show Abstracthide AbstractThe superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. The developmental principles directing the generation of SC cell-type diversity are however not understood. Here we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic MADM (Mosaic Analysis with Double Markers)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally-related cell lineages revealed that radial glial progenitor (RGP) cells in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at single RGP/cell level of the mammalian SC ontogeny Overall design: scRNA-Seq of neurons originating from a single radial glia progenitor (clone) labeled at E10 using MADM and Fz10-CreER or Sox2-CreER