Developmental emergence of cortical neurogliaform cell diversity

GABAergic interneurons are key regulators of cortical circuit function. Among the dozens of reported transcriptionally distinct subtypes of cortical interneurons, neurogliaform cells (NGC) are unique: are recruited by long-range excitatory inputs and the primary source of slow cortical inhibition. Despite their functional importance, the developmental emergence and cellular diversity of NGCs remains unclear. Here, combining single-cell transcriptomics, genetic fate-mapping, electrophysiological morphological characterization, we reveal that discrete molecular subtypes of NGCs, with distinctive functional and anatomic profiles, populate the neocortex. Furthermore, we show that NGC subtypes emerge gradually through development, as incipient differential molecular signatures are apparent in Preoptic Area (POA) born NGC precursors. By identifying NGC developmentally-conserved transcriptional programs, we report that the transcription factor Tox2 constitutes an identity hallmark across NGC subtypes. Using CRISPR-Cas9-mediated genetic loss-of-function, we show that Tox2 is critical for NGC development: POA-born cells lacking Tox2 fail to differentiate into NGCs. Together, these results reveal that NGCs are born from a spatially restricted pool of Tox2+ POA precursors, after which intra-type diverging molecular programs are gradually acquired post-mitotically and result in functionally and molecularly discrete NGC cortical subtypes. Overall design: Two scRNA-seq collection types were used: microfluidic-based scRNA-seq for experiments involving FACS (P15, P30 and E14.5 Hmx3-Cre::Htr3a-GFP; R26R-tdTOMfl/fl ; E14.5 FlashTag) and droplet-based for experiments on WT cells (E14.5). For each time-point, several biological replicates where included (3 to 5). Analyzed cell populations include diverse subtypes of interneurons at several ages and differentiation stages.