Multimodal Hox5 activity generates motor neuron diversity [RNA-seq]

Motor neurons (MNs) are the final output of circuits driving fundamental behaviors, such as respiration and locomotion. Hox proteins are essential in generating the MN diversity required for accomplishing these functions, but the transcriptional mechanisms that enable Hox paralogs to assign distinct MN subtype identities despite their promiscuous DNA binding motif are not well understood. Here we show that Hoxa5 can modify chromatin accessibility in all mouse spinal cervical MN subtypes and engages TALE co-factors to directly bind and regulate subtype-specific genes. We identify a paralog-specific interaction of Hoxa5 with the phrenic MN-specific transcription factor Scip and show that heterologous expression of Hoxa5 and Scip is sufficient to suppress limb-innervating MN identity. We also demonstrate that phrenic MN identity is stable after Hoxa5 downregulation and identify Klf proteins as potential regulators of phrenic MN maintenance. Our data identify multiple modes of Hoxa5 action that converge to induce and maintain MN identity. Overall design: Comparative gene expression analysis (RNA-seq data) for cervical and thoracic motor neurons at e12.5. Each cervical or thoracic region contains three biological replicates.

运动神经元（Motor Neurons, MNs）是调控呼吸、运动等基础生命行为的神经环路的最终输出单元。同源盒蛋白（Hox proteins）在产生实现上述功能所需的运动神经元多样性过程中发挥关键作用，但尽管Hox旁系同源蛋白具有非特异性DNA结合基序，其介导不同运动神经元亚型身份特化的转录调控机制仍未被充分阐明。本研究发现，Hoxa5可重塑所有小鼠脊髓颈段运动神经元亚型的染色质可及性，并招募TALE辅因子（TALE co-factors）直接结合并调控亚型特异性基因。本研究鉴定出Hoxa5与膈神经运动神经元特异性转录因子Scip之间存在旁系同源特异性相互作用，并证实异位表达Hoxa5与Scip足以抑制肢体支配型运动神经元的身份特征。本研究还证实，在Hoxa5表达下调后，膈神经运动神经元的身份特征仍保持稳定，并鉴定出Krüppel样因子（Klf proteins）是维持膈神经运动神经元身份的潜在调控因子。本研究的数据揭示了Hoxa5发挥作用的多种模式，这些模式协同作用以诱导并维持运动神经元的身份特征。实验整体设计：针对胚胎发育第12.5天（e12.5）的颈段与胸段运动神经元开展比较基因表达分析（RNA测序，RNA-seq）。每个颈段或胸段分组均包含3次生物学重复。