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. Overall design: 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（proneural transcription factor）可构建分子级联反应，以调控神经元多样性的形成。其中一类促神经转录因子Atoh1可分化为小脑兴奋性神经元，以及脑干中超过30种与听觉、呼吸及平衡功能密切相关的独特神经核团。尽管学界已对Atoh1谱系（Atoh1-lineage）衍生的神经元进行了定性描述，但调控其命运决定的转录程序，以及其神经元多样性的完整范围仍未明确。本研究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）以及ATOH1的DNA结合分析。该高分辨率数据集不仅揭示了特定脑干核团的全新分子标记物，还证实转录异质性的祖细胞需要ATOH1以完成正常迁移。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（unipolar brush cell）祖细胞，该类细胞此前被认为是某一亚型髓母细胞瘤（medulloblastoma）的起源细胞。综上，本研究数据为发育中小鼠后脑的研究提供了前所未有的见解，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。
实验设计：通过荧光激活细胞分选（FACS）富集小鼠后脑内被TdTomato和/或绿色荧光蛋白（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平台开展单细胞RNA测序。对胚胎期（E9.5、E10.5、E11.5、E12.5、E13.5、E14.5及E16.5）各阶段的单细胞转录组图谱进行了比较分析。