We performed immunofluorescence to validate the clusters we observed. At D24, ASCL1 and NKX2-1 co-localized in a subset of cells, as expected for the ipMGE.24.DLL1 cluster (Figure 4B). We also observed expression of SP8 in a subset of Cit+ neurons, which made up the nLGE clusters at D24 (Figure 4C). At D54, co-localization of OLIG2 and ASCL1 was observed in presumptive ipMGE cells (Figure 4D), while CORT expression was present in Cit+ nCTX cells (Figure 4E). One Cit− cluster expressed CRYAB at D100, while six were positive for HOPX. These genes were expressed in mutually exclusive clusters by gene expression (Figure 4A) and antibody staining (Figure 4F). RBFOX3 was expressed in a limited number of nCTX clusters, and we observed its expression via immunofluorescence in a subset of Cit+ cells (Figure 4G).
