Live Imaging of Skin Regeneration

总结 组织的稳态和再生是通过不断的自我更新和分化来维持的。 干细胞未能平衡这些过程与不同和共同的公共卫生问题有关， 癌症和退行性疾病。发现调节干细胞决定的机制是 根本上重要，但由于无法完整地研究这些动态过程， 哺乳动物。我的团队克服了这个障碍，利用独特的可访问性的皮肤和我们的小说 活体成像方法，以在活体动物中真实的时间跟踪上皮细胞。到目前为止， 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的 干细胞行为之间的关系。这导致了我们概念上的创新模型，即干细胞是 在分化的连续体上，基于组织需求通过连续体协调它们的进展。 尽管取得了这些进展，细胞命运的决定如何在组织水平上进行分子协调仍然是一个问题。 不清楚 在这里，我们调整我们的活体成像技术，以可视化关键的分子事件-变化， 转录，染色质结构和信号通路激活-并确定其在群体水平上的作用 细胞命运决定的协调。我们假设，一个全球机制的基础上的地方协调， 干细胞状态，这种机制是必不可少的，以确保皮肤的稳态。为了验证这个假设，我们 将决定如何启动分化（目标1）。我们在基底层发现了一群细胞， 表达茎标记和分化标记，但不显示分化的形态学标志。以确定 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水平， 可塑性、染色质重排和细胞外调节因子。在目标2中，我们将定义细胞间信号传导 协调干细胞和分化行为的模式。钙信号作为一个信号整合剂， 我们的初步数据表明，钙离子在表皮分化的晚期和伤口愈合中起重要作用， 信号传导在基底表皮中是普遍的和动态的。为了确定钙信号是如何与 协调干细胞的命运决定，我们将揭示在基础钙信号的高阶动力学 层，并确定钙信号如何调节细胞周期的协调，自我更新， 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、转录组学、小鼠遗传学和机器学习。这项研究意义重大，因为我们 期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 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.