sci-Plex captures the in vitro temporal dynamics of neurogenic reprogramming from MG.

A) Schematic of the sci-Plex experimental design for assaying reprogramming MG isolated from P11 mice. B) Combined UMAP of cells from the Timecourse and Pulse experiments. Cells are colored by cell type. C) UMAP displaying the pseudotime scores that were calculated for the MG to Bipolar trajectory. D) Schematic depicting the timing and duration of Ascl1 OE in the Timecourse and Pulse experiments. E) Histograms displaying the frequency of cells with each pseudotime score across Ascl1 OE conditions. The vertical black lines are at pseudotime score 16. The yellow region corresponds to the MG, the green region corresponds to the ProL cells, and the purple region reflects cells from the Transition to BP cell state. F) Stacked bar plot of the cell type composition across all Ascl1 OE conditions. The colors represent the cell types as indicated. Only the MG and MG-derived cell types are included. G) Gene expression plots along pseudotime for genes of interest. Each point represents an individual cell’s expression of the indicated gene. The cells are colored by cell type as in F. H) Gene expression heatmap for the top 250 DEGs as assessed across a pseudotime score of 10-20. The dashed line is at pseudotime score 16. All cells with a non-infinite pseudotime score are ordered by pseudotime score along the x-axis. Genes were clustered by k-means into 3 clusters.

sci-Plex as a screen to identify small molecules that affect Ascl1-dependent MG reprogramming.

A) Bar plot representing the distribution of targets for the 92 compounds included in the screen. B) Schematic of the experimental design for the small molecule screen. C) UMAP of the MG and MG-derived cell types. Cells are colored by cell type. D) UMAP from C with cells colored by collection. E) Dot plot of the genes used to define the MG-derived cell types from the screen. Dot size indicates the percent of cells that express the gene of interest. The color indicates the log10 mean UMIs per cell. F) Quantification of the fold change in Neuron cell counts between each indicated condition and the Ascl1 only control. The plots from Collection 1 and Collection 2 used only the control wells collected on their respective days. The dose of each compound is indicated by the size of the dot. G) Quantification of the total cell counts for each treatment. The compound’s dose is indicated by the size of the dot. H) Plots displaying the fold change of Neuron (red) and ProL (orange) cell counts compared to Ascl1 only across all doses for the top hits from the screen. All conditions in which at least 20 cells were recovered are displayed.

Validation of sci-plex hits in an in vivo neuronal regeneration paradigm.

A) Schematic of the transgenic mouse used to induce Ascl1 and GFP specifically in MG. B) Experimental paradigm for testing the in vivo dynamics of Ascl1-dependent MG reprogramming. C) Dot plot of the genes used to define the major cell types found in the reprogramming cells in vivo across all timepoints. The size of the dot indicates the percent of cells that express the gene of interest. The color indicates the log10 mean UMIs per cell. D) An integrated UMAP of the cells recovered from in vivo reprogramming 5, 9, or 21 days after NMDA treatment. Cells are colored by cell type. E) Stacked bar plot of the cell type composition across in vivo Ascl1 OE durations. The colors represent the cell types as in D. Only the MG and MG-derived cell types are included. F) Heatmap of the in vivo expression of genes related to the pathways regulated by the top small molecules hits from the in vitro screen. Reprogramming cells are ordered along pseudotime. The row normalized z-score was calculated from size factor normalized gene expression counts. G) Experimental paradigm for testing the in vivo effect of hit compounds on reprogramming. H) Representative sections of retina after Ascl1 only, Ascl1/NMDA/TSA or Ascl1/NMDA/TSA/Metformin treatment. Immunostaining for GFP (green) and Otx2 (purple) show MG-derived cells (GFP+) expressing the neuronal marker Otx2. Scale bar =50μm. I) Quantification of the percentage of GFP+ MG-derived cells that express the neuronal marker in Ascl1/NMDA/TSA (control) versus the addition of Metformin (p=0.017). Statistical significance was determined using an unpaired t-test. Height of the bar indicates the mean, and the error bars indicate the standard deviation. J) Quantification of the percentage of GFP+/Otx2+ cells in control versus DBZ treatment (p=0.0009). Statistical significance was determined using an unpaired t-test. Height of the bar indicates the mean, and the error bars indicate the standard deviation. K) Representative widefield image of GFP+ MG-derived neurons (GFP+/Otx2+) showing the widespread stimulation of neurogenesis in metformin treated retinas. Scale bar is 100μm.