Revealing regulatory networks mediating human in vitro neurogenesis
Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. By overexpressing neurogenic transcription factors in human induced pluripotent stem cells, we obtained postmitotic neurons in high purity, homogeneity and speed, allowing us to study transcriptomic changes over the entire time course of neurogenesis. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. We also used these induced neurons to study the brain-enriched miR-124, which has been assigned as a key player of neuronal differentiation via its complex but little understood regulation of thousands of annotated targets. To systematically chart its regulatory functions, we used gene editing to disrupt all six miR-124 alleles. Using RNA-induced-silencing-complex precipitation, we identified 98 high-confidence direct miR-124 targets. By performing advanced transcription-factor-network analysis, we identified indirect miR-124 effects on apoptosis, neuronal subtype differentiation, and the regulation of previously uncharacterized zinc finger transcription factors. Our data emphasize the need for combined experimental- and system-level analyses to comprehensively disentangle and reveal gene regulatory networks, including their involvement in the neurogenesis of diverse neuronal cell types found in the human brain.