To investigate the mechanism by which FOXG1 could affect the overproduction of GABAergic neurons, we compared cell proliferation in ASD- and control-derived organoids by BrdU incorporation with or without FOXG1 RNAi. Quantification of double-labeled BrdU+/Ki67+ cells at TD11 revealed no general changes in proliferation between proband- and control-derived organoids (Figure 7A, B and Figure S6 A, B). However, there was a significant increase in the number of DLX2+ cells that incorporated BrdU in proband-derived organoids, as well as an increased proportion of BrdU+ cells that colocalized DLX1/2. Both effects were precluded by FOXG1-knockdown (Figure 7A–C). Furthermore, at TD31, proband-derived organoids showed a greatly increased proportion of DLX+ and GAD1+ cells that had incorporated BrdU at TD11 (Figure 7D–H), an effect that was also greatly attenuated by FOXG1 RNAi, which lowered the proportion of BrdU+/DLX+ and BrdU+/GAD1+ cells in ASD-derived organoids to levels comparable to those of the unaffected control (Figure 7D–H). Moreover, GAD1+ cells at TD31 were not aberrantly entering the cell cycle, suggesting that GABAergic neurons were terminally differentiated in the organoids (Figure S 6C, D). Taken together, these
