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

(A) Schematic of AAV vectors used in this study. (B) Experimental design. (C-F) Analysis of EdU incorporation. Uninjured mouse eyes were injected with (C) AAV-GFAP-mCherry-non target (NT) shRNA (control), (D) AAV-GFAP-mCherry-p27 shRNA, (E) AAV-GFAP-cyclin D1, and (F) AAV-GFAP-cyclin D1-p27 shRNA (CCA) at P6 and harvested at P18 after 5-day EdU intraperitoneal injection. Retinal sections were co-labeled for MG marker Sox9. (G) Quantification of EdU+ Sox9+ cells. AAV-GFAP-mCherry-NT shRNA control virus (n=8), AAV-GFAP-mCherry-p27shRNA (n=11), AAV-GFAP-cyclin D1 (n=8), and CCA (n=14). (H) Quantification of the percentages of EdU+GFP+ and EdU-GFP+ cells in the area with efficient virus infection in the Glast-CreERT2; Sun1:GFP mouse retina injected with the control or CCA vector. (I) Quantification of the total GFP+ MG in the CreERT2; Sun1:GFP retina infected by CCA. Retinal area (250 µm in width) with the most efficient virus infection in the middle retina region was selected for quantification. (J) Quantification of EdU+ Sox9+ cells in the young (CCA injection at P6, n=13), adult (CCA injection at P28, n=17), and aged retinas (CCA injection at P255-P347, n=7). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Data are presented as mean ± SEM. * P<0.05; *** P<0.001; ns=not significant by one-way ANOVA analysis with Tukey’s post-hoc test (G, J) and two-tail unpaired student t-test (I).

MG proliferation driven by CCA is self-limiting

(A) Time-course analysis of MG proliferation after CCA injection. EdU was administered for two consecutive days, starting from different days after the CCA injection, and samples were harvested for analysis one day after the second EdU injection. Data are presented as mean ± SEM, n≥4. (B) Analysis of EdU and BrdU labeled cells. (C) Quantification of the number of EdU+BrdU-, EdU-BrdU+ and EdU+ BrdU+ cells. Data are presented as mean ± SEM. n≥4. (D) Representative retinal sections of Glast-CreERT2; Sun1:GFP mice at one week, two weeks, and four months post CCA injection. (E) Quantification of MG daughter cell (EdU+GFP+) distribution in each retinal layer. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Data are presented as mean ± SEM, n=3.

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

(A) Schematic illustration of scRNA-seq experiment. (B) UMAP plot of scRNA-seq data for MG treated with CCA, CCANT, and control virus, with clusters identified by the known marker gene expression. (C) Expression of retinal cell markers in different cell clusters. (D) Split UMAP plots of the control, CCA, and CCANT groups. (E) The proportion of cell clusters in the control, CCA, and CCANT groups. (F) Heatmap of top DEG genes between cell clusters. Cells are shown in columns, and genes are in rows. Color scale denotes Z score of the normalised gene expression levels. (G) The violin diagram shows the expression of IFN pathway, MG and Rod genes in different cell clusters. (H) Feature plots of cell cycle regulator (Mik67, Mcm5), glial (Glul, Kcnj10), gliosis (Gfap), cell proliferation (Btg2), and rod (Rho and Nrl) normalised gene expression in different cell clusters. CCA, cell cycle activator; CCANT, cell cycle activator + NMDA + TSA.

Gnat1 and Rho mRNA are expressed in the ONL and OPL MG

(A-B) Gnat1 and Rho mRNA in situ hybridization in the control (A) and the Glast- CreERT2;Sun1:GFP mouse retinas harvested at three weeks post CCA injection (B). (C-F) Magnified views of the highlighted regions in (A) and (B). (G) The number of Gnat1 mRNA dots per GFP+ cell and (H) the average pixel level of Rho mRNA per GFP+ cell. n=3 mice, data are presented as mean ± SEM. ns=not significant, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by one-way ANOVA with Tukey’s post hoc test (G, H).

Glul mRNA level is decreased in the ONL and OPL MG

(A-B) Glul mRNA in situ hybridization. (C-D) Magnified views of the highlighted regions in (A) and (B). (E) The average pixel level of Glul mRNA per GFP+ cell. n=3 mice, data are presented as mean ± SEM. ns=not significant, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by one-way ANOVA with Tukey’s post hoc test (E).

CCA induces de novo genesis of Otx2+ BP-like and HuC/D+ AC-like cells from MG

(A-B) Representative retinal sections of Glast-CreERT2; tdTomato mice at four months post injection of CCA. Sections were immunostained with EdU and Otx2. (C-D) Magnified views of the highlighted regions in (B). (E) Quantification of tdT+EdU+Otx2+/ tdT+EdU+%. (F-G) Representative retinal sections immunostained with EdU and HuC/D. (H-I) Magnified views of the highlighted regions in (F) and (G). (J) Quantification of tdT+EdU+HuC/D+/tdT+EdU+%. n=3 mice, data are presented as mean ± SEM. *p<0.05; **p<0.01, by unpaired two-tailed Student’s t-test (E, J).

CCA does not lead to retinal neoplastic transformation

(A-C) Optomotor and electroretinography (ERG) tests were performed on wild type mice at 1 year after CCA injection (n≥4). Visual acuity by the optomotor test (A), and b-wave amplitudes of the scotopic ERG under different light intensity in (B) and photopic ERG under 30 cd × s/m2 in (C), respectively. Data are presented as mean ± SEM. ns=not significant by two-tail unpaired Student’s t-test (A-C). (D-E) Sox9 immunofluorescence in untreated control mice (D) and WT mice one year after CCA injection (E). (F-G) Zoom-in images of the indicated area in (D) and (E). (H) Quantification of the number of Sox9+ cells in retinas at two weeks or one year after CCA injection and in age-corresponding WT control retinas. (I) Quantification of the number of Sox9+ cells in each retinal layer. Data are presented as mean ± SEM. ns=not significant, **p ≤0.01, ***p ≤0.001 by one-way ANOVA with Tukey’s post hoc test (H) and two-tail unpaired Student’s t-test (I).