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很有前景的治疗方法是使用细胞疗法来替代失去的运动神经元。开发这种治疗方法的第一步将是创造一种策略，指导替代神经元形成功能性运动神经元回路。在胚胎发育过程中，运动神经元回路是通过产生多个运动神经元亚型而形成的，每个运动神经元亚型都指向特定的肌肉目标。差异基因表达区分了不同的运动神经元亚型，并有助于这些神经元的不同轴突寻路。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规范和轴突引导的遗传机制知之甚少。本提案的目的是鉴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回路形成中的功能。利用一种称为4-硫脲嘧啶（4-TU）标记的新技术，结合高通量rna测序（RNA-seq），初步结果揭示了与胸运动神经元相比，臂运动神经元中丰富的许多基因。4-TU标记利用尿嘧啶磷酸核糖基转移酶（UPRT）的细胞类型特异性表达来标记发育中的脊髓特定细胞亚群中新合成的rna。由于肱脊髓含有外侧运动柱（LMC）神经元，而胸脊髓没有，因此在肱脊髓中富集的基因可能在LMC神经元中富集。目的1将首先通过检查14个臂运动神经元基因的表达来研究LMC规范和轴突引导。在此基础上，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。LMC规格将通过LMC特异性标记物的免疫染色检查。LMC轴突寻路将通过GFP标记运动轴突和逆行标记实验来评估。Aim 2将通过在这两种运动神经元亚型中进行4-TU标记和RNA-seq实验，鉴定在内侧LMCm神经元和内侧运动柱神经元中差异表达的新基因。通过运动神经元亚型特异性基因的共免疫染色检测5个推定LMCm和5个推定MMCm基因的表达。LCMm和MMCm特异性基因将用于功能获得和功能丧失实验。通过LMCm或MMCm特异性标记物的免疫染色，标记运动神经元轴突，并通过逆行标记来评估运动神经元亚型规范和轴突寻路。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首次公正检查。

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.