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

描述（由申请人提供）：在哺乳动物中，最初的感觉信息检测，如来自外部物体和身体的触觉、本体感受、热刺激和疼痛刺激，是由初级感觉神经元进行的。这些神经元的细胞体位于背根神经节（DRG）。它们的轴突能够支配外周和脊髓的特定目标。我们的长期研究目标是了解初级感觉神经元投射模式如何建立的分子机制，并确定不同感觉神经元亚群的神经回路。最近，我们发现了一个名为Mrgs的G蛋白偶联受体（gpcr）大家族，在DRG的初级感觉神经元中特异性表达。有趣的是，不同的Mrgs在不同的感觉神经元亚群中表达。我们已经产生了几种敲入小鼠系，其中不同的轴突示踪剂，包括法酰化绿色荧光蛋白和人胎盘碱性磷酸酶，插入到不同的Mrg位点。通过分析这些小鼠，我们发现表达不同Mrgs的感觉神经元在周围和脊髓中都具有高度不同和特定的投射模式。一个显著的例子是MrgB4+神经元的轴突只支配有毛的皮肤，而不支配无毛的皮肤。据我们所知，MrgB4是第一个能够区分毛状皮肤神经支配神经元和无毛状皮肤神经支配神经元的标记物。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。MrgB4+纤维在背角片层的末端形成一个不寻常的不连续带，表明MrgB4+纤维聚集在片层内的特定区域。MrgB4+轴突投射模式的高特异性提出了该GPCR是否在外周和脊髓中建立这种特异性模式的问题。本文拟研究MrgB4在中枢神经系统高阶感觉神经元轴突引导及其神经回路中的功能。目的一是分析MrgB4纯合突变小鼠毛状皮肤和脊髓中MrgB4+纤维的投射模式，从而确定MrgB4是否起到轴突引导或靶识别的作用。在Aim II中，我们将确定毛发生长是否参与多毛皮肤感觉神经支配的建立和维持。为了实现这一目标，我们将在无毛敲除小鼠背景下研究MrgB4+纤维的投射模式。目的III，我们希望利用一种跨神经元的顺行示踪剂小麦胚芽凝集素（WGA）来绘制MrgB4+神经元的神经回路。我们将生成MrgB4-WGA敲入小鼠，我们将对WGA的表达模式进行详细分析，以鉴定由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，将对理解与体感觉系统中异常布线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，对感觉神经回路的研究将极大地促进我们了解感觉信息的传递和处理方式，这对于开发治疗慢性疼痛等感觉功能异常疾病的新药至关重要。

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.