There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis.

Striatal medium-sized spiny neurons (MSNs), composed of striatonigral and striatopallidal neurons, are derived from the lateral ganglionic eminence (LGE). We find that the transcription factor Sp9 is expressed in LGE progenitors that generate nearly all striatal MSNs and that Sp9 expression is maintained in postmitotic striatopallidal MSNs. Sp9-null mice lose most striatopallidal MSNs because of decreased proliferation of striatopallidal MSN progenitors and increased Bax-dependent apoptosis, whereas the development of striatonigral neurons is largely unaffected. ChIP qPCR provides evidence that Ascl1 directly binds the Sp9 promoter. RNA-seq and in situ hybridization reveal that Sp9 promotes expression of Adora2a, P2ry1, Gpr6, and Grik3 in the LGE and striatum. Thus, Sp9 is crucial for the generation, differentiation, and survival of striatopallidal MSNs.

Immature neurons generated by the subpallial MGE tangentially migrate to the cortex where they become parvalbumin-expressing (PV+) and somatostatin (SST+) interneurons. Here, we show that the Sp9 transcription factor controls the development of MGE-derived cortical interneurons. SP9 is expressed in the MGE subventricular zone and in MGE-derived migrating interneurons. Sp9 null and conditional mutant mice have approximately 50% reduction of MGE-derived cortical interneurons, an ectopic aggregation of MGE-derived neurons in the embryonic ventral telencephalon, and an increased ratio of SST+/PV+ cortical interneurons. RNA-Seq and SP9 ChIP-Seq reveal that SP9 regulates MGE-derived cortical interneuron development through controlling the expression of key transcription factors Arx, Lhx6, Lhx8, Nkx2-1, and Zeb2 involved in interneuron development, as well as genes implicated in regulating interneuron migration Ackr3, Epha3, and St18. Thus, Sp9 has a central transcriptional role in MGE-derived cortical interneuron development.