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的发育是必不可少的。细胞间通讯是基于化学刺激-包括信号蛋白-调节组织中的细胞行为。协调发育的信号蛋白的一个重要家族是Wnt信号家族。Wnts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信号传导对于早期生命的发育（例如胚胎发生）、器官发育、伤口愈合和再生是至关重要的。我们知道，一个相对较小和特定的细胞群控制和分布控制发育的Wnt蛋白。相邻的更大的细胞群然后对信号做出反应。因此，Wnt功能依赖于Wnt蛋白从生产细胞到靶细胞的精确递送。目前，Wnt蛋白如何在细胞之间转运以激活信号传导尚不清楚。因此，我们不明白信息是如何从一个细胞传递到另一个细胞的。该提案旨在首次了解信息如何在细胞之间传递。在准备这项提案时，首席科学家揭示了Wnt蛋白存在一种完全出乎意料的细胞间转运机制。特定的指状细胞膜突起-称为cytonemes -将Wnt蛋白携带到其尖端并将其运输到相邻细胞。与靶细胞接触后，Wnt蛋白被应答细胞吸收。该过程导致靶细胞中的信号激活。在这些信号突起上转运的Wnt蛋白的数量受损导致发育期间的严重后果，导致组织畸形和严重的发育困难。因此，了解这个新发现的运输系统的管理系统是了解Wnt如何发挥作用，阐明Wnt信号在胚胎发育和组织稳态的功能是至关重要的。这些知识将为能够操纵Wnt蛋白转运以控制再生和疾病中Wnt信号级联的活性提供基础。基于我们的初步工作，我们提出Wnt蛋白控制它们自己的转运机制：我们假设Wnt触发一组特定的受体，激活细胞素的形成，并且从这些“信号细胞指”传递到靶细胞的Wnt信号的量对于信号激活的水平至关重要。我们将使用我们建立的最先进的遗传策略，结合研究团队开创的先进成像技术，测量活斑马鱼胚胎中转运的Wnt蛋白量。这将是第一次让我们了解这种信号是如何运作的。在这个项目结束时，我们将确定Wnt产生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传导。此外，超分辨率成像实验将使我们能够量化Wnt信号传导位点的信号传导成分，从而了解Wnt信号传导的实际机制。我们相信这些发现将对基础细胞和发育生物学产生重大影响，并加深对细胞间通讯和组织发育的理解。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。因此，我们设想，从长远来看，这些结果将为开发新的工具提供信息，以在发育、伤口愈合和再生过程中操纵Wnt信号通路，用于治疗人类疾病。

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.