GABA precursors arose in a segregated fashion in an area of the organoids and did not colocalize with TBR1+ excitatory neuron precursors (Figure S5). The number of cells immunoreactive for ASCL1/MASH1 and NKX2.1 (two TFs expressed by GABAergic progenitor cells) and the neurotransmitter GABA was also increased in ASD-derived organoids (Figure S5). Moreover, western blot analyses further confirmed increased protein expression of GAD1/2 in ASD organoids (Figure S5Q, R). Along with the upregulation of GSX2, ASCL1, DLX1, DLX2, DLX5, DLX6 and DLX6-AS1, which is the top upregulated gene at TD11 (Table S2) and the increase in GABA transporter immunostaining (Figure 3H, I), these cellular analyses strongly suggest an overproduction of progenitors and neurons of the GABAergic lineage as well as an altered balance between the number of excitatory and inhibitory neurons in organoids derived from probands (Figure 4K).
