A single cell transcriptomic map of the developing Atoh1-lineage uncovers neural fate decisions and neuronal diversity in the hindbrain. [CUT&RUN]

Proneural transcription factors set up the molecular cascade to orchestrate neuronal diversity. One such transcription factor, Atoh1, gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although the neurons that arise from the Atoh1-lineage have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA-sequencing and ATOH1 DNA binding in Atoh1-lineage neurons of the developing mouse hindbrain. This high-resolution dataset revealed new markers for specific brainstem nuclei and demonstrated transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable proliferating unipolar brush cell progenitor in the mouse Atoh1-lineage that was described in humans as the origin of one medulloblastoma subtype. Collectively, our data reveal unprecedented insight into the developing mouse hindbrain and provide markers for functional assessment of less studied neuronal populations. Overall design: Atoh1 binding profiles in the hindbrain of Atoh1GFP/GFP E12.5 and E14.5 mouse embryos. Atoh1-lineage neurons labeled by GFP as well as GFP-negative cells in mouse hindbrains were enriched by FACS, followed by CUT&RUN and sequencing on the NovaSeq 6000.