Dox treatment (Figure 4E). Given the observation that NGN2 expression could affect NGN2-iN fate specification, we revisited the time course data for NGN2-iN development. The expression of NGN2 remained nearly consistent during NGN2-iN development, with a slight increase of NGN2 expression at weeks 4 and 5 (Figures 4F and S3B). Interestingly, the expression level of NGN2 is consistently lower in GPM6A+ cells, independent of the duration of development (Figure 4G). Supporting this data, the proportion of GPM6A+ cells also decreased with longer NGN2-iN culture (Figure 4H). This could imply that prolonged expression of NGN2 potentially steers NGN2-iN fate away from CNS lineages. Altogether, these data show that the duration of NGN2 induction impacts the proportion of neuron subtypes that emerge in this single factor reprogramming paradigm.
