Quantitative analysis of cytoneme-based Wnt trafficking and signalling in vivo

细胞间的通讯对于调节所有多细胞生物体的发育是必不可少的。细胞间的通讯建立在化学刺激的基础上--包括调节组织中细胞行为的信号蛋白。协调发育的一个重要的信号蛋白家族是Wnt信号家族。WNT调节重要的细胞过程，包括细胞分裂的速度；细胞的命运或如何分化成不同的形式；以及细胞如何运动。因此，WNT信号对早期生命的发育(如胚胎发育)、器官发育、伤口愈合和再生至关重要。我们知道，相对较小和特定的一组细胞控制和分配控制发育的Wnt蛋白。然后，相邻的更大的细胞群对信号做出反应。因此，WNT的功能依赖于WNT蛋白从产生细胞到靶细胞的准确输送。目前，Wnt蛋白如何在细胞间转运以激活信号转导尚不清楚。因此，我们不理解消息是如何从一个单元传递到另一个单元的。这项提议旨在第一次了解信息是如何在细胞之间传递的。在为这一提议做准备的过程中，首席科学家揭示了Wnt蛋白存在一种完全意想不到的细胞间转运机制。特定的指状细胞膜突起--称为细胞素--将Wnt蛋白带到顶端，并将它们输送到邻近的细胞。与靶细胞接触后，反应细胞摄取Wnt蛋白。这一过程导致靶细胞中的信号激活。这些信号突起上运输的Wnt蛋白数量的损害会导致发育过程中的严重后果，导致组织畸形和严重的发育困难。因此，了解控制这一新发现的运输系统的系统对于了解Wnt如何在胚胎发育和组织动态平衡中阐明Wnt信号功能是至关重要的。这一知识将为能够操纵Wnt蛋白运输以控制再生和疾病中Wnt信号级联的活动提供基础。在我们初步工作的基础上，我们提出了Wnt蛋白控制自己的运输机制：我们假设Wnt触发了一组特定的受体，激活了细胞素的形成，并且从这些信号转导细胞手指传递到靶细胞的Wnt信号量对于信号激活的水平是至关重要的。我们将使用我们成熟的、最先进的遗传策略，结合研究团队首创的先进成像技术来测量活斑马鱼胚胎中运输的Wnt蛋白的数量。这将第一次让我们了解这一信号是如何运作的。到这个项目结束时，我们将确定产生Wnt的细胞如何控制这些信号突起的出现。我们将进一步确定Wnt信号蛋白如何从突起运输到靶细胞的受体，以启动相互信号。此外，超分辨率成像实验将使我们能够量化信号位置上的WNT信号成分，从而了解WNT信号的实际机制。我们相信，这些发现将对基础细胞和发育生物学以及对细胞间通讯和组织发育的更深入理解产生重大影响。通过这种方式，我们的目标是控制脊椎动物组织中Wnt信号网络的时空激活动力学。我们预计，从长远来看，这一结果将有助于开发新的工具，在人类疾病的发育、伤口愈合和再生过程中操纵Wnt信号通路。

Cell-to-cell communication is essential for regulation of development of all multicellular organisms. Intercellular communication is based on chemical stimuli - including signalling proteins - which regulate the cellular behaviour in a tissue. An important family of signalling proteins that orchestrate development is the Wnt signalling family. Wnts regulate vital cellular processes including how fast cells divide; the fate of cells or how to differentiate into different forms; and how cells move. Wnt signalling is therefore fundamental to the development of early life (e.g. embryogenesis), organ development, wound healing, and regeneration. We know that a relatively small and specific group of cells control and distribute Wnt proteins controlling development. Adjacent, larger groups of cells then respond to the signal. Wnt function is therefore dependent on precise delivery of Wnt proteins from producing cells to target cells. Currently, how Wnt proteins are transported between cells to activate signalling is unknown. As such we do not understand how the message is delivered from one cell to another. This proposal aims to understand, for the first time, how the message is conveyed between cells. In preparation for this proposal, the lead scientist has revealed the existence of a completely unexpected cell-to-cell transport mechanism for Wnt proteins. Specific finger-like cell membrane protrusions - called cytonemes - carry Wnt proteins to their tips and transport them to neighbouring cells. After contact with the target cell Wnt proteins are taken up by the responding cells. This process leads to signal activation in a target cell. Impairment of the number of Wnt protein transported on these signal protrusions leads to severe consequences during development, leading to malformation of tissues and severe developmental difficulties. Understanding the systems that govern this newly identified transport system is therefore fundamental for understanding how Wnt functions to elucidate Wnt signal function during embryogenesis and tissue homeostasis. This knowledge will provide the foundations to be able to manipulate Wnt protein transport to control the activity of Wnt signalling cascades in regeneration and diseases. Based on our preliminary work, we propose that the Wnt proteins control their own transport mechanisms: We hypothesize that Wnt triggers a specific set of receptors, which activates formation of cytonemes, and the amount of Wnt signals handed over from these "signalling cell fingers" to the target cell is crucial for the level of signal activation. We will use our established, state-of-the-art, genetic strategies, combined with advanced imaging techniques pioneered by the research team to measure the amount of Wnt protein transported in a living zebrafish embryo. This will for the first time allows us to understand how this signalling operates. By the end of this project, we will determine how Wnt-producing cells control the emergence of these signalling protrusions. We will further have identified how Wnt signalling proteins traffic from the protrusions to the receptor of the target cell to initiate reciprocal signalling. Furthermore, super-resolution imaging experiments will allow us to quantify Wnt signalling components at the signalling sites and thus understand the actual mechanism of Wnt signalling. We believe that these findings will have a significant impact on basic cell and developmental biology and a deeper understanding of cell-cell communication and tissue development. In this way, we aim to control the spatiotemporal activation dynamics of Wnt signalling networks in vertebrate tissue. We envisage that in the longer term the results will, therefore, inform the development of novel tools to manipulate Wnt signalling pathways during development, wound healing and regenerative processes for the treatment of human disease.