Neocortical temporal patterning by a two-layered regulatory structure

In the developing neocortex, a diverse array of neurons with defined types and abundances are systematically generated by a limited population of radial glial progenitors (RGPs) as they undergo successive fate changes. How this temporal patterning is regulated at the molecular level remains largely unknown. Here we present an in-depth single-cell multi-omics assay for deep characterization of regulatory programs, along with an analytical framework that integrates joint dynamics in single-cell transcription and chromatin structure to interrogate the regulation of temporal identities and fate progression of RGPs. We found that RGP temporal patterning is governed by a two-layered regulatory structure consisting of global regulators acting upon a cascade of transcription factor (TF) hubs. This cascade proceeds not by confining cascaded TF expression to specific temporal windows, but through synergistic transcriptional and epigenetic dynamics that temporally modulate TF regulatory activity. Furthermore, global regulators exhibit a progressively increasing expression pattern, and our computational modeling analysis found that the gradient of this pattern specifies the duration of each cascading stage and, subsequently, the abundance of specific progeny output. Together, this two-layered regulatory structure offers a mechanistic control of temporal patterning and emphasizes the importance of global regulators in managing temporal programs.