Role for dynamic protrusions in epithelial patterning

成年苍蝇的背部有一排漂亮的刚毛。长期以来，这一直被用作理解生物学模式形成的简单模型。当对活的苍蝇胚胎进行成像时，很明显，这种刚毛的模式随着时间的推移，通过一个自组织的过程发展起来，从混乱的开始，到有序的最终状态。值得注意的是，即使受到大范围扰动的挑战，这种模式也能实现。操纵果蝇遗传学和拍摄苍蝇发育实况的可能性，使蝇胸成为探索这一生物模式形成过程中一般原理的理想模型。这一领域的基础性发现可能会影响我们对各种过程的理解，如人类内耳或肠道组织的生成。在最近对这一过程的研究中，我们发现构成结节（一种扁平组织）的细胞有许多手指状的突起（丝状足），这些突起跨越几个细胞直径。它们在组织下面形成了一个密集的细胞-细胞接触网，使非相邻细胞能够相互接触，更重要的是，可以远距离交换信息。像大多数细胞突起一样，它们是动态的，平均寿命不到10分钟。正因为如此，随着时间的推移，随着刚毛模式的建立，细胞与不同的邻居接触，使每个细胞在决定自己的命运之前，能够确保自己与附近的其他细胞正确地定位。抑制突出的形成导致图案严重破坏，其中刷毛之间的平均分离减少。这些数据表明，丝状突起在发送调节鬃毛图案的信号方面起着重要作用。这引起了科学界的兴趣，因为许多研究人员怀疑突起可能在它们自己的研究系统中介导远距离信号。在这里，为了建立这一新发现，我们的目标是确定支撑突起形成、动力学和突起介导的信号传导的分子机制。我们将采用纳米技术和基因技术结合的方法来确定动态丝状足在有序和有图案的上皮生成中的作用。首先，我们将使用高倍光学显微镜对发育中的果蝇中的单个细胞进行成像，特别注意其突起的动力学和形态。通过将信号标记与丝状伪足标记一起使用，我们将能够通过测量与丝状伪足长度和与其他细胞接触的持续时间相关的参数，看到是什么使细胞分化成刚毛和那些没有刚毛的细胞。接下来，我们将寻找调节丝状突起动力学的蛋白质。为了做到这一点，我们将减少候选蛋白质的数量，并询问它们的消耗是否影响丝状形状和/或动力学。通过这种方式，我们期望识别丝状足的物理成分和调节其组装的蛋白质，这些蛋白质作用于细胞-细胞信号传导事件的下游。为了找出这些突起的作用，并理解单个丝状足和整个组织模式之间的联系，我们将破坏特定组织区域的丝状足动力学，并询问在组织的扰动区域中刚毛间距如何变化，将结果与该过程的计算模型中的模拟结果进行比较。最后，我们将询问丝状动力学是否有助于突变或环境冲击后这种模式过程的鲁棒性。我们相信这项工作将使我们更好地理解这种依赖于细胞-细胞接触动态变化的新型模式。我们预计这将对发育、体内平衡和疾病期间的其他模式过程以及组织工程产生广泛的影响。

The back of an adult fly carries a beautiful array of bristles. This has long been used as a simple model to understand pattern formation in biology. When imaged live fly embryos it is clear that this pattern of bristles develops over time through a process of self-organisation, which from messy beginnings yields a well-ordered final state. Remarkably, such patterns can be achieved even when challenged with wide range of perturbations. The possibility to manipulate fly genetics and to image fly development live make the fly thorax (notum) an ideal model with which to explore general principles in this process of biological pattern formation. Fundamental discoveries in this area are likely to have implications for our understanding of processes as diverse as the generation of the organisation in the human inner ear or the gut.In a recent study of this process we discovered that the cells that make up the notum (a flat tissue) have numerous fingerlike protrusions (filopodia) that span several cell diameters. These formed a dense web of cell-cell contacts underneath the tissue that enabled non-neighbouring cells to contact one another and, importantly, to exchange information at a distance. Like most cellular protrusions, these are dynamic, with an average lifetime of less than 10 minutes. Because of this, cells contact different neighbours over time as the pattern of bristles is being established, enabling each cell to ensure that it is correctly positioned with respect to other cells in the vicinity before deciding on its fate. Inhibition of protrusion formation resulted in patterning that was severely disrupted and in which the average separation between bristles was decreased. These data suggest that filopodial protrusions play an important role in sending the signals that regulate bristle patterning. This generated interest across the scientific community because many researchers suspect protrusions may mediate long range signalling in their own systems of study.Here, in order to build on this novel finding, we aim to identify the molecular mechanisms that underpin protrusion formation, dynamics, and protrusion mediated signalling in flies. We will use approaches taken from nanotechnology together with genetic techniques to determine the role of the dynamic filopodia in the generation of a well-ordered and patterned epithelium. First, we will image individual cells in developing flies using high power light microscopy, paying particular attention to the dynamics and morphology of their protrusions. By using markers of signalling together with markers of filopodia, we will be able to see what differentiates cells that become bristles from those that do not by measuring parameters related to filopodial length and duration of contact with other cells. Next, we will search for proteins that regulate the dynamics of filopodial protrusions. To do this, we will reduce the number of candidate proteins and ask if their depletion affects filopodial form and/or dynamics. In this way we expect to identify proteins that are physical components of the filopodia and proteins that regulate their assembly acting downstream of cell-cell signalling events. To find out what these protrusions do and to understand the link between individual filopodia and whole tissue patterning we will disrupt filopodia dynamics in specific tissue regions and ask how bristle spacing changes across the perturbed region of tissue, comparing results with simulations in a computational model of the process. Finally, we will ask if filopodial dynamics contribute to the robustness of this patterning process following mutation or environmental shock. We believe this work will provide us with a better understanding of this new type of patterning that relies on dynamic changes in cell-cell contacts. We anticipate this having broad implications for other patterning processes during development, homeostasis and disease, and for tissue engineering.