Live Imaging and Genetic Dissection of Basement Membrane Development and Repair

基底膜是一层薄薄的细胞外蛋白质（细胞外基质），几乎覆盖了人体所有的上皮细胞。这种特殊的细胞“柏油路”是其覆盖细胞功能所必需的，并且异常的基底膜在许多病理中起作用。尽管有这种临床意义，我们对基底膜是如何形成的知之甚少。此外，尽管它在任何类型的组织损伤中都会受到一定的损伤，但我们对基底膜如何修复一无所知。基底膜由许多不同的成分组成，如IV型胶原蛋白，它们通过酶促反应连接以产生最终稳定的结构。这些成分的产生和组织也被认为需要许多不同的细胞类型。由于这种复杂性，一个完整的了解基底膜需要它的检查在一个活的有机体，直到最近一直在实验上是不可行的。在这个建议中，我们将利用我们的能力，活的图像基底膜的发展和修复在一个活的有机体。果蝇（Drosophila melanogaster）正在成为一种广泛使用的模型系统，以了解基底膜，因为这种动物具有与人类相同的各种细胞外基质蛋白。此外，我们实验室的初步数据显示，胚胎发生过程中的基底膜形成可以在动物发育过程中实时成像。在这个建议中，我们将利用我们的能力，生活图像基底膜的发展，沿着与我们的能力，在苍蝇敲除几乎任何感兴趣的基因，充分剖析的机制，基底膜的形成和repair.In的第一个目标，我们将通过延时显微镜观察的时间进程的基底膜形成。随后，我们将使用策略来专门去除假设的基底膜成分和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子。这一分析将使我们能够突出基底膜形成背后的分子机制以及不同细胞在其产生中的相对需求。在随后的目的中，我们将研究基底膜修复反应。来自实验室的初步数据显示，上皮细胞和下面的基底膜可以通过激光消融在果蝇中特异性地受损;这导致愈合反应，从而在几个小时的过程中基底膜孔被密封。我们将研究这种反应，并确定涉及基底膜修复的细胞和分子机制。我们的分析表明，基底膜愈合是一个活跃的细胞过程，需要招募果蝇巨噬细胞（果蝇炎性细胞），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，果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。这些数据表明，受损的基底膜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.