Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic representations of the intravitreal injection of AAV 7m8 -GFAP-cyclinD1-p27 Kip1 shRNA-WPRE (CCA). (b) The overview process of MG proliferation induced by CCA. (c) Representative Sox9 and EdU immunostaining on retinal sections from Glast-Cre ERT2 ;Sun1:GFP mice treated with CCA at P28 and collected 4 months post-treatment. C1-C2 are the magnified views of the highlighted regions. The white arrows refer to GFP+ EdU+ Sox9- cells. (d) Representative Otx2 immunostaining on retinal sections from Glast-Cre ERT2 ;Sun1:GFP mice treated with CCA at P28 and collected 4 months post-treatment. D1-D2 are the magnified views of the highlighted regions. The white arrows refer to GFP+ Otx2+ cells. (e) Hes1 mRNA in situ hybridization in the control and the Glast-Cre ERT2 ;Sun1:GFP mouse retinas harvested at four months post CCA injection. (f) Magnified views of the highlighted regions in (e). n=3 mice.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Injection, shRNA, Immunostaining, In Situ Hybridization, Control

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic of Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mouse used in this study. (b) Schematic illustration of the experiment examining how Rbpj deletion affects neurogenesis in retinal progenitor cells. Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice were received TAM injection at postnatal day 1 (P1) and harvested at P12. (c) Representative immunostaining of Otx2 and Sox9 on retinal. (d) Percentage of tdT+ rod photoreceptor cells in overall tdT+ cells. (e) Percentage of tdT+ Sox9+ cells in overall tdT+ cells. n≥3 mice, ns. not significant, **** P < 0.0001, by one-way ANOVA with Tukey post hoc test.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Injection, Immunostaining

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Representative immunostaining of Otx2 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Percentage of tdT+ Otx2+ cells in overall tdT+ cells. n≥3 mice, ns=not significant, * P < 0.05, ** P < 0.01, by one-way ANOVA with Tukey post hoc test.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Immunostaining, Injection

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of MG dedifferentiation and reprogramming experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice in different timepoints post TAM injection. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (b). (d) Percentage of GFP+ Sox9- cells in overall GFP+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (e) Percentage of GFP+ Otx2+ cells in overall GFP+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, ** P < 0.01, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Immunostaining, Injection

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of the experiment examining MG proliferation. (b) Representative immunostaining of Otx2 and EdU on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice harvested at 4 months post TAM injection.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Immunostaining, Injection

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of the neurogenesis assessment experiment. (b) Representative immunostaining of EdU and Otx2 on retinal sections. The white arrows refer to tdT+ Otx2+ cells. (c) Magnified views of the highlighted regions in (b). The white arrows refer to tdT+ Otx2+ cells. (d) Percentage of tdT+ Otx2+ cells in overall tdT+ cells. 3W: 3 weeks, 4M: 4 months, n=3 mice, data are presented as mean ± SEM. ns=not significant, *** P < 0.001, by one-way ANOVA with Tukey’s post hoc test. (e) Percentage of tdT+ Otx2+ cells in overall tdT+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, * P < 0.05, *** P < 0.001, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (f) Representative immunostaining of EdU and Otx2 or Crx on retinal sections. (g) Percentage of tdT+ EdU+ Otx2+ cells in ONL tdT+ EdU+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (h) Percentage of tdT+ EdU+ Crx+ cells in ONL tdT+ EdU+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Percentage of tdT+ EdU+ Otx2+ cells in overall tdT+ EdU+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (b) Representative immunostaining of EdU and Nrl on retinal sections. (c) Percentage of tdT+ EdU+ Nrl+ cells in ONL tdT+ EdU+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, *** P < 0.001, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of the snRNA-seq experiment. To induce Rbpj deletion and GFP expression in MG, we administered TAM for 7 consecutive days in Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice from P28. At P36, CCA was injected into the CCA and Rbpj KO+CCA groups, while control and Rbpj KO mice were uninjected by any AAV vector. 3-4 retinas from mice at indicated ages were pooled together for nuclei extraction, and MG nuclei were then isolated by GFP signal via FACS for snRNA-seq. (b) UMAP plot of snRNA-seq data from four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA) at different timepoints, and separated UMAP based on different timepoints. clusters were identified based on known marker gene expression. (c) Proportions of cell clusters within Ctrl, CCA, Rbpj KO and + groups at different timepoints. (d) Feature plots highlighting the cluster of quiescent MG ( Kcnj10 ), proliferating MG ( Mki67 ), reactivated MG ( Gfap ), MGPC ( Dll1 , Ascl1 ), transitional MG ( Mt-Nd4 ), AC-like MG ( Elavl3 ), and BC-like MG ( Otx2 ). (e) Heatmap showing the expression of top 50 differentially expressed genes (DEGs) of 3-week reactivated MG between CCA and Rbpj KO+CCA (p<0.05). (f) Heatmap showing the expression of top 50 DEGs of 4-month MGPC between Rbpj KO and Rbpj KO+CCA (p<0.05).

