of Müller glia in the Ccnd1^−/− retina

Retinal cells were birthdated by injecting pregnant animals with 2 doses of EdU at E18.5 and retinas were collected at P14. (A–H) P14 wild type and Ccnd1^−/− retina sections were triple labeled for EdU (green), SOX9 (red) and anti-GS (white). Panels B and F show merged images for EdU and SOX9. Insets denoting enlarged areas of retinal sections (C, D, G and H) point to EdU⁺, SOX9⁺, GS⁺ triple labeled cells for further confirmation of Müller glia identity. Closed arrows point to EdU^high, SOX9⁺, GS⁺ cells. Open arrows point to outlined EdU^low, SOX9⁺, GS⁺ cells in D and H. Scale bars: 100 μm (A,B,E,F); 20 μm (C,D,G,H).

Figure 7. Precocious production of bipolar cells in the Ccnd1^−/− retina

Retinal cells were birthdated by injecting pregnant animals with 2 doses of EdU at E18.5 and retinas were collected at P14. (A–D) Wild type and (E–H) Ccnd1^−/− retinal sections were triple labeled for EdU (green), VSX2 (red) and PKCα (white). All images were captured in the central retina. B and F are merged images for EdU and VSX2. Insets denote areas of retinal sections enlarged to point out EdU⁺, VSX2⁺, PKCα⁻ cone bipolar cells and/or Müller glia (arrowheads) and EdU⁺, VSX2⁺, PKCα⁺ rod bipolar cells (arrows). Note the abundance of EdU^high, VSX2⁺ cells in Ccnd1^−/− retinal sections compared to wild type sections. Outlined cell in D is a weakly labeled EdU^low rod bipolar cell. EdU^high rod bipolar cells were rare in the mutant samples (Table 2) Scale bars: 100 μm (A,B,E,F ); 20 μm (C,D,G,H).

Figure 8. GFAP expression in the Ccnd1^−/− retina at P14

(A–C) Ccnd1^−/− retinal section stained for DAPI (A) and GFAP (B). Merged images in C. The hypocellular area of the ONL demarcates a zone of photoreceptor degeneration initially described by Ma et al. (1998). In contrast to the pattern of ectopic proliferation, which extends across the retina at earlier ages, GFAP expression within the retina is confined to the dysplastic zones. (D–F) Wild-typeretinal section stained for DAPI (D) and GFAP (E). Merged images in F. GFAP is expressed predominantly along the basal boundary of the retina and is expressed in astrocytes, which is also true in the mutant retina. The punctate staining in the inner and outer plexiform layers is due to secondary antibody cross-reactivity.

Figure 9. Late proliferating cells in the Ccnd1^−/− retina are neurogenic

Retinal cells were birthdated by a single EdU injection at P6 and retinas were collected at P14. (A,B) Low power images of P14 wild type and Ccnd1^−/− retina sections showing EdU labeled cells. Retinas are located within the dashed lines. (C–F) Representative central regions of Ccnd1^−/− retinal sections were triple labeled for EdU, SOX9 and GS to identify birthdated Müller glia. Merged images in F. Open arrow points to a birthdated Müller glial cell (EdU^high, SOX9⁺, GS⁺) and closed arrow points to a birthdated non-Müller glial cell (EdU^high, SOX9⁻, GS⁻). (G–J) Representative central regions of Ccnd1^−/− retina sections tripled labeled for EdU, the pan-bipolar marker VSX2, and the rod bipolar marker PKCα to identify birthdated cone and rod bipolar cells. Merged images in J. Double head arrow points to a birthdated cone bipolar cell (EdU⁺, VSX2⁺, PKCα⁻). Arrowhead points to a birthdated rod bipolar cell (EdU⁺, VSX2⁺, PKCα⁺). (K–M) P14 central retina section double-labeled for EdU and general photoreceptor cell marker RCVRN to identify birthdated photoreceptor cells. Open arrow points to a birthdated photoreceptor cell (EdU⁺, RCVRN⁺). Scale bars: 100 μm (A,B); 20 μm (C–J; K–M).

Figure 10. Model of extended histogenesis in the Ccnd1^−/− retina

A subset of RPCs are restricted in their proliferative potential and depend on Ccnd1 to stay in the cell cycle (dark gray box in wild type). Without Ccnd1, these RPCs prematurely exit from the cell cycle and adopt a precursor fate (asterisk marked cells in wild type and Ccnd1^−/−). A larger cohort of RPCs depend on CCND1 to maintain their rate of cell cycle progression (light gray boxes in wild type and Ccnd1^−/−). Without Ccnd1, the cell cycle time of mutant RPCs is slower. and linger in the mutant retina. They eventually exit from the cell cycle to make neurons and glia, even after neurogenesis has ceased in the wild type retina.