Live Imaging and Genetic Dissection of Basement Membrane Development and Repair

基底膜是连接在一起的细胞外蛋白质(细胞外基质)的一层薄层，位于人体几乎所有上皮细胞的下方。这种特殊的细胞“柏油碎石”是其上层细胞发挥功能所必需的，而异常的基底膜在许多病理过程中发挥着作用。尽管有这种临床相关性，但我们对基底膜是如何形成的知之甚少。此外，尽管基底膜在任何类型的组织损伤中都会受到一定的损害，但我们对基底膜是如何修复的一无所知。基底膜由许多不同的成分组成，如IV型胶原，它们通过酶反应连接起来，产生最终的稳定结构。这些部件的生产和组织也被认为需要许多不同类型的细胞。由于这种复杂性，对基底膜的完全了解需要在活的有机体中进行检查，而直到最近，这在实验上都是不可行的。在这个建议中，我们将利用我们的能力来活生生地成像基底膜在活的有机体中的发育和修复。果蝇(果蝇)正在成为一个广泛使用的模型系统来了解基底膜，因为这种动物有一种与人类相同的细胞外基质蛋白。此外，我们实验室的初步数据显示，胚胎发育过程中基底膜的形成可以在动物发育过程中实时成像。在这项建议中，我们将利用我们的能力来实时成像基底膜的发育，以及我们在果蝇中敲除几乎任何感兴趣的基因的能力，以全面剖析基底膜形成和修复的机制。在第一个目标中，我们将通过时间推移显微镜来表征基底膜形成的时间过程。随后，我们将使用策略来特定地去除假设的基底膜成分以及在胚胎中被认为参与基底膜发育的各种细胞内形成基底膜所需的因素。这一分析将使我们能够突出基底膜形成的分子机制以及不同细胞在其生产过程中的相对需求。在接下来的目标中，我们将研究基底膜的修复反应。来自实验室的初步数据显示，激光消融可以特异性地破坏果蝇的上皮和下层基底膜；这会导致愈合反应，在几个小时的过程中，基底膜孔被封闭。我们将描述这种反应，并确定基底膜修复所涉及的细胞和分子机制。我们的分析表明，基底膜修复是一个活跃的细胞过程，需要苍蝇巨噬细胞(果蝇炎症细胞)的招募，我们将直接测试巨噬细胞在修复损伤中的功能。此外，初步数据显示，苍蝇巨噬细胞直接对基底膜的损伤(而不是对覆盖的上皮细胞的损伤)做出反应，我们将直接进行测试。这一数据表明，基底膜受损可能在炎症反应中发挥重要作用，这将具有广泛的临床意义。

The basement membrane, a thin layer of linked extracellular proteins (extracellular matrix), underlies nearly all epithelial cells in the human body. This specialised cellular "tarmac" is necessary for the function of its overlying cells, and an abnormal basement membrane plays a role in a number of pathologies. Despite this clinical relevance, we know little about how the basement membrane is formed. Furthermore, despite its certain damage during any type of tissue injury, we know nothing about how the basement membrane is capable of repair. The basement membrane is composed of a number of different components, such as Collagen Type IV, which are linked by enzymatic reactions to yield a final stable structure. The production and organisation of these components is also thought to require a number of different cell-types. Due to this complexity, a complete understanding of the basement membrane requires its examination within a living organism, which until recently has been experimentally unfeasible.In this proposal we will exploit our ability to live image basement membrane development and repair within a living organism. Fruit flies (Drosophila melanogaster) are becoming a widely utilised model system to understand the basement membrane as this animal has an assortment of extracellular matrix proteins identical to humans. Furthermore, preliminary data from our laboratory has revealed that basement membrane formation during embryogenesis can be imaged live during animal development. In this proposal we will exploit our ability to live image basement membrane development, along with our capacity in flies to knockout virtually any gene of interest, to fully dissect the mechanisms of basement membrane formation and repair.In the first Objective we will characterise the timecourse of basement membrane formation by time-lapse microscopy. We will subsequently use strategies to specifically remove hypothesised basement membrane components and factors required for its formation within the various cells in the embryo thought to be involved in basement membrane development. This analysis will allow us to highlight the molecular mechanisms behind basement membrane formation and the relative requirement of different cells in its production.In the subsequent Objective we will examine the basement membrane repair response. Preliminary data from the laboratory has revealed that the epithelium and underlying basement membrane can be specifically damaged in the fly by laser ablation; this leads to a healing response whereby over the course of a few hours the basement membrane hole is sealed. We will characterise this response and determine the cellular and molecular mechanisms involved in basement membrane repair. Our analysis suggests that basement membrane healing is an active cellular process that requires recruitment of fly macrophages (Drosophila inflammatory cells), and we will directly test the function of macrophages in repairing the damage. Furthermore, preliminary data reveals that the fly macrophages directly respond to damage to the basement membrane (rather than damage to the overlying epithelial cells), which we will directly test. This data suggests that a damaged basement membrane may be playing a significant role in inflammatory responses, which will have wide reaching clinical implications.