AAV-mediated overexpression of Oct4 induces neurogenesis in wildtype mouse Müller glia following NMDA-induced excitotoxicity. (A) Schematic of the transgenic construct used to specifically label Müller glia with Sun1-GFP expression. (B) Schematic of the GFAP AAV constructs and experimental workflow. (C) Representative images of retinas immunolabeled for GFP, mCherry, Otx2, and HuC/D. White arrowheads indicate GFP-positive Müller glia-derived neurons expressing neuronal markers Otx2 or HuC/D. (D) Quantification of mean percentage ± SD of GFP-positive Müller glia-derived neurons expressing either Otx2 or HuC/D. Significance was determined via two-way ANOVA with Tukey’s multiple comparison test: ****p < 0.0001. Each data point was calculated from an individual retina. TAM, tamoxifen; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

Oct4 overexpression enhances neurogenesis observed in Rbpj-deficient Müller glia. (A) Schematic of the transgenic constructs used to induce loss of function of Rbpj specifically in Müller glia. (B) Schematic of the GFAP AAV constructs and experimental workflow. (C) Representative images of retinas immunolabeled for GFP, mCherry, Otx2, and HuC/D. White arrowheads indicate GFP-positive Müller glia-derived neurons expressing neuronal markers Otx2 or HuC/D. (D) Quantification of mean percentage ± SD of GFP-positive Müller glia-derived neurons expressing either Otx2 or HuC/D. Significance was determined via two-way ANOVA with Tukey’s multiple comparison test: ****p < 0.0001. TAM, tamoxifen; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

ScRNA-Seq analysis of Müller glia and Müller glia-derived neurons derived from Rbpj-deficient Müller glia following Oct4 overexpression. (A) Schematic of the scRNA-seq experimental pipeline. (B) UMAP plot showing the clustering of GFP-positive cells from retinas infected with GFAP-mCherry and GFAP-Oct4-mCherry AAV constructs. (C) Stacked bar plots showing the proportion of cells in each cluster across two sample groups. (D) Feature plots highlighting the cluster of Müller glia (Hes5), neurogenic MGPCs (Neurog2, Insm1, Ascl1), bipolar cells (Otx2), and amacrine cells (Elavl3). (E) Heatmap showing the expression of top differentially expressed genes (DEGs) for Rbpj-deficient Müller glia cell cluster from retinas infected with GFAP-mCherry and GFAP-Oct4-mCherry AAV constructs. (F) Heatmap showing the expression of top differentially expressed genes (DEGs) for MGPC cell cluster from retinas infected with GFAP-mCherry and GFAP-Oct4-mCherry AAV constructs.

Integrated snRNA/scATAC-Seq analysis of control and Rbpj-deficient Müller glia with Oct4 overexpression.

(A) Schematic of the multiomic scRNA/ATAC-seq experimental pipeline. (B) UMAP plot of multiomic datasets showing the clustering of GFP+ cells from control and Rbpj-deficient Müller glia from GFAP-mCherry and GFAP-Oct4-mCherry AAV infected retinas. (C) UMAP plot showing the identity of cell clusters determined by marker gene expression. (D) Stacked bar plots represent the proportion of cells in each cluster across different sample groups. (E) Feature plots highlighting the cluster of Müller glia (Hes1), neurogenic MGPCs (Neurog2, Sox4), bipolar cells (Otx2), Pou5f1- and Rfx4-expressing cells. (F) Heatmap showing expression of DEGs along the neurogenesis trajectory. (G) Heatmap showing differential motif activity along the neurogenesis trajectory. (H) Heatmaps of DEGs differentially expressed between Oct4 and mCherry control samples in the wildtype and Rbpj-deficient Müller glia cell clusters. Venn diagram showing unique and common DEGs between Oct4 and mCherry control in the wildtype and Rbpj-deficient Müller glia clusters. (I) Increased chromatin accessibility regions associated with the Oct4 and Rfx4 loci observed following Oct4 overexpression in both wildtype and Rbpj-deficient Müller glia. (J) Top-ranked enriched motifs in chromatin regions showing increased accessibility following Oct4 overexpression in both wildtype and Rbpj-deficient Müller glia cell clusters.

Oct4/pou5f3 mRNA is not detectably expressed in Müller glia after injury.

Expression of pluripotency factors in resting or injured retinas by scRNA-Seq and bulk RNA data in (a) zebrafish and (b) mouse after retinal injury from Hoang et al., 2020 and in (c) Nfia/b/x;Rbpj-deficient Müller glia from Le et al., 2024.

Immunohistochemical analysis of glial, AAV reporter, and additional molecular markers at 1- and 2-weeks following NMDA in wildtype Müller glia.

