Cellular and molecular regulators of melanocyte regeneration

项目总结： 拟议的研究试图分离和鉴定黑素细胞干细胞(McSCs)，并鉴定 控制MCSC介导的黑素细胞再生的信号和途径。MCSC基因的表达将 用单细胞RNA测序(ScRNAseq)进行了表征。在斑马鱼中，我们将执行纵向 在再生过程中对McSCs进行采样，以揭示细胞内和外的参与 决定着MCSC的命运。白癜风患者样本的scRNAseq也将被用来识别人类 并评估这些细胞中活跃的信号。初步研究确定了新的候选人 MCSC生物学中的通路，包括NGFR信号转导。这个途径很耐人寻味，因为NGFR是 黑色素瘤起始细胞，最近被发现是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抑制剂疗法都具有耐药性。我们的数据引出了这样的假设 NGFR信号支持McSCs的细胞状态，这种活性被肿瘤中的黑色素瘤细胞所依赖 维持和抗药性。斑马鱼的功能研究将用于询问NGFR和其他 候选路径。被怀疑在MCSC命运执行、KIT和巨噬细胞- 也将使用类似的方法进行调查。组合路径操作将是 以评估这些不同的途径如何相互依赖来调节再生。ScRNAseq 将为靶向途径研究提供信息，作为确定新的MCSC调节信号的资源，以及 洞察跨物种的MCSC信号的保护。这一系列实验将会 提供对黑素细胞干细胞的详细了解，以及在 再生过程。 这项建议的具体目标是： 目标1：确定McSCs和其他细胞在 再生 假设：再生的特征是广泛的转录变化，反映细胞状态的变化和 对组织损伤作出反应的调节器的参与。 Subaim 1A：绘制在黑素细胞再生过程中发生的转录和细胞变化 斑马鱼 Subaim 1B：利用白癜风和正常皮肤的scRNAseq确定人的McSCs及其后代 目标2：确定并功能分析参与MCSC维护和 黑素细胞再生 假设：细胞信号系统网络协调调节MCSC的维持、激活和 命运之刑。 Subaim 2A：确定在McSCs及其后代中活动发生变化的信号通路 在再生过程中 Subaim 2B：功能分析参与MCSC维持和激活的通路 Subaim 2C：研究巨噬细胞和其他中介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