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Expressing, Injection, Control, Plasmid Preparation, Extraction, Isolation, Marker, Gene Expression

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Pseudotime trajectory analysis showing the MG reprogramming process. (b) Dynamic cell type distribution along the pseudotime. (c) Change of gene expression level along the pseudotime. MG (Hes5, Kcnj10, Slc1a2, Sox9, Hes1, Aqp1, Rlbp1); Reactivated MG (Mx2, Vim); Proliferating MG (Mcm5, Mki67); MGPC (Neurog2, Dll1, Ascl1, Eya2); AC-like (Elavl4, Caln1, Rbfox3); photoreceptor cell (Prom1); RGC (Tubb3); BC-like (Otx2, Car10, Cabp5). (d) Gene expression trends along pseudotime. (e) Separated gene expression trends along pseudotime.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of the snATAC-seq experiment. (b) Proportions of cell clusters within Ctrl, CCA, Rbpj KO and Rbpj KO+CCA groups. (c) UMAP plot of snATAC-seq data from four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA). (d) Feature plots highlighting the cluster of quiescent MG (Kcnj10), KO MG (Hes5), active MG (Bal3), MGPC (Dll1), AC-like MG (Caln1), BC-like MG (Otx2). (e) Dot plot showing gene expression and cell percentages for quiescent MG, KO MG, active MG, MGPC, AC-like MG, BC-like MG. (f) Volcano plot showing the differential gene expression of MG and active MG within CCA treatment group (p<0.05). Red dots indicate the genes upregulated in active MG and blue dots indicate genes downregulated in active MG. (g) WikiPathways enrichment analysis of upregulated genes in active MG (p<0.05). (h) Increased chromatin accessibility at the Neurod2 locus was observed in the active MG. The black arrow shows the transcription direction.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of the experiment assessing long-term cell survival. (b) Representative immunostaining of EdU and Otx2 on retinal sections. The white arrows refer to tdT+ Otx2+ cells. (c) Percentage of tdT+ Otx2+ cells in overall tdT+ cells, n=3 mice, 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. (d) Percentage of tdT+ EdU+ Otx2+ cells in overall tdT+ EdU+ cells, ns=not significant, ** P < 0.01, by one-way ANOVA with Tukey’s post hoc test. (e) Number of EdU+ cells per 500 µm, ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

doi: 10.64898/2026.03.09.710684

Figure Lengend Snippet: (a) Schematic illustration of ZO1 staining experiment. (b) Representative immunostaining of Otx2 and ZO1 on retinal sections from Glast-Cre ERT2 ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice at 9 months post TAM injection. The white arrows refer to tdT+ Otx2+ cells.

Article Snippet: Primary antibodies used in this study included goat anti-Otx2 antibody (1:200, AF 1979; R&D systems), mouse anti-HuC/D (1:200, A21271; Thermo Fisher Scientific), rabbit anti-Sox9 antibody (1:500, AB5535; Millipore), rabbit anti-mCAR (1:200, AB15282, Millipore), rabbit anti-Nrl (1:150, AF2945, R&D Systems), mouse anti-PKCα (1:200, sc-8393, Santa Cruz), rabbit anti-Crx (1:500, PA5-32182, Thermo Fisher Scientific), goat anti-Sox2 (1:500, AF2018, R&D Systems), rabbit anti-Pax6 (1:500, AB2237, Millipore), rabbit anti-ZO1 (1:500, 61-7300, Thermo Fisher Scientific), rabbit anti-Rbpms (1:500, ab152101, Abcam).

Techniques: Staining, Immunostaining, Injection