Dissecting Gene Regulatory Networks Governing Human Cortical Cell Fate

Human cortical neurogenesis involves conserved and specialized developmental processes during a restricted window of prenatal development. Radial glia (RG) neural stem cells shape cortical cell diversity by giving rise to excitatory neurons, oligodendrocytes, and astrocytes, as well as olfactory bulb interneurons (INs) and a recently characterized population of cortical INs. Complex genetic programs orchestrated by transcription factor (TF) circuits govern the balance between self-renewal and differentiation, and between different cell fates. Despite progress in measuring gene regulatory network activity during human cortical development, functional studies are required to evaluate the roles of TFs and effector genes in human RG lineage progression. Here we establish a human primary culture system that allows sensitive discrimination of cell fate dynamics and apply single cell clustered regularly interspaced short palindromic repeats interference (CRISPRi) screening to examine the transcriptional and cell fate consequences of 44 TFs active during cortical neurogenesis. We identified multiple TFs, with novel roles in cortical neurogenesis, including ZNF219, previously uncharacterized, that represses neural differentiation and NR2E1 and ARX that have opposing roles in regulating RG lineage plasticity and progression across developmental stages. We also uncovered convergent effector genes downstream of multiple TFs enriched in neurodevelopmental and neuropsychiatric disorders and observed conserved mechanisms of RG lineage plasticity across primates. We further uncovered a postmitotic role for ARX in safeguarding IN subtype specification through repressing LMO1. Our study provides a framework for dissecting regulatory networks driving cell fate consequences during human neurogenesis. Overall design: (1) 2D Libraries: Perturb-seq targeting 44 TFs on differentiation D0 and D7: Primary human culture derived from cortical RG were transduced with CRISPRi library targeting 44 TFs with capture sequence on each guide expanded for 7 days (D0) and differentiated for 7 days (D7). Cells were sorted based on GFP expression and captured using 10X Chromium v3.1 HT kit for single cell RNA sequencing. (2) HM2D libraries: Perturb-seq targeting 5 TFs with lineage tracing on differentiation D7: Primary human or macaque culture derived from cortical RG were transduced with CRISPRi library targeting 5 TFs with capture sequence on each guide and STICR static barcode library, expanded for 7 days and differentiated for 7 days. Cells were sorted based on GFP and mCherry co-expression and captured using 10X Chromium v3.1 HT kit. (3) Slice culture libraries: Perturb-seq targeting 5 TFs with lineage tracing on differentiation D12-14: The germinal zone of the developing human brain was locally transduced with CRISPRi library targeting 5 TFs and STICR static barcode library, cultured for 12-14 days. Cells were sorted based on GFP and mCherry co-expression and captured using 10X Chromium v3.1 HT kit. (4) Double knockdown libraries: Perturb-seq targeting ARX and LMO1 on differentiation D7: Primary human culture derived from cortical RG were transduced with CRISPRi library targeting ARX and LMO1 expanded for 7 days (D0) and differentiated for 7 days (D7). Cells were sorted based on GFP and mCherry co-expression and captured using Illumina Single Cell CRISPR Library kit.