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.

项目摘要/摘要 SOXB1转录因子，在维持干细胞效力和有机体中起着突出作用 开发在成人组织中表达，并在再生过程中具有关键作用。虽然Soxb1 基因与成年动物的多种过程有关，它们在调节方面的作用机制 干细胞和体内再生知之甚少。该领域的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体稀缺。我们建议使用 Planarian Schmidtea地中海作为研究Soxb1基因在组织中的功能的模型 再生。平面人能够从非常小的身体碎片中再生完全的蠕虫，这是 成人多能干细胞赋予的能力。我的实验室发现抑制 S. Mediterranea Soxb1基因SOXB1-2导致动物表现出惊人的癫痫发作。 分子分析表明，Soxb1-2在平质干细胞中表达，是再生所必需的 以及维持平面主义者表皮和感觉神经元种群。但是，机制 基础SOXB1-2+干细胞分化仍然很大程度上未知。我们假设Soxb1-2功能 作为一个先驱转录因子，它是用于获得外胚层细胞命运及其持续的干细胞 感觉神经元亚群的分化和功能是必需的。 SOXB1-2的分析 功能将为干细胞调节所需的保守基因靶标提供见解 哪个末端分化细胞在一生中保持命运。 AIM 1将确定哪种干细胞和 通过挖掘> 100,000新的单细胞基因，分化的细胞类型在地中海链球菌中表达SOXB1-2 转录组。我们将创建可以是Soxb1-2+细胞发育轨迹的预测 通过高通量原位杂交结合了实验性评估，结合了已建立的细胞类型特异性 标记。 AIM 2将在不同的感觉神经元中识别由SOXB1-2调节并与Soxb1-2共表达的基因 通过手术分离感觉器官区域后，通过进行RNA测序实验来进行种群 控制和SOXB1-2 RNAi治疗的平面官。将差异表达的基因与单细胞进行比较 转录组确定细胞类型特异性，并通过原位杂交验证。此外，我们会的 建立ATAC-SEQ或采用芯片序列方法来识别SOXB1-2的直接基因组靶标 平质干细胞。 AIM 3将使用RNAi实验来分析SOXB1-2调节的基因 赋予专门的感觉细胞命运和功能。定义还需要哪些基因来恢复特定 感觉，新型的行为测定将用于建立损害感觉的基因敲低 诸如化学和机械敏化之类的模态。给定表达SOXB1基因的细胞类型的广泛范围 在哺乳动物中，拟议的工作将提供有关其持续转录如何共同调节的见解 维持细胞类型特异性基因模块对于正常组织稳态或修复必不可少的。