(a) Schematic of the transgenic construct used to specifically label Müller glia with Sun1-GFP expression. (b) Schematic of the GFAP AAV constructs and experimental workflow. Representative images of retinas immunolabeled for GFP, mCherry and (c-d) Sox2, (e-f) EdU/Iba1, (g-h) Ascl1, (i-j) Oct4, (k-l) Otx2. White arrowheads indicate GFP-positive cells expressing selected markers. Yellow arrowheads indicate GFP-positive cells that do not co-label with Sox2 and EdU/Iba1+ cells that do not co-label with GFP+ cells. Quantification of mean percentage ± SD of GFP-positive Müller glia co-labeled with (m) EdU and (n) Otx2. Significance was determined via two-way ANOVA with Tukey’s multiple comparison test: ***p < 0.001, *p < 0.05. Each data point was calculated from an individual retina. TAM, tamoxifen; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

Immunohistochemical analysis of glial, AAV reporter, and additional molecular markers of Müller glia-derived neurons at 5 weeks following NMDA following Oct4 transduction in wildtype and Rbpj-deficient Müller glia.

(a-h) Representative images of wildtype retina immunolabeled for GFP, mCherry, and (a) Oct4, (b) Ascl1, (c) Sox2, (d) Scgn, (e) Cabp5, (f) Isl1, (g) Nrl, (h) EdU. (i-k) Representative images of Rbpj cKO retina immunolabeled for GFP, mCherry, and (i) Oct4, (j) Sox9, (k) EdU. White arrowheads indicate GFP-positive cells co-labeled with the relevant marker. Yellow arrowheads indicate GFP-positive Müller glia that did not co-labeled with Müller glia markers. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

Immunohistochemical analysis of glial, AAV reporter, and additional molecular markers at 1- and 2-weeks following NMDA in Rbpj-deficient Müller glia.

(a) Schematic of the transgenic constructs used to induce loss of function of Rbpj specifically in Müller glia. (b) Schematic of the GFAP AAV constructs and experimental workflow. Representative images of retinas immunolabeled for GFP, mCherry and (c-d) Sox2, (e-f) EdU/Iba1, (g-h) Ascl1, (i-j) Oct4, (k-l) Otx2. White arrowheads indicate GFP-positive cells expressing selected markers. Yellow arrowheads indicate GFP-positive cells that do not co-label with Sox2 and EdU/Iba1+ cells that do not co-label with GFP+ cells. (m) Quantification of mean percentage ± SD of GFP-positive Müller glia-derived neurons expressing Otx2. Significance was determined via two-way ANOVA with Tukey’s multiple comparison test: *p < 0.05. Each data point was calculated from an individual retina. TAM, tamoxifen; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

AAV-mediated overexpression of Oct4 induces neurogenesis in injured Notch1/2-deficient Müller glia.

(a) Schematic of the transgenic mice used to induce deletion of Notch1/2 specifically in Müller glia. (b) Schematic of the experimental workflow. (c) Representative images of retinas immunolabeled for GFP, mCherry, Otx2, and HuC/D. White arrowheads indicate GFP-positive Müller glia-derived neurons expressing neuronal markers Otx2 or HuC/D. Quantification of mean percentage ± SD of GFP-positive Müller glia-derived neurons expressing either Otx2 or HuC/D. Significance was determined via two-way ANOVA with Tukey’s multiple comparison test: ****p < 0.0001. TAM, tamoxifen; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

Oct4 overexpression does not enhance neurogenesis in Nfia/b/x-deficient Müller glia because GFAP minipromoter is not active in these cells.

(a) Schematic of the transgenic mice used to induce deletion of Nfia/b/x specifically in Müller glia. (b) Schematic of the experimental workflow. (c) Representative images of retinas immunolabeled for GFP, mCherry, Otx2, and HuC/D. White arrowheads indicate mCherry expression in Sox2+ Müller glia that are not Sun1-GFP+. Quantification of mean percentage ± SD of GFP-positive Müller glia-derived neurons expressing either Otx2 or HuC/D. TAM, tamoxifen; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

ScRNA-Seq analysis of Müller glia-derived neurons from Rbpj-deficient Müller glia following Oct4 overexpression.

(a) UMAP plot showing GFP-positive cells from retinas infected with GFAP-mCherry and GFAP-Oct4-mCherry AAVs. (b) Expression of glial and neuronal markers. (c) Top DEGs of different cell types generated by Oct4 overexpression in Rbpj cKO.

Pseudotime analysis showing the transition from resting to neurogenic MGPCs to bipolar cells.

(a) Top DEGs across different retinal cell types. (b) Pseudotime trajectory of neurogenesis from Müller glia to bipolar cells. (c) Heatmap showing differential chromatin accessible regions at different pseudotime stages along the neurogenesis trajectory.