GSE208199
Induction of neurons from mouse ESC by the bHLH factors Ascl1 and Ngn2 shows distinct genetic dependencies, pluripotency exit, and cell cycle shutdown. [Ascl1_Ngn2_RNAseq_timecourse]
Two closely related proneural bHLH transcription factors, Ascl1 and Ngn2, both can induce mouse embryonic stem cells to differentiate into induced neurons. Although Ascl1 and Ngn2 induced transcriptional programs partially overlap, it is not known whether the differences translate into the distinct mechanisms. We found that both transcription factors induce mutually exclusive side populations by binding and inducing different lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we revealed differential genetic dependencies between Ascl1 and Ngn2. Specifically, Ascl1 is more dependent on factors including Tcf7l1, that regulate pluripotency and cell cycle. In the absence of the pluripotency network repressor Tcf7l1 Ascl1 is still able to repress the core pluripotency genes, however failed to exit cell cycle. Overexpression of Cdkn1c induced cell cycle exit and restored the generation of neurons. This study illuminates two different mechanistic approaches to convert ESC to induced neurons: rapid shutdown of the initial state while installing a terminal cell identity versus repurposing the initial gene regulatory network to allow generation of neurons via intermediate states. Understanding the precise mechanism and the variety of cell states induced is essential to design efficient cell replacement therapeutic applications or precise tools for disease modelling. Overall design: Ascl1 or Ngn2 overexpression in mouse embryonic stem cells over period of 6 days and sorted for Mapt-Venus positive (neurons) or negative cells (non-neurons)