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