Engrailed transcription factors direct embryonic medial excitatory cerebellar nuclei neuron identity and survival

In the cerebellum, the excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the circuit. The medial eCN (eCNm) were recently divided into molecularly defined subdomains in the adult, however how they are established during development is not known. We define molecular subdomains of the eCNm using scRNA-seq and spatial expression analysis during embryogenesis. Our analysis shows that the eCNm are transcriptionally divergent from the rest of the eCN by E14.5. We previously showed that conditional loss of the homeobox genesEn1andEn2leads to death of a subset of embryonic eCNm. We demonstrate that loss ofEn1/2in embryonic eCNm results in cell death of a specific posterior eCNm molecular subdomain and loss of TBR2 expression in an anterior subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the two other cerebellar excitatory neuron types. Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage. Atoh1-lineage neurons of the cerebellum labeled by tdTomato in control or Engrailed 1/2 knockout E14.5 embryos were isolated by FACS, and scRNA-seq using the 10X Genomics' Chromium Next GEM Single Cell 3' Reagent Kits (v3.1). Samples were then sequenced on a NovaSeq 6000.在小脑中，三类小脑核的兴奋性神经元（excitatory neurons of the three cerebellar nuclei, eCN）构成了该神经环路的主要输出通路。近期有研究在成年个体中基于分子特征将内侧eCN（eCNm）划分为多个分子定义的亚结构域，但其在发育过程中的建立机制仍不明晰。本研究借助单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与空间表达分析，对胚胎发育阶段的内侧eCN（eCNm）开展分子亚结构域鉴定。分析结果显示，在胚胎发育第14.5天（E14.5），内侧eCN（eCNm）的转录组特征已与其余小脑核兴奋性神经元产生显著分化。我们前期研究证实，同源框基因En1与En2的条件性敲除会导致部分胚胎期内侧eCN（eCNm）神经元死亡。本研究进一步证明，胚胎期内侧eCN（eCNm）中En1/2的缺失会引发特定后侧内侧eCN（eCNm）分子亚结构域的细胞死亡，并使前侧亚结构域丧失TBR2的表达，同时还会降低突触相关基因的表达水平。我们还揭示了EN1/2在另外两类小脑兴奋性神经元谱系中，同样发挥介导TBR2表达、调控神经元分化与存活的保守功能。综上，本研究明确了胚胎期内侧eCN（eCNm）的分子多样性，并揭示了EN1/2在小脑兴奋性神经元谱系中的保守调控作用。本研究通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）分离了对照或Engrailed 1/2敲除的E14.5胚胎中被tdTomato标记的Atoh1谱系小脑神经元，随后使用10X Genomics公司的Chromium Next GEM Single Cell 3' Reagent Kits（v3.1）完成单细胞RNA测序，最后在NovaSeq 6000测序平台上对样本进行测序。