Transcriptional Dynamics of Motor Neuron Maturation in vivo and in vitro

Mouse spinal motor neurons are specified in the embryo on embryonic day 9.5 (E9.5), but motor function doesn't fully mature till the third week of postnatal life (P14-P21) and the degenerative disease Amyotrophic Lateral Sclerosis only affects motor neurons in adulthood (age of onset differs between models). In order to better understand motor neuron maturation, we have generated a temporal map of motor neuron transcriptional states at E10.5, E13.5, P4, P13, P21, P56, 2yr. In addition we have also profiled stem cell derived motor neurons that were co-cultured with astrocytes at DIV0, DIV7, DIV28. We performed RNA-seq and ATAC-seq on motor neurons or motor neuron nuclei at all in vivo and in vitro timepoints. In vivo mouse motor neurons were purified at E10.5 from HB9-GFP mice by dissociating and FACs sorting for GFP+ motor neuorns. At E13.5-2yr, motor neuron nuclei were purified from Chat-Cre;SUN1-gfp-myc mice. For in vitro experiments, embryonic stem cells were derived from Chat-Cre;SUN1-gfp-myc mice, differentiated into motor neurons and co-cultured with astrocytes. Nuclei were purified at DIV0 (before co-culture), DIV7, and DIV28. In addition, to understand the role of activity in controlling gene expression, motor neurons were co-cultured with astrocytes for 28 days in the presence of TTX.小鼠脊髓运动神经元在胚胎发育第9.5天（E9.5）即已特化，但运动功能直至出生后第三周（P14-P21）才完全成熟；而退行性疾病肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis, ALS）仅在成年阶段累及运动神经元，不同疾病模型的发病年龄存在差异。为深入解析运动神经元的成熟过程，我们构建了运动神经元转录状态的时序图谱，涵盖体内（in vivo）的E10.5、E13.5、P4、P13、P21、P56及2年龄时间节点。此外，我们还对与星形胶质细胞共培养的干细胞源性运动神经元进行了组学表征，采样节点为体外（in vitro）培养第0天（DIV0）、第7天（DIV7）及第28天（DIV28）。我们对所有体内、体外时间节点的运动神经元或运动神经元细胞核进行了RNA测序（RNA-seq）及转座酶可及性测序（ATAC-seq）。体内小鼠运动神经元的纯化流程如下：E10.5时期的样本取自HB9-GFP转基因小鼠，通过组织解离并经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分选GFP阳性的运动神经元；E13.5至2年龄时期的样本，则从Chat-Cre;SUN1-gfp-myc双转基因小鼠中纯化运动神经元细胞核。体外实验所用的胚胎干细胞取自Chat-Cre;SUN1-gfp-myc小鼠，经诱导分化为运动神经元后与星形胶质细胞共培养，我们分别在共培养第0天（共培养前）、第7天及第28天纯化运动神经元细胞核。此外，为解析神经活动对基因表达的调控作用，我们将运动神经元与星形胶质细胞共培养28天，同时施加河豚毒素（Tetrodotoxin, TTX）以阻断神经活动。