COMPETITIVE INHIBITION BY SP9 TRANSCRIPTION FACTOR CO-BINDING BALANCES PRODUCTION OF D1 AND D2 MEDIUM SPINY NEURONS

In the developing mammalian brain, a series of fate transitions orchestrated by intrinsic and extrinsic gene regulation occur in the ganglionic eminence to give rise to a diversity of GABAergic cell types. Dysregulation of this process is linked to psychiatric disease including autism spectrum disorders and schizophrenia. Recent work has established that loss of the transcription factor SP9 results in a dearth of Drd2-expressing MSNs (D2 MSNs) in the striatum (Zhang et al. 2016). To uncover SP9’s role, we performed a CRISPR-Cas9 mediated sparse single-cell gene knockout of Sp9 in the ganglionic eminence of transgenic Cas9 mice by co-electroporating piggyback transposon single guide RNA (tCROP-seq) with a heritable lineage barcode (TrackerSeq). ScRNA-seq analysis corroborated loss of D2 MSNs, with enrichments in Drd1 expressing MSNs (D1 MSNs) and Tszh1 intercalated cells of the amygdala (ITCs), suggesting a potential fate switch upon Sp9 depletion. We leveraged the lineage history of cells to identify upstream gene regulatory modules controlling cell fate multifurcations from a shared progenitor state to D2 MSNs, D1 MSNs, and ITCs, revealing that SP9 may work together with partner transcription factors to bias progenitor states towards D2 MSNs fates. By integrating interaction assays, chromatin profiling, and reporter analyses, we uncover regulatory principles by which developmental transcription factors balance alternative differentiation trajectories. These findings provide insight into how proportional representation of MSN subtypes is established during development, supporting a model of competitive transcription factor inhibition capable of balancing two differentiation programs to produce equal ratios of MSN cell types.