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. 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.

促神经转录因子可构建分子级联反应，以调控神经元多样性的形成。其中一类转录因子Atoh1（Atoh1），可产生小脑兴奋性神经元，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立神经核团。尽管学界已对Atoh1谱系来源的神经元进行了定性描述，但驱动其命运抉择的转录调控程序，以及其神经元多样性的完整范围仍未明确。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。该高分辨率数据集不仅揭示了特定脑干神经核团的新型标记物，还证实转录组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谱系中发现了大量增殖性单极刷细胞祖细胞——该类细胞在人类中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标记物。本研究检测了Atoh1GFP/GFP基因型小鼠胚胎（胚胎发育第12.5天，即E12.5；第14.5天，即E14.5）后脑组织中的ATOH1结合谱：通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）富集小鼠后脑内的GFP标记的Atoh1谱系神经元与GFP阴性细胞，随后开展CUT&RUN实验，并在NovaSeq 6000测序平台上完成测序。