Genetic analysis of cell fate specification in the mammalian dorsal spinal cord.

描述(申请人提供)：哺乳动物脊髓中神经元回路的正确形成首先需要可靠地指定神经元。这种特性发生在胚胎发育过程中，通过传递给神经管的幼稚细胞的信号，引导它们成为腹侧(运动)或背侧(感觉)神经元。虽然我们对指定腹侧神经元的信号了解很多，但我们对背侧神经元指定的理解仍有很大差距。小鼠的正向遗传筛选揭示了几种生物学背景下哺乳动物特有的新机制，包括神经管模式。这类筛选表明，构建纤毛所需的基因是几个信号通路所必需的，包括Sonic Hedgehog(Shh)。例如，Hennin(HNN)突变破坏了纤毛轴丝的结构，导致纤毛异常短。HNN突变小鼠由于Shh信号中断而出现异常的腹侧神经管模式。此外，它们还显示出新的背侧模式缺陷，我的初步结果表明，这是由于背侧神经管中的BMP信号中断所致。由于Shh信号在HNN突变体的背侧神经管中被正确抑制，我推测HNN突变体的纤毛缺陷扰乱了背侧神经管中的BMP信号，导致了观察到的背侧模式缺陷。在目标1中，我提出了一系列实验来检验这一假设。在目标2中，我提出了一种基因筛查来识别背部神经元指定所必需的新的或未知的基因。我将加入一种专门标记背部神经管的GFP标记，以直接可视化破坏背部模式的突变。一旦突变系建立，我将整合高通量重测序技术来快速克隆基因，使我能够立即专注于新基因，以确定背神经管规范中相关的体内参与者。这里提出的工作有可能通过揭示与许多发育过程有关的保守信号通路的新信息来改善人类健康。胚胎发育过程中这些通路的中断会导致最常见的一类出生缺陷，神经管缺陷。在成年人中，这些通路的中断与肿瘤的形成和癌症有关。 公共卫生相关性：当脊髓的胚胎前体神经管未能完全闭合时，常见的出生缺陷，如脊柱裂，就会发生。神经管中的细胞必须接收来自神经管外部的各种信号，才能使脊髓正常发育。我们的研究将发现和研究哺乳动物的基因，这些基因是细胞接收和响应这些关键信号所必需的。

DESCRIPTION (provided by applicant): Proper formation of neuronal circuits in the mammalian spinal cord first requires that neurons be reliably specified. This specification occurs during embryonic development via signals imparted to naive cells of the neural tube, directing them to become ventral (motor) or dorsal (sensory) neurons. Though we know a great deal about the signals that specify ventral neurons, large gaps remain in our understanding of dorsal neuron specification. Forward genetic screens in the mouse have revealed novel mammalian- specific mechanisms in several biological contexts, including neural tube patterning. Such screens have shown that genes required to build cilia are necessary for several signaling pathways, including Sonic hedgehog (Shh). For instance, the hennin (hnn) mutation disrupts the structure of the ciliary axoneme, resulting in abnormally short cilia. hnn mutant mice have abnormal ventral neural tube patterning due to disrupted Shh signaling. In addition, they show novel dorsal patterning defects, and my preliminary results suggest that this is due to disrupted BMP signaling in the dorsal neural tube. Since Shh signaling is repressed correctly in the dorsal neural tube of hnn mutants, I hypothesize that the cilia defect of hnn mutants disrupts BMP signaling in the dorsal neural tube, leading to the observed dorsal patterning defects. In Aim 1, I propose a series of experiments to test this hypothesis. In Aim 2, I propose a genetic screen to identify novel or unsuspected genes necessary for dorsal neuron specification. I will incorporate a GFP marker that specifically labels the dorsal neural tube to directly visualize mutations that disrupt dorsal patterning. Once mutant lines are established, I will integrate high-throughput resequencing technology to rapidly clone the genes, enabling me to focus immediately on novel genes to identify the relevant in vivo players in dorsal neural tube specification. The work proposed here has the potential to improve human health by uncovering new information about conserved signaling pathways that are involved in many developmental processes. Disruption of these pathways during embryogenesis can cause the most common class of birth defects, neural tube defects. In adults, disruption of these pathways is implicated in tumor formation and cancer. PUBLIC HEALTH RELEVANCE: Common birth defects such as spina bifida occur when the embryonic precursor to the spinal cord, the neural tube, fails to completely close. Cells in the neural tube must receive various signals from outside the neural tube in order for proper development of the spinal cord to occur. Our research will discover and study the genes in mammals that are necessary for cells to receive and respond to these critical signals.