Cellular and molecular regulators of melanocyte regeneration

项目概要： 拟议的研究试图分离和表征黑素细胞干细胞（McSC），并确定黑素细胞干细胞（McSC）的功能。 控制McSC介导的黑素细胞再生的信号和途径。McSC基因表达将是 使用单细胞RNA测序（scRNAseq）表征。在斑马鱼中， 在再生过程中对McSCs进行取样，以揭示细胞内在和外在的通路， 决定McSC的命运还将进行来自白癜风患者的样品的scRNAseq以鉴定人类白癜风。 并评估这些细胞中活跃的信号传导。初步研究发现了新的候选人 McSC生物学中的通路，包括NGFR信号传导。这种途径很有趣，因为NGFR是一种标志物， 黑色素瘤起始细胞，并且最近发现作为去分化黑色素瘤细胞的定义特征， 对免疫检查点抑制剂和BRAF抑制剂疗法都有抗性。我们的数据表明 NGFR信号支持McSCs细胞状态，并且这种活性被肿瘤中的黑色素瘤细胞所吸收， 维持和耐药性。斑马鱼的功能研究将用于询问NGFR和其他 候选路径。怀疑在McSC命运执行、KIT和巨噬细胞中重要的途径- 介导的，也将使用类似的方法进行探讨。组合途径操作将是 进行评估这些不同的途径如何相互依赖，以调节再生。scRNAseq 将为靶向通路研究提供信息，作为识别新的McSC调控信号的资源， 提供了跨物种的McSC信号的保护的见解。这种实验组合将 提供了黑素细胞干细胞的详细了解和控制，指导他们在 再生过程 这项建议的具体目标是： 目的1：定义在生长过程中McSCs和其他细胞的转录变化和细胞轨迹 再生 假设：再生的特征是广泛的转录变化，反映了细胞状态的变化， 响应于组织损伤的调节器的接合。 Subaim 1A：绘制黑素细胞再生过程中发生的转录和细胞变化， 斑马鱼 Subaim 1B：使用白癜风和正常皮肤的scRNAseq定义人类McSCs及其后代 目的2：确定和功能分析参与McSC维持的信号通路， 黑素细胞再生 假设：一个细胞信号系统网络协调调节McSC的维持、激活和增殖。 命运的执行。 Subaim 2A：识别在McSCs及其后代中活动发生变化的信号通路 在再生期间 Subaim 2B：功能分析参与McSC维持和激活的途径 Subaim 2C：研究巨噬细胞和其他中间细胞类型在再生中的作用

PROJECT SUMMARY: The proposed studies seek to isolate and characterize melanocyte stem cells (McSCs) as well as identify the signals and pathways that govern McSC-mediated melanocyte regeneration. McSC gene expression will be characterized using single-cell RNA sequencing (scRNAseq). In zebrafish we will perform longitudinal sampling of McSCs during regeneration to uncover pathways, both cell intrinsic and extrinsic, involved in determining McSC fates. scRNAseq of samples from vitiligo patients will also be performed to identify human McSCs and assess signaling active in these cells. Preliminary studies have identified novel candidate pathways in McSC biology, including NGFR signaling. This pathway is intriguing because NGFR is a marker of melanoma initiating cells and was recently found as a defining feature of dedifferentiated melanoma cells that are resistant to both immune checkpoint inhibitor and BRAF inhibitor therapies. Our data lead to the hypothesis that NGFR signaling supports the McSCs cell state and this activity is co-opted by melanoma cells in tumor maintenance and drug resistance. Functional studies in zebrafish will be used to interrogate NGFR and other candidate pathways. Pathways suspected to be important in McSC fate execution, KIT and macrophage- mediated, will be also probed using similar methodologies. Combinatorial pathway manipulations will be performed to assess how these different pathways depend on each other to regulate regeneration. scRNAseq will inform the targeted pathway studies, serve as a resource to identify new McSC-regulatory signals, and provide insight into the conservation of McSC signaling across species. This combination of experiments will provide a detailed understanding of melanocyte stem cells and the controls that guide them during the regeneration process. The specific aims of this proposal are: Aim 1: Define transcriptional changes and cellular trajectories of McSCs and other cells during regeneration Hypothesis: Regeneration is characterized by broad transcriptional changes that reflect cell state changes and engagement of regulators in response to tissue injury. Subaim 1A: Map the transcriptional and cellular changes that occur during melanocyte regeneration in zebrafish Subaim 1B: Define human McSCs and their descendants using scRNAseq of vitiligo and normal skin Aim 2: Determine and functionally analyze signaling pathways involved in McSC maintenance and melanocyte regeneration Hypothesis: A network of cellular signaling systems coordinately regulates McSC maintenance, activation and fate execution. Subaim 2A: Identify signaling pathways whose activities change in McSCs and their descendants during regeneration Subaim 2B: Functionally analyze pathways involved in McSC maintenance and activation Subaim 2C: Investigate the roles of macrophages and other intermediary cell types in regeneration