Comprehensive spatiotemporal mapping of single-cell lineages in developing mouse brain by CRISPR-based barcoding

A fundamental interest in developmental neuroscience is to map the complete single-cell lineages within the brain. We developed a CRISPR-based lineage specific tracing (CREST) method for clonal tracing in Cre mice. We used two complementary strategies based on CREST method to map single-cell lineages in developing mouse ventral midbrain (vMB). Applying snapshotting CREST (snapCREST), we constructed a spatiotemporal lineage landscape of developing vMB, and identified six progenitor archetypes that could represent the principal clonal fates of individual vMB progenitors. We uncovered three distinct clonal lineages in the floor plate that specified glutamatergic, dopaminergic, or both neurons, and identified Pbx1 as a key regulator in fate specification of dopaminergic neurons in sub-cluster and clonal level. We further created pandaCREST to associate the transcriptomes of progenitor cells in vivo with their differentiation potentials. We identified multiple origins of dopaminergic neurons and demonstrated that a transcriptome-defined progenitor type comprises heterogeneous progenitors, each with distinct clonal fates and molecular signatures. Therefore, the CREST method and strategies allow comprehensive single-cell lineage analysis that could offer new insights into the molecular programs underlying neural specification.