Molecular mechanisms underlying sensory neuron regeneration and function

项目摘要/摘要 SoxB1转录因子，在维持干细胞能力和生物体功能方面发挥重要作用 在成体组织中表达，并在再生过程中起关键作用。虽然SoxB1 基因涉及成年动物的不同过程，它们的作用机制是调节 干细胞和体内的再生还知之甚少。该领域的一个主要障碍是大多数模型 生物体的再生能力有限或干细胞数量稀少。我们建议使用 以稻纵卷尾藻为模型研究SoxB1基因在组织中的功能 再生。行星动物能够从非常小的身体碎片中再生出完整的蠕虫， 由成体多能干细胞群体所赋予的能力。我的实验室发现抑制 地中海链霉菌SoxB1基因soxB1-2使动物表现出惊人的癫痫样运动。 分子分析表明，soxb1-2在脊椎动物干细胞中表达，是再生所必需的。 以及维持浮游生物的表皮和感觉神经元群。然而，这种机制 潜在的soxB1-2+干细胞分化在很大程度上仍不清楚。我们假设soxB1-2的功能 作为一种先驱转录因子，启动干细胞以获得外胚层细胞的命运及其持续 感觉神经元亚群的分化和功能需要活性。SoxB1-2的分析 功能将提供对干细胞调节所需的保守基因靶点和机制的洞察 哪些终末分化的细胞在一生中维持着它们的命运。目标1将确定哪个干细胞和 挖掘100,000个新的单细胞基因在稻瘟病菌分化细胞中表达soxB1-2 抄本。我们将创建soxB1-2+细胞发育轨迹的预测， 高通量原位杂交结合已建立的细胞类型特异性 记号笔。目标2将在不同的感觉神经元中鉴定受soxB1-2调控并与soxB1-2共表达的基因 通过外科手术分离感觉器官区域后进行RNA测序实验 对照和soxB1-2 RNAi处理的浮游生物。差异表达的基因将与单细胞进行比较 转录本以确定细胞类型特异性，并通过原位杂交进行验证。此外，我们还将 建立ATAC-SEQ或采用CHIP-SEQ方法鉴定SoxB1-2的直接基因组靶点 脊椎动物干细胞。Aim 3将使用RNAi实验来分析所需的soxB1-2调控基因 赋予特殊的感觉细胞命运和功能。定义哪些基因是恢复特定基因所必需的 感觉，新的行为分析将被用来确定损害感觉的基因敲除 像化疗和机械感觉这样的方式。鉴于表达SoxB1基因的细胞类型繁多 在哺乳动物中，这项拟议的工作将为深入了解其持续转录是如何共同调节的。 维持细胞类型特定的基因模块对于正常的组织稳态或修复是必不可少的。

Project Summary/Abstract SoxB1 transcription factors, which play prominent roles in maintaining stem cell potency and organismal development, are expressed in adult tissues and have key roles in regenerative processes. Although SoxB1 genes have been implicated in diverse processes in adult animals, their mechanism of action in regulating stem cells and regeneration in vivo is poorly understood. A major obstacle in the field is that most model organisms have limited regenerative capacity or scarce stem cell populations. We propose to use the planarian Schmidtea mediterranea as a model to investigate the function of SoxB1 genes in tissue regeneration. Planarians are capable of regenerating complete worms from very small body fragments, an ability that is conferred by a population of adult pluripotent stem cells. My laboratory discovered that inhibiting the S. mediterranea SoxB1 gene, soxB1-2, causes animals to exhibit striking seizure-like movements. Molecular analysis revealed that soxB1-2 is expressed in planarian stem cells and is required for regeneration and maintenance of epidermal and sensory neuron populations in planarians. However, the mechanism underlying soxB1-2+ stem cell differentiation remains largely unknown. We hypothesize that soxB1-2 functions as a pioneer transcription factor that primes stem cells for acquiring ectodermal cell fates and its sustained activity is required for differentiation and function of sensory neuron subpopulations. Analysis of soxB1-2 function will provide insights into conserved gene targets required for stem cell regulation and mechanisms by which terminal differentiated cells maintain their fates throughout life. Aim 1 will determine which stem cell and differentiated cell types express soxB1-2 in S. mediterranea by mining >100,000 new single-cell gene transcriptomes. We will create predictions of soxB1-2+ cell developmental trajectories that can be experimentally assessed with high-throughput in situ hybridization combined with established cell-type specific markers. Aim 2 will identify genes regulated by and co-expressed with soxB1-2 in distinct sensory neuron populations by performing RNA-sequencing experiments after surgically isolating sensory organ regions from control and soxB1-2 RNAi-treated planarians. Differentially expressed genes will be compared to single cell transcriptomes to determine cell type-specificity, and validated by in situ hybridization. Additionally, we will establish an ATAC-seq or employ a ChIP-seq approach to identify direct genomic targets of SoxB1-2 in planarian stem cells. Aim 3 will use RNAi experiments to analyze soxB1-2-regulated genes that are required to confer specialized sensory cell fate and function. To define which genes are required for restoring specific senses, novel behavioral assays will be employed to establish the gene knockdowns that impair sensory modalities like chemo- and mechanosensation. Given the wide range of cell types that express SoxB1 genes in mammals, the proposed work will offer insights into how its sustained transcription co-regulates maintenance of cell type-specific gene modules indispensable for normal tissue homeostasis or repair.