Molecular mechanisms underlying sensory neuron regeneration and function

感觉神经元再生和功能的分子机制

• 批准号: 10385485
• 负责人: Ricardo M. Zayas
• 金额: $ 7.33万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10385485
• 关键词: ATAC-seq; Adult; Afferent Neurons; Animal Model; Animals; Behavioral Assay; Cell Differentiation process; Cell Maintenance; Cells; ChIP-seq; Development; Ectoderm Cell; Exhibits; Fresh Water; Gene Targeting; Genes; Genetic Transcription; Genomics; Homeostasis; Impairment; In Situ Hybridization; Laboratories; Life; Maintenance; Mammals; Mining; Modality; Modeling; Molecular; Molecular Analysis; Movement; Natural regeneration; Nervous system structure; Normal tissue morphology; Operative Surgical Procedures; Organ; Planarians; Play; Pluripotent Stem Cells; Population; Process; RNA Interference; Regenerative capacity; Regulation; Role; Seizures; Sensory; Specificity; Tissues; Work; cell type; differential expression; experimental study; in vivo; insight; knock-down; mature animal; novel; organ growth; regeneration function; regenerative; repaired; stem cell differentiation; stem cell model; stem cell population; stem cell self renewal; stem cells; tissue regeneration; transcription factor; transcriptome; transcriptome sequencing

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.

项目概要/摘要 SoxB1 转录因子，在维持干细胞效力和有机体方面发挥着重要作用 发育中，在成体组织中表达，并在再生过程中发挥关键作用。虽然SoxB1 基因与成年动物的多种过程有关，它们的作用机制是调节 对干细胞和体内再生知之甚少。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群稀缺。我们建议使用 以地中海涡虫为模型研究 SoxB1 基因在组织中的功能 再生。涡虫能够从非常小的身体碎片中再生出完整的蠕虫， 成体多能干细胞群赋予的能力。我的实验室发现抑制 地中海链霉菌 SoxB1 基因 soxB1-2 会导致动物表现出惊人的癫痫样运动。 分子分析表明 soxB1-2 在涡虫干细胞中表达并且是再生所必需的 以及涡虫表皮和感觉神经元群的维持。然而，该机制 潜在的 soxB1-2+ 干细胞分化仍然很大程度上未知。我们假设 soxB1-2 起作用 作为先驱转录因子，启动干细胞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分析 功能将提供对干细胞调节和机制所需的保守基因靶点的见解 终末分化细胞在一生中维持其命运。目标 1 将确定哪种干细胞和 通过挖掘超过 100,000 个新的单细胞基因，分化的细胞类型在地中海南海中表达 soxB1-2 转录组。我们将创建 soxB1-2+ 细胞发育轨迹的预测 通过高通量原位杂交结合已建立的细胞类型特异性进行实验评估 标记。目标 2 将鉴定不同感觉神经元中受 soxB1-2 调节并与其共表达的基因 通过手术分离感觉器官区域后进行 RNA 测序实验 对照和 soxB1-2 RNAi 处理的涡虫。差异表达基因将与单细胞进行比较 转录组以确定细胞类型特异性，并通过原位杂交进行验证。此外，我们将 建立 ATAC-seq 或采用 ChIP-seq 方法来识别 SoxB1-2 的直接基因组靶标 涡虫干细胞。目标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。定义恢复特定功能所需的基因 感官，将采用新的行为测定来建立损害感官的基因敲除 化学和机械感觉等方式。鉴于表达 SoxB1 基因的细胞类型广泛 在哺乳动物中，拟议的工作将深入了解其持续转录如何共同调节 维持正常组织稳态或修复所必需的细胞类型特异性基因模块。