Transcriptional Regulation of Motor Neuron Subtype Development

描述（由申请人提供）：运动神经元回路控制脊椎动物的呼吸、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替代丢失的运动神经元。开发这种治疗方法的第一步将是创造一种指导替代神经元形成功能性运动神经元电路的策略。在胚胎发育过程中，运动神经元电路是通过产生多个运动神经元亚型形成的，每个亚型都投射到特定的肌肉目标。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差异轴突寻路。从完整的脊髓中分离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特化和轴突导向的遗传机制知之甚少。该提案的目标是确定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电路形成中的功能。使用称为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，许多基因在肱肌运动神经元中富集。4-TU标记使用尿嘧啶磷酸核糖转移酶（UPRT）的细胞类型特异性表达来标记发育中脊髓中特定细胞亚群中新合成的RNA。因为臂脊髓含有侧向运动柱（LMC）神经元而胸脊髓不含有，所以在臂脊髓中富集的基因可能在LMC神经元中富集。Aim 1将通过首先检查14个臂运动神经元基因的表达来研究LMC的特化和轴突引导。从这些，我将使用被证实在LMC神经元中特异性表达的基因进行功能获得和功能丧失实验。将通过LMC特异性标志物的免疫染色检查LMC规格。通过用GFP标记运动轴突和通过逆行标记实验来评估LMC轴突寻路。目的2通过4-TU标记和RNA-seq实验，筛选出内侧LMCm（medial LMCm）和内侧运动柱（medial Motor Column，MMCm）神经元中差异表达的基因。将通过运动神经元亚型特异性基因的共免疫染色检查5种推定的LMCm和5种推定的MMCm基因的表达。然后将LCMm和MMCm特异性基因用于功能获得和功能丧失实验。将通过LMCm或MMCm特异性标志物的免疫染色、标记运动神经元轴突和通过逆行标记来评估运动神经元亚型特异性和轴突寻路。这些实验将是第一个无偏见的检查运动神经元亚型特异性基因表达在发育中的胚胎。

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.