EXPRESSION PROFILES FOR RET-POSITIVE DRG NEURONS

描述（由申请人提供）：这是一个研究项目，旨在生成小鼠发育和出生后期间背根感觉神经节（DRG）中特定伤害感受器群体的表达谱，作为nGUDMAP项目的一部分。DRG在神经化学、药理学、形态学和分子特征等多个水平存在广泛的异质性。还有内脏和躯体感觉神经系统的交叉致敏。这种异质性和多样性的分子基础，其在正常和异常的泌尿生殖感觉功能中的意义是缺乏的，并且是理解伤害感受机制和设计合理方法来治疗诸如疼痛性膀胱综合征/间质性膀胱炎等病症的障碍。在个体发育过程中描绘不同类型的DRG神经元的基因表达模式和分子网络是克服这一障碍的必要的第一步。我们研究了一种依赖于胶质细胞源性神经营养因子（GDNF）家族配体（GFLs）的伤害感受器亚型。GFLs激活伤害感受器和低阈值机械感受器亚组中的受体酪氨酸激酶Ret。GFL-RET下游信号传导的激活对于轴突维持、存活、伤害感受和疼痛敏感性是重要的。DRG中的Ret阳性感觉神经元在小鼠和人类的膀胱损伤模型中表现出分子、神经化学和结构变化，例如膀胱疼痛综合征或间质性膀胱炎（IC/PBS），它们参与躯体和内脏感觉，表达与躯体和内脏疼痛有关的关键离子通道（TrpV 1、TrpA 1），并且GFLs可以产生镇痛和痛觉过敏。我们将描绘视网膜反应阳性的DRG感觉神经元中表达的基因。我们已经制造了创新的Ret报告小鼠，其首次用绿色荧光蛋白组成性或条件性地标记Ret表达细胞。我们将这些与FACS和RNA-Seq结合使用，以实现三个小目标：1）为了产生Ret+ DRG感觉神经元在妊娠早期中期至出生后期间的表达谱，2）为了产生Ret+ Nav1.8+伤害感受器在与上述相同的时间点的表达谱，和3）使用Ret-报告基因雄性和雌性出生后小鼠产生Ret+ L 6-S1 DRG感觉神经元的表达谱以丰富膀胱内脏传入的分离。我们的初步数据为DRG和膀胱传入神经中的Ret+感觉神经元提供了证据，它们正常激活TrpV 1和TrpA 1离子通道，使用FACS成功分离它们，并与生物信息学家合作获得基因组技术以生成RNA-Seq数据并将其存入GUDMAP数据库。与其他GUDMAP项目成员和社区的互动将提供协同作用，这将加速与盆腔内脏感觉系统相关的发现和机制项目。

DESCRIPTION (provided by applicant): This is a Research Project to generate expression profiles of a specific population of nociceptors in the dorsal root sensory ganglia (DRG) during development and postnatal period in mice as a part of the nGUDMAP project. There is extensive heterogeneity in DRG at several levels including neurochemistry, pharmacology, morphology, and molecular profiles. There is also cross- sensitization of visceral and somatic sensory nervous systems. The molecular basis for this heterogeneity and diversity, its implication in normal and abnormal genitourinary sensory function is lacking and is an obstacle to understanding mechanisms of nociception and devising rational approaches to treat conditions such as painful bladder syndrome/interstitial cystitis. Delineating gene expression patterns and molecular networks in distinct types of DRG neurons during ontogeny is an essential first step to overcome this obstacle. We study a subtype of nociceptor that depends on glial cell line-derive neurotrophic factor (GDNF) family ligands (GFLs) for postnatal survival. GFLs activate receptor tyrosine kinase Ret in nociceptors and a subset of low threshold mechanoreceptors. Activation of GFL-RET downstream signaling is important for axon maintenance, survival, nociception, and pain sensitivity. Ret-positive sensory neurons in the DRG show molecular, neurochemical and structural changes in bladder injury models in mice and in humans in conditions such as painful bladder syndrome or interstitial cystitis (IC/PBS), they participate in both somatic and visceral sensation, express key ion channels that are implicated in somatic and visceral pain (TrpV1, TrpA1), and GFLs can produce analgesia and hyperalgesia. We will delineate genes expressed in Ret-positive DRG sensory neurons. We have made innovative Ret-reporter mice that constitutively or conditionally label Ret-expressing cells with green fluorescence protein for the first time. We will use these in conjunction with FACS and RNA-Seq to accomplish three miniobjectives: 1) To generate expression profiles of Ret+ DRG sensory neurons in early midgestation through postnatal period, 2) To generate expression profiles of Ret+ Nav1.8+ nociceptors at the same time points as above, and 3) Generate expression profiles of Ret+ L6-S1 DRG sensory neurons to enrich isolation of bladder visceral afferents using Ret- reporter male and female postnatal mice. Our preliminary data provide evidence for Ret+ sensory neurons in the DRG and in bladder afferents, their normal activation of TrpV1 and TrpA1 ion channels, their successful isolation using FACS and access to genomic technology in collaboration with bioinformaticists to generate RNA-Seq data and depositing it in the GUDMAP database. Interactions with members of other GUDMAP projects and the community will provide synergies that will accelerate discovery and mechanism based projects related to pelvic visceral sensory system.