02622nas a2200385   4500000000100000000000100001008004100002260001200043653001000055653002500065653001700090653001400107653003000121653003100151653001100182653001400193653002600207653001800233100002200251700003400273700001800307700001500325700001900340700001800359700001800377700002200395700001500417700001700432700002200449245007400471300001200545490000800557520165700565022001402222       2023                            d  c2023-0910aBrain10aCell Differentiation10aCell Lineage10aChromatin10aGene Expression Profiling10aGene Expression Regulation10aHumans10aorganoids10aTranscription Factors10aTranscriptome1 aJonas Simon Fleck1 aSophie Martina Johanna Jansen1 aDamian Wollny1 aFides Zenk1 aMakiko Seimiya1 aAkanksha Jain1 aRyoko Okamoto1 aMalgorzata Santel1 aZhisong He1 aJ. Gray Camp1 aBarbara Treutlein00aInferring and perturbing cell fate regulomes in human brain organoids  a365-3720 v6213 aSelf-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.  a1476-4687