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. 2022 Nov 23;8(47):eabq7219. doi: 10.1126/sciadv.abq7219

Fig. 4. Islet1 and Pou4f2 coinduction stimulates RGC-like neurons from MG in vitro.

Fig. 4.

(A) Schematic of transgenic construct to induce IPA in all primary MG in vitro by doxycycline. (B) Paradigm for inducing Ascl1-mediated neurogenesis in vitro. (C to E) Representative images of EdU+ MG-derived neurons expressing neuronal markers.) (C) EdU+ (white) MG-derived cell expressing Tuj1 (red). (D) MG-derived neuron expressing EdU (white), Neurofilament M (NFM; red), and the GFP transgene reporter (GFP). ( (E) MG-derived neuron expressing Calbindin (red) colabeling with EdU (white), DAPI, and GFP. (F to H) UMAP plots of cultured MG reprogrammed with Ascl1 (F) and IPA (G), integrated with cells from in vivo IPA regeneration model (H) as reference. (I) Stacked bar plot showing composition of neuronal clusters in each sample. BC, bipolar cell. (J) Feature plots highlighting differentially expressed genes in neuronal clusters of either reprogramming strategy. (K) Highlighted cells of the Ascl1 and IPA datasets used for downstream DGE analysis in (L). (M) Heatmap of genes differentially expressed in either the Ascl1 or IPA condition. WT, untreated cultured MG included for baseline values. Statistics for differential gene analysis: Wilcoxon Mann-Whitney test for significance (P < 0.05). Scale bars, 50 μm. Mouse schematic was made with Biorender.com.