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

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 previously described 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. Atoh1-lineage neurons labeled by TdTomato and/or GFP in mouse hindbrain were enriched by FACS, followed by scRNA-seq with 10X chromium 3' v3.1 kit and NovaSeq 6000. Single-cell transcriptomic profiles across embryonic stages (E9.5, E.10.5, E11.5, E12.5, E13.5, E14.5, and E16.5) were compared促神经转录因子可构建分子级联反应，以统筹神经元多样性的生成。其中一类转录因子Atoh1可分化为小脑兴奋性神经元，以及脑干中与听觉、呼吸、平衡功能密切相关的30余种独特神经核团。尽管学界已对Atoh1谱系来源的神经元完成定性描述，但驱动其命运决定的转录调控程序，以及其多样性的完整覆盖范围仍未明确。本研究针对发育中小鼠后脑的Atoh1谱系神经元，开展了单细胞RNA测序（single-cell RNA-sequencing）与ATOH1 DNA结合位点分析。这套高分辨率数据集不仅揭示了针对特定脑干神经核团的全新分子标记，还证实具有转录异质性的神经前体细胞需依赖ATOH1才能完成正常迁移。此外，本研究在小鼠Atoh1谱系中发现了一类大量增殖的单极刷细胞前体——该类细胞此前被认为是某一亚型髓母细胞瘤的起源。综上，本研究数据为发育中小鼠后脑的相关研究提供了前所未有的认知，并为研究较少的神经元群体的功能评估提供了分子标记物。本研究通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）富集了小鼠脑干中被TdTomato和/或绿色荧光蛋白（green fluorescent protein, GFP）标记的Atoh1谱系神经元，随后使用10X Chromium 3' v3.1试剂盒与NovaSeq 6000测序平台完成单细胞RNA测序，并对胚胎发育各阶段（E9.5、E10.5、E11.5、E12.5、E13.5、E14.5及E16.5）的单细胞转录组图谱进行了对比分析。