Etv1 controls the establishment of non-overlapping motor innervation of neighboring facial muscles during development

The motor neurons innervating the muscles of facial expression are organized into somatotopic hindbrain clusters termed subnuclei. Each of the medial, intermediate, dorsolateral, and lateral subnuclei gives rise to a specific branch of the facial motor nerve (cranial nerve VII). How subnucleus-specific gene expression could mediate the accurate development of facial nerve projections was not well understood. We isolated specific facial motor subnuclei from E16.5 hindbrain cryosections using laser capture microdissection, purified the total RNA and analyzed it on Affymetrix Mouse Genome 430 2.0 arrays. E16.5 mouse hindbrains were fresh frozen in optimal cutting compound (OCT), cryosectioned onto PEN-foil covered glass slides, stained with cresyl violet to identify facial motor neuron cell bodies, and individual subnuclei were isolated on a Zeiss Palm Microbeam system. cDNA libraries were prepared using Nugen WT-Ovation Pico RNA amplification system and analyzed using an Affymetrix Mouse Genome 430 2.0 array支配面部表情肌的运动神经元在菱脑内形成具有躯体拓扑分布特征的簇状结构，这类结构被称为亚核。内侧亚核、中间亚核、背外侧亚核与外侧亚核各自发出面神经运动支（颅神经VII）的特定分支。亚核特异性基因表达如何介导面神经投射的精准发育，此前尚未得到充分阐明。本研究通过激光捕获显微切割（laser capture microdissection）技术，从E16.5天小鼠胚胎菱脑冰冻切片中分离特定的面运动神经元亚核，提取总RNA后使用Affymetrix Mouse Genome 430 2.0芯片进行转录组分析。将E16.5小鼠胚胎菱脑置于最优切割包埋剂（optimal cutting compound, OCT）中快速冷冻，随后冰冻切片至覆有聚萘二甲酸乙二醇酯（PEN）箔的载玻片上，经焦油紫（cresyl violet）染色以识别面运动神经元胞体，再使用Zeiss Palm Microbeam系统分离单个亚核；随后使用Nugen WT-Ovation Pico RNA扩增系统制备cDNA文库，最后通过Affymetrix Mouse Genome 430 2.0芯片完成检测分析。