Role for dynamic protrusions in epithelial patterning

一只成年苍蝇的背部有一排美丽的刷毛。长期以来，这一直被用作一个简单的模型来理解生物学中的模式形成。当拍摄到活体苍蝇胚胎的图像时，很明显，这种刷毛模式是通过一个自我组织的过程来发展的，这个过程从混乱的开始产生了一个有序的最终状态。值得注意的是，即使在受到广泛干扰的情况下，这种模式也可以实现。操控苍蝇遗传学和活体成像苍蝇发育的可能性使苍蝇胸腔成为探索这一生物模式形成过程中一般原理的理想模型。这一领域的基本发现可能会对我们理解不同的过程产生影响，比如人类内耳或肠道中组织的产生。在最近对这一过程的研究中，我们发现组成鼻孔的细胞(扁平组织)有许多指状突起(丝状足突)，跨越几个细胞直径。这些细胞在组织下形成了密集的细胞-细胞接触网络，使不相邻的细胞能够相互联系，更重要的是，可以远距离交换信息。像大多数细胞突起一样，这些突起是动态的，平均寿命不到10分钟。正因为如此，随着时间的推移，随着刷毛模式的建立，细胞会接触不同的邻居，使每个细胞在决定自己的命运之前，能够确保自己相对于附近的其他细胞正确定位。抑制突起的形成导致图案被严重破坏，其中刷毛之间的平均间隔减小。这些数据表明，丝状突起在发出调节刷毛图案的信号方面发挥了重要作用。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他们自己的研究系统中介导远程信号。在这里，为了建立这一新的发现，我们的目标是识别支撑突起形成、动力学和突起介导的苍蝇信号的分子机制。我们将使用纳米技术的方法和基因技术来确定动态丝状足细胞在生成有序和有图案的上皮细胞中的作用。首先，我们将使用高倍光学显微镜对发育中的果蝇的单个细胞进行成像，特别关注它们突起的动力学和形态。通过使用信号标记和丝状伪足标记，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时间相关的参数来区分成为刚毛的细胞和没有成为刚毛的细胞。下一步，我们将寻找调节丝状突起动态的蛋白质。为了做到这一点，我们将减少候选蛋白质的数量，并询问它们的耗尽是否影响丝足形态和/或动力学。通过这种方式，我们希望识别丝状伪足的物理成分的蛋白质，以及在细胞-细胞信号事件下游作用的调节其组装的蛋白质。为了找出这些突起的作用，并了解单个丝状伪足和整个组织图案之间的联系，我们将扰乱特定组织区域的丝状伪足动态，并询问整个组织扰动区域的刷毛间距是如何变化的，将结果与该过程的计算模型中的模拟进行比较。最后，我们将询问丝轴动力学是否有助于在突变或环境冲击后这种图案化过程的稳健性。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图案。我们预计这将对发育过程中的其他构图过程、动态平衡和疾病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.