Transcriptional Regulation of Motor Neuron Subtype Development

描述(申请人提供)：脊椎动物的运动神经元回路控制呼吸、协调运动和交感神经系统活动。运动神经元退行性疾病的一种有希望的治疗方法是使用细胞疗法来取代丢失的运动神经元。开发这种疗法的第一步将是创建一种策略，指导替换神经元形成功能运动神经元电路。在胚胎发育期间，运动神经元回路是通过产生多个运动神经元亚型来形成的，每个亚型都投射到特定的肌肉靶点。差异基因表达区分不同的运动神经元亚型，并有助于这些神经元的不同轴突路径。从完整的脊髓中分离出单个运动神经元亚型是极其困难的。由于这一挑战，人们对运动神经元亚型规范和轴突指导的遗传机制知之甚少。本研究的目的是找出在不同运动神经元亚型中差异表达的新基因，并研究它们在运动神经元回路形成中的功能。使用一种名为4-硫尿嘧啶(4-TU)标记的新技术，结合高通量RNA测序(RNA-seq)的初步结果显示，与胸部运动神经元相比，臂运动神经元中存在大量丰富的基因。4-TU标记使用UPRT酶的细胞类型特异性表达来标记在发育中的脊髓细胞的特定亚群中新合成的RNA。由于臂丛脊髓含有外侧运动柱(LMC)神经元，而胸髓不含，因此在臂丛脊髓中丰富的基因很可能在LMC神经元中丰富。AIM1将通过首先检测14个臂运动神经元基因的表达来研究LMC的特性和轴突引导。由此，我将使用在LMC神经元中特异性表达的基因进行功能获得和功能丧失的实验。LMC规格将通过LMC特异性标记物的免疫染色进行检查。LMC轴突的寻径将通过用GFP标记运动轴突和通过逆行标记实验来评估。目的2通过对内侧LMC(LMCm)神经元和内侧运动柱(MMCM)神经元进行4-TU标记和RNA-SEQ实验，寻找在这两种运动神经元中差异表达的新基因。5个可能的LMCM和5个可能的MMCM基因的表达将通过运动神经元亚型特异性基因的共同免疫染色来检测。LCMM和MMCM的特异性基因随后将用于功能获得和功能丧失的实验。运动神经元亚型规范和轴突寻找将通过LMCM或MMCM特异性标记的免疫染色、运动神经元轴突的标记和逆行标记来评估。这些实验将是对发育中的胚胎中运动神经元亚型特异性基因表达的第一次无偏见检查。

DESCRIPTION (provided by applicant): Motor neuron circuits control respiration, coordinated movement and sympathetic nervous system activity in vertebrates. A promising treatment for motor neuron degenerative diseases is using cell therapies to replace lost motor neurons. The first step in developing such a treatment will be to create a strategy for directing replacement neurons to form functional motor neuron circuits. During embryonic development, motor neuron circuits are formed through the generation of multiple motor neuron subtypes, which each project to specific muscle targets. Differential gene expression distinguishes distinct motor neuron subtypes and contributes to the differential axon pathfinding of these neurons. It is extremely difficult to isolate individual motor neuron subtypes from intact spinal cords. Because of this challenge, there is relatively little known about the genetic mechanisms that underlie motor neuron subtype specification and axon guidance. The goal of this proposal is to identify novel genes that are differentially expressed in different motor neuron subtypes, and to investigate their functions in the context of motor neuron circuit formation. Preliminary results using a novel technique called 4-Thiouracil (4-TU) tagging, combined with high-throughput RNA-sequencing (RNA-seq), have revealed numerous genes that are enriched in brachial motor neurons compared to thoracic motor neurons. 4-TU tagging uses the cell-type-specific expression of the enzyme Uracil Phosphoribosyl Transferase (UPRT) to label newly synthesized RNAs in specific subsets of cells in the developing spinal cord. Because the brachial spinal cord contains Lateral Motor Column (LMC) neurons and the thoracic spinal cord does not, genes that are enriched in the brachial spinal cord are likely enriched in LMC neurons. Aim1 will investigate LMC specification and axon guidance by first examining the expression of 14 brachial motor neuron genes. From these, I will use genes that are confirmed to be specifically expressed in LMC neurons for gain of function and loss of function experiments. LMC specification will be examined by immunostaining for LMC-specific markers. LMC axon pathfinding will be assessed by labeling motor axons with GFP and through retrograde labeling experiments. Aim 2 will identify novel genes that are differentially expressed in medial LMC (LMCm) neurons and Medial Motor Column (MMCm) neurons, by performing 4-TU tagging and RNA-seq experiments in these two motor neuron subtypes. The expression of 5 putative LMCm and 5 putative MMCm genes will be examined through co-immunostaining for motor neuron subtype specific genes. LCMm and MMCm specific genes will then be used for gain of function and loss of function experiments. Motor neuron subtype specification and axon pathfinding will be assessed by immunostaining for LMCm or MMCm specific markers, labeling motor neuron axons, and through retrograde labeling. These experiments will be the first unbiased examination of motor neuron subtype specific gene expression in developing embryos.