Understanding Skin Tissue Repair in Live Mammals

皮肤保护我们的身体免受环境的伤害，它在受伤时的修复能力直接关系到 无论是疾病还是生存。如果不能正确修复受伤组织，可能会导致慢性伤口，这是 伴随着严重的并发症甚至死亡。多种细胞类型，如上皮细胞和成纤维细胞， 必须协调他们的行为，以实现损伤修复。到目前为止，这些过程主要是在 基因同源的小鼠模型。然而，健康的皮肤含有许多含有体细胞的克隆 突变，包括致癌的RAS突变，这是鳞状细胞癌的主要原因。 遗传多样性的上皮细胞如何影响损伤修复尚不清楚。这项提议的目标是 揭示在修复过程中，细胞行为是如何在单细胞和组织规模水平上得到适当协调的。这个 解决这些基本问题的关键障碍在于无法研究这些动态过程 在一只完整的哺乳动物体内。为此，我的实验室建立了一种活体策略，直接可视化和 操控活体小鼠皮肤上皮细胞和成纤维细胞。我们以前曾使用此策略来定义 上皮细胞和成纤维细胞在动态平衡中的作用及其复杂的时空组织 修复过程中的上皮细胞行为。我们假设上皮细胞和成纤维细胞使用灵活的行为 以使遗传马赛克皮肤的伤口能够得到适当的愈合。 我们将首先定义马赛克上皮如何对损伤做出反应(目标1)。我们的初步结果显示， 马赛克HRAS突变皮肤上皮以正常速度愈合，不会诱发肿瘤，但WT和HRAS 突变细胞在修复过程中表现出不同的行为。我们假设WT细胞的行为灵活性 赋予他们遏制HRAS突变邻居的能力，并最终实现正常的损伤修复。为了测试这一点， 我们将定义上皮细胞的行为，并评估信号通路在损伤修复中的作用 HRAS-马赛克皮肤上皮。然后我们将研究成纤维细胞在与上皮细胞接触过程中的作用 损伤修复(目标2)。我们假设真皮上部的成纤维细胞和 表皮中的上皮细胞协调细胞的行为和修复。为了测试这一点，我们将定义行为 在WT上皮细胞和HRAS镶嵌上皮存在的情况下，成纤维细胞的功能。我们将实现 这两个目标都是通过将活体显微镜与基因和药物操作相结合来实现的 体内修复行为或驻留细胞类型。 拟议的实验将使我们能够剖析 使用尖端技术的集成方法，修复过程中不同的细胞活动、种群和相互作用 成像技术、基因操作、细胞生物学和单细胞测序。考虑到许多方面 损伤修复在其他器官中广泛保守，我们的发现也将与其他组织相关，并将 为提高各类患者的损伤修复水平提供了重要依据。

Skin protects our body against the environment, and its ability to repair upon injury is directly connected to both disease and survival. Failure to properly repair injured tissue can result in chronic wounds, which are associated with severe complications and even death. Multiple cell types, like epithelial cells and fibroblasts, must coordinate their behaviors to achieve injury repair. These processes have so far mainly been studied in genetically homogeneous mouse models. However, healthy skin contains many clones harboring somatic mutations, including oncogenic Ras mutations which are the leading cause of Squamous Cell Carcinoma. How genetically diverse epithelial cells affect injury repair is unknown. The goal of this proposal is to unravel how cell behaviors are properly orchestrated on the single-cell and tissue-scale level during repair. The critical barrier to addressing these fundamental questions lies in the inability to study these dynamic processes in an intact mammal. To this end, my laboratory has established an in vivo strategy to directly visualize and manipulate epithelial cells and fibroblasts in the skin of live mice. We have previously used this strategy to define the roles of epithelial cells and fibroblasts in homeostasis, and the complex spatiotemporal organization of epithelial cell behaviors during repair. We hypothesize that epithelial cells and fibroblasts use flexible behaviors to enable proper wound healing of genetically mosaic skin. We will first define how a mosaic epithelium responds to injury (Aim 1). Our preliminary results show that a mosaic Hras mutant skin epithelium heals at a normal rate and does not induce tumors, but that WT and Hras mutant cells exhibit different behaviors during repair. We hypothesize that the behavioral flexibility of WT cells confers the ability to contain their Hras mutant neighbors and ultimately achieve normal injury repair. To test this, we will define the epithelial cell behaviors and evaluate the roles of signaling pathways during injury repair of an Hras-mosaic skin epithelium. We will then examine the roles of fibroblasts in contact with epithelial cells during injury repair (Aim 2). We hypothesize that direct communication between fibroblasts in the upper dermis and epithelial cells in the epidermis coordinates cell behaviors and repair. To test this, we will define the behaviors and functions of fibroblasts in the presence of WT epithelial cells and an Hras-mosaic epithelium. We will achieve both these aims by combining intravital microscopy with genetic and pharmacological manipulations of distinct repair behaviors or resident cell types in vivo. The proposed experiments will allow us to dissect the coordination and functional significance of distinct cell activities, populations, and interactions during repair using an integrated approach of cutting-edge imaging technology, genetic manipulation, cell biology, and single cell sequencing. Given that many aspects of injury repair are widely conserved in other organs, our findings will be relevant to other tissues as well, and will provide an important foundation to improve injury repair in a variety of patients.