Genetic and Molecular Analysis of sensory nerve innervation in hairy skin and spi

描述(申请人提供)：在哺乳动物中，来自外部物体和身体的触觉、本体感觉、温度和疼痛刺激等感觉信息的最初检测是由初级感觉神经元执行的。这些神经元的胞体位于背根节(DRG)。它们的轴突能够支配外周和脊髓中的特定靶点。我们研究的长期目标是了解初级感觉神经元投射模式建立的分子机制，并确定不同亚群感觉神经元的神经回路。最近，我们发现了一个G蛋白偶联受体(GPCRs)大家族，称为MRGs，在DRG的初级感觉神经元中特异表达。有趣的是，不同的MR在不同的感觉神经元亚群中表达。我们已经建立了几个敲入小鼠系，其中不同的轴突示踪剂，包括法尼化绿色荧光蛋白和人胎盘碱性磷酸酶，被插入到不同的MRG基因座。通过对这些小鼠的分析，我们发现表达不同mRGs的感觉神经元在外周和脊髓都有非常明显和特定的投射模式。一个明显的例子是，mrgB4+神经元的轴突只支配有毛的皮肤，而不是无毛的皮肤。据我们所知，MRGB4是第一个能够区分有毛皮肤支配神经元和无毛皮肤支配神经元的标志物。有趣的是，大多数MRgB4+纤维终止于毛囊的特定区域。此外，MRgB4+轴突在脊髓背角的投射也非常独特。在背角板层中的MRgB4+纤维终末形成了一条不寻常的不连续带，这表明MRgB4+纤维汇聚到板层内的特定区域。MRgB4+轴突投射模式的高度特异性提出了一个问题，即这种GPCR是否在外周和脊髓建立这种特定模式中发挥了作用。在此，我们拟研究MRgB4在中枢神经高阶感觉神经元轴突引导及其神经环路中的作用。目的分析mrgB4纯合子突变小鼠毛皮和脊髓中mrgB4+纤维的投射模式，以确定mrgB4在轴突引导或靶点识别中的作用。在AIM II中，我们将确定毛发生长是否与多毛皮肤感觉神经的建立和维持有关。为了实现这一目标，我们将研究无毛基因敲除小鼠背景下MRgB4+纤维的投射模式。目的利用一种跨神经元顺行示踪剂小麦胚凝集素(WGA)，对MRgB4+神经元的神经回路进行定位。我们将产生mrgB4-WGA敲入小鼠，并对WGA表达模式进行详细分析，以确定WGA标记的mrgB4+初级感觉神经元的二级(可能是三级)神经元。我们相信，研究感觉神经如何在皮肤和脊髓中建立适当的投射的分子机制，将对了解与躯体感觉系统异常连接相关的神经病理疾病，如某些慢性疾病具有广泛的意义。此外，感觉神经回路的研究将极大地帮助我们了解感觉信息是如何传递和处理的，这将是开发治疗慢性疼痛等感觉功能异常疾病的新药所必需的。

DESCRIPTION (provided by applicant): In mammals, the initial detection of sensory information, such as tactile, proprioceptive, thermal, and pain stimuli from both external objects and the body, is carried out by primary sensory neurons. The cell bodies of these neurons locate in dorsal root ganglia (DRG). Their axons are able to innervate specific targets in the periphery and the spinal cord. The long-term objective of our research is to understand the molecular mechanism of how the projection patterns of primary sensory neurons are established and to determine the neural circuits of different subpopulation of sensory neurons. Recently, we have identified a large family of G protein coupled receptors (GPCRs), called Mrgs, expressed specifically in primary sensory neurons in DRG. Interestingly, different Mrgs are expressed in different subpopulations of sensory neurons. We have generated several knock-in mouse lines, in which different axonal tracers including farnesylated green fluorescent protein and human placenta alkaline phosphatase were inserted into different Mrg loci. By analyzing these mice, we found that sensory neurons expressing different Mrgs have highly distinct and specific projection patterns both in the periphery and the spinal cord. One striking example is that the axons of MrgB4+ neurons only innervate hairy skin but not glabrous skin. To our knowledge, MrgB4 is the first marker which can segregate hairy skin innervating neurons from glabrous skin innervating neurons. Interestingly, most of MrgB4+ fibers terminate at a specific region of hair follicles. Furthermore, the projection of MrgB4+ axons in the dorsal horn of the spinal cords is also very unique. The MrgB4+ fibers terminations in the dorsal horn lamina layer form an unusual discontinuous band, suggesting that MrgB4+ fibers converge to specific regions within the lamina layer. The high specificity of MrgB4+ axon projection pattern raises the question of whether this GPCR play a role in the establishment of such specific pattern in the periphery and the spinal cord. Here we propose to study the function of MrgB4 in axon guidance of sensory neurons and their neural circuits in the higher order of the CNS. Aim I is to analyze the projection patterns of MrgB4+ fibers in hairy skin and spinal cord of MrgB4 homozygous mutant mice, from which we would like to determine whether MrgB4 plays roles in axon guidance or target recognition. In Aim II, we will determine whether hair growth is involved in establishment and maintenance of sensory innervations in hairy skin. To achieve this goal, we will study the projection patterns of MrgB4+ fibers in hairless knockout mouse background. Aim III we would like to map the neural circuit of MrgB4+ neurons using a trans-neuronal anterograde tracer, wheat germ agglutinin (WGA). We will generate MrgB4-WGA knock-in mice and we will carry out a detailed analysis of the pattern of WGA expression to identify the second- (possibly third-) order neurons to MrgB4+ primary sensory neurons labeled by WGA. We believe that studying the molecular mechanism of how sensory nerves establish proper projections in the skin and the spinal cord will have broad implications for understanding neuropathological diseases associated with abnormal wiring in the somatosensory system such as those in certain chronic conditions. In addition, the study of sensory neural circuits will greatly facilitate our understanding of how sensory information is transmitted and processed, which will be essential for developing novel drugs to treat diseases with abnormal sensory function such as chronic pain.