Live Imaging of Skin Regeneration

摘要 组织动态平衡和再生是通过持续的自我更新和分化来维持的 干细胞。未能平衡这些过程与各种常见的公共卫生问题有关，例如 癌症和退化性疾病。发现调节干细胞决定的机制是 从根本上说很重要，但由于无法完整地研究这些动态过程而受到阻碍 哺乳动物。我的团队通过利用皮肤的独特可及性和我们的小说克服了这一障碍 实时追踪活体动物上皮细胞的活体成像方法。到目前为止，我们已经使用了这个 一种全面识别表皮内所有命运决定的方法，使我们能够揭示复杂性 干细胞行为之间的关系。这导致了我们在概念上的创新模型，干细胞是 在分化的连续体上，根据组织需求在连续体中协调它们的进展。 尽管取得了这些进展，但细胞命运的决定是如何在组织水平上进行分子协调的仍然存在 不清楚。 在这里，我们调整了我们的活体成像技术来可视化关键的分子事件-变化到 转录、染色质结构和信号通路激活--并确定它们在种群水平上的作用 协调细胞命运的决定。我们假设，全球机制是地方协调的基础 干细胞状态，这一机制是确保皮肤动态平衡的关键。为了检验这一假设，我们 将决定如何启动差异化(目标1)。我们确定了基底层的一群细胞-- 表达干标记和分化标记，但不显示分化的形态标志。要确定 如果这些细胞处于从干细胞到分化的可逆转变中，我们将询问它们的转录 可塑性、染色质重排和细胞外在调节因子。在目标2中，我们将定义细胞间信号 协调干细胞和分化行为的模式。钙信号在细胞周期中起信号整合的作用 在表皮分化的后期和伤口愈合中，我们的初步数据显示，钙 信号在基表皮中普遍存在且动态变化。为了定义钙信号如何与 协调干细胞命运的决定，我们将揭示钙信号的高阶动力学基础 并确定钙信号如何调节细胞周期的协调、自我更新和 基底细胞的分化。为了实现这些目标，我们将使用尖端成像的综合方法 技术、转录学、老鼠遗传学和机器学习。这项研究具有重要意义，因为我们 有望揭示协调干细胞自我更新和分化的全球分子机制 活的哺乳动物。我们的发现可能会推动相关领域的创新，因为干细胞的许多原理 生物学在其他器官中是保守的。

Summary Tissue homeostasis and regeneration are sustained by continuous self-renewal and differentiation of stem cells. Failure to balance these processes is associated with diverse and common public health issues, such as cancer and degenerative diseases. Discovering the mechanisms that regulate stem cell decisions is fundamentally important but has been stymied by the inability to study these dynamic processes in an intact mammal. My group overcomes this obstacle by leveraging the unique accessibility of the skin and our novel intravital imaging approaches to track epithelial cells in real time in live animals. We have so far used this approach to comprehensively identify all fate decisions within the epidermis, allowing us to uncover complex relationships between stem cell behaviors. This has led to our conceptually innovative model that stem cells are on a continuum of differentiation, coordinating their progression through the continuum based on tissue demand. Despite these advances, how cell fate decisions are molecularly orchestrated on the tissue-level remains unclear. Here, we adapt our intravital imaging techniques to visualize key molecular events – changes to transcription, chromatin structure, and signaling pathway activation – and determine their role in population-level coordination of cell fate decisions. We hypothesize that a global mechanism underlies the local coordination of stem cell states, and that this mechanism is essential to ensure skin homeostasis. To test this hypothesis, we will determine how differentiation is initiated (Aim 1). We identified a population of cells in the basal layer that co- express stem- and differentiation-markers but do not display morphological signs of differentiation. To determine if these cells are in a reversible transition from stemness to differentiation, we will interrogate their transcriptional plasticity, chromatin rearrangements, and cell-extrinsic regulators. In Aim 2, we will define intercellular signaling patterns that coordinate stem cell and differentiation behaviors. Calcium signaling acts as a signal integrator in the late stages of epidermal differentiation and in wound-healing, and our preliminary data show that calcium signaling is prevalent and dynamic in the basal epidermis. To define how calcium signaling intersects with the coordination of stem cell fate decisions, we will uncover the high-order dynamics of calcium signaling in the basal layer and determine how calcium signaling regulates the coordination of cell cycles, self-renewal, and differentiation of basal cells. To achieve these aims, we will use an integrated approach of cutting-edge imaging technology, transcriptomics, mouse genetics, and machine learning. This research is significant because we expect to uncover global molecular mechanisms that coordinate stem cell self-renewal and differentiation in a live mammal. Our findings will likely drive innovation in related fields, given that many principles of stem cell biology are conserved in other organs.