Simultaneous p27Kip1 downregulation and cyclin D1 overexpression drive robust MG proliferation in the uninjured mouse retina

(A) Schematic representations of AAV vectors used in this study. AAV-GFAP-GFP-non-target (NT) shRNA for control, AAV-GFAP-mCherry-p27 shRNA for p27kip1 knockdown (KD), AAV-GFAP-cyclin D1 for cyclin D1 overexpression (OE), and AAV-GFAP-cyclin D1-p27 shRNA for p27kip1 KD and cyclin D1 OE. (B) Experimental design. Mice received an intravitreal AAV injection on postnatal day 6 (P6), designated as D0, and daily EdU injections intraperitoneally from D7 to D11. (C-F) Analysis of EdU incorporation with Sox9 colabeling in uninjured mouse eyes injected with indicated viruses. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (G) Quantification of EdU+Sox9+ cells per 250 µm high infection areas. Control (n=8 eyes), p27kip1 KD (n=11 eyes), cyclin D1 OE (n=8 eyes), CCA (n=14 eyes). (H) Quantification of EdU+Sox9+ cells per 250 µm high infection areas in retinas injected with CCA at indicated ages: P6 (n=13), P28 (n=17), and P255-P347 (n=7). Five EdU injections were given intraperitoneally from D7 to D11 post-CCA injection, and retinas were harvested for EdU analysis on D12. Data are presented as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001, ns = not significant (one-way ANOVA with Tukey’s post-hoc test) (G-H).

MG proliferation induced by CCA is self-limiting

(A) Time-course analysis of MG proliferation following CCA injection. EdU was administered for two consecutive days, starting at various days post-CCA injection, with samples harvested one day after the second EdU injection. Data are presented as mean ± SEM (n≥4). (B) Analysis of cells labeled with EdU and BrdU. (C) Quantification of the percentages of EdU+BrdU−, EdU−BrdU+ and EdU+BrdU+ cells of the total Sox9+ cells. Data are presented as mean ± SEM (n=3). ***P<0.001, ****P<0.0001 (one-way ANOVA with Tukey post hoc test). (D) Experimental design. (E) A representative image of sparsely labeled MG in the Glast-CreERT2;tdTomato mouse retina. (F) Quantification of the numbers of 1-cell, 2-cell, and larger clones in ten 250 µm hotspot areas from four retinas infected with CCA. (G-I) Eye samples from the time-course EdU analysis (A) were stained for cyclin D1 and Sox2. Representative retinal sections from uninjected eyes (G), mice with EdU injections at D1-2 and harvest at D3 (H), and mice with EdU injections at D11-12 and harvest at D13 (I). Arrowheads point out the EdU+ cells that are negative for cyclin D1 staining. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

scRNA-seq analysis of MG at three weeks post-CCA treatment

(A) Schematic illustration of the scRNA-seq experiment. (B) UMAP plot of scRNA-seq data for all MG combined from three groups with control, CCA, or CCANT treatment, with clusters identified based on known marker gene expression. (C) Violin diagram showing expression of retinal cell markers in different cell clusters. (D) Split UMAP plots of control, CCA, and CCANT groups. (E) Proportions of cell clusters within control, CCA, and CCANT groups. (F) Heatmap of top DEGs between cell clusters. Cell clusters are shown in columns, and genes are in rows. Color scale denotes Z score of the normalized gene expression levels. (G) Violin diagram illustrating the expression of IFN pathway genes, MG genes and rod genes across different cell clusters. (H) Feature plots showing normalized gene expression of Stat1, Stat2, Stat3, Gbp6, Irgm1, and Igtp in different cell clusters. CCA, cell cycle activator; CCANT, CCA + NMDA + TSA.

Glul mRNA levels decrease in the MG that migrated to the ONL and OPL

(A) Experimental design. (B) In situ hybridization showing Glul mRNA expression. Control retina did not receive any AAV injection. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (C) Magnified views of the highlighted cells in panel (B). (D) Average pixel intensity of Glul mRNA per GFP+ cell. Pixel intensity of MG treated with CCA was normalized to the average pixel intensity of MG in the uninjected eye of the same animal. n=60 GFP+ cells in each retinal layer from three mice, data are presented as mean ± SEM. ****P<0.0001 (unpaired two-tailed Student’s t-test) (D).

Temporary loss of Sox9 in a subset of MG following CCA treatment

(A) Experimental design. (B) Representative retinal sections of Glast-CreERT2; Sun1:GFP mice without virus injection (control) or mice at three weeks and four months post-CCA injection. Arrowheads highlight Sox9−GFP+ cells. (C-D) Magnified views of the boxed regions in (B). Arrowheads highlight Sox9−GFP+ cells. (E) Quantification of Sox9−GFP+ cells as a percentage of total GFP+ cells in each retinal layer. n=3 mice, data are presented as mean ± SEM. ns=not significant, *P<0.05 (unpaired two-tailed Student’s t-test). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

CCA induces de novo genesis of Otx2+ cells from MG

(A) Experimental design. (B) Representative retinal sections of Glast-CreERT2; tdTomato mice without AAV injection (control) or mice at four months post-CCA injection. Sections were co-stained for EdU and Otx2. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (C-D) Magnified views of the highlighted cells in (B). Arrowhead highlights a tdT+EdU+ MG in the OPL that is negative for Otx2 (C), while Arrowhead highlights a tdT+EdU+Otx2+ MG in the INL. (E) Quantification of tdT+EdU+Otx2+ cells as a percentage of total tdT+EdU+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **P<0.01 (one-way ANOVA with Tukey’s post hoc test).

CCA does not lead to retinal neoplastic transformation

(A) Experimental design. (B-C) Optomotor and electroretinography (ERG) tests were performed on wild type mice with one eye injected with CCA and the other eye as control (without AAV injection) at one-year post-CCA injection. Visual acuity by the optomotor test (B), b-wave amplitudes of the scotopic ERG under different light intensity in (C), and b-wave amplitudes of the photopic ERG under 30 cd × s/m2 in (D). Data are presented as mean ± SEM. ns=not significant (paired two-tailed Student’s t-test) (B-D). (E-H) Immunostaining for MG marker Sox9. (F,H) Zoomed-in images of the boxed areas in (E) and (G). (I) Quantification of the numbers of Sox9+ cells in control retinas versus CCA-treated retinas at two weeks or one-year post-CCA injection and in age-matched wild type control retinas. Data are presented as mean ± SEM. ns=not significant, ***P<0.001 (one-way ANOVA with Tukey’s post hoc test). (J) Quantification of the number of Sox9+ cells in each retinal layer. Data are presented as mean ± SEM. ns=not significant, **P<0.01 (unpaired two-tailed Student’s t-test).