Figure 5.

PAX6 Acts as Master Regulator to Control Reprogramming of Glutamatergic and Cholinergic Neurons

(A) Computational mapping of the Smart-seq time course onto the 10X Genomics UMAP. Smart-seq cells are colored based on 10X Genomics cluster membership and positioned based on the five nearest neighbors.

(B) Visualization of scaled expression values of exogenous TFs that showed significant enrichment (Fisher's exact test, p < 0.05) in glutamatergic and/or cholinergic clusters on 2D UMAPs.

(C) Neuronal subtype-specific co-expression modules on the basis of significant associations (Fisher's exact test, p < 0.05) with exogenous TFs shown in (B). Exogenous TFs associated with genes showing highest CS in each module are shown in black, all other exogenous TFs are shown in gray. Neuronal subtype-specific genes are colored. Direct targets of exogenous TF are indicated with dashed lines based on TF with chromatin immunoprecipitation sequencing (ChIP-seq) evidence, highlighted with green borders.

(D) Validation of novel combinations of exogenous TFs by quantification of immunostainings for VGlut1, CHAT, TH, and GABA of CHi (light gray), fibroblasts infected with the complete TF-pool (dark gray) and fibroblasts infected with novel combinations (color) at 21 dpi. n = 4 independent experiments, unpaired Student's t test. Error bars represent mean + SD.

(E and F) The generation of repetitive action potentials in induced neurons infected with DLX2, NEUROG2, PAX6, ZIC1 (E) or DLX1, ISL1, NEUROG2, or PAX6 (F). Representative traces in the presence (upper panel) or absence (lower panel) of extracellular Na⁺ were recorded using the current-clamp protocol.

(G and H) Boxplots showing the log2-transformed TPM values of neurogenic and neuronal subtype-specific genes (G) and fibroblast-specific genes (H) in cells with (+) or without (−) exogenous PAX6 (top), NEUROG2 (middle), and DLX1 (bottom). Boxplots are colored based on −log10-transformed p values.

(I) Edge-normalized network summarizing the associations of exogenous TFs with glutamatergic, cholinergic, GABAergic, and dopaminergic modules.