Regulation of Notch signalling during development of a model stem cell niche

生物体在其一生中的健康在很大程度上取决于成人健康所依赖的组织和器官的正常发育。因此，多细胞生物的细胞和身体结构的多样性是如何从进化过程中高度保守的少数发育信号中产生的，这是非常奇怪的。了解这种复杂性是如何产生的是一个重要的研究目标，并将产生相当大的影响，特别是在BBSRC的优先领域“健康老龄化的整个生命周期”。这是因为相同的发育信号继续用于成年人，以确保器官和组织的适当更新和修复。因此，了解这些信号被控制以影响细胞行为的正常机制至关重要。Notch受体介导一种这样的关键发育信号，其在发育的许多方面起着许多关键作用，并且是影响健康老龄化的许多疾病（包括癌症）的关键参与者。因此，Notch信号传导在开发中被利用和调节的方式引起了相当大的和广泛的兴趣。Notch被邻近细胞上的膜结合配体激活，因此它通常介导短程信号。我们已经确定了Notch激活的替代方法，其不需要Notch配体，而是依赖于Notch从细胞表面的内化和内部细胞器内的激活。我们现在希望使用果蝇来研究在什么样的生理环境中，这种新形式的信号激活可能在发育过程中被利用，以及它如何与配体依赖性Notch活性一起发挥作用来调节组织形成。为了实现这一目标，我们已经确定了两种形式的Notch信号的需求，用于发育包括成体卵巢中生殖系干细胞的小生境的特化细胞。这些都是维持成人持续产卵所必需的。卵巢干细胞生态位证实了成年人适当发育调节的重要性，因为它是更新组织和维持体内平衡所需的干细胞。小生境是一个专门的环境，提供锚定和信号，调节干细胞群体的大小，维持和分化，这些小生境的适当发展对成年人的健康至关重要。卵巢生态位是一种理想的发育模型，因为它可以在其发育的不同阶段作为活组织解剖，使我们能够在体内进行Notch内吞摄取的测定。构成发育中的小生境的不同细胞群可以通过对不同标记物进行染色而容易地区分。发育中的卵巢也很容易通过遗传操作从不同阶段的不同细胞中去除特定的基因功能，并允许我们对将继续形成成年生态位和相关结构的细胞进行谱系追踪。因此，我们将能够检查在不同的细胞，包括发展中的卵巢和检查的贡献，每个信号的小生境形成的Notch信号的不同形式的要求。此外，我们已经确定了一个细胞连接蛋白ZO-1作为卵巢生态位大小和干细胞能力的调节剂，从细胞培养数据中，我们假设ZO-1作为一个特定的负调节剂的配体非依赖性形式的Notch信号。我们将使用卵巢发育模型来测试我们的ZO-1对Notch作用的模型。ZO-1是第一个确定的Notch激活的替代模式的上游调节因子，其可能将组织形态发生与细胞命运调节联系起来。因此，这项工作将具有广泛的意义和影响。因为我们的研究利用果蝇作为模式生物，从而有助于BBSRC的优先领域“替代，改进和减少使用动物的研究”。

The well being of the organism throughout its life is highly dependent on the proper development of the tissues and organs on which adult health depends. It is therefore extraordinary how the diverse variety of cells and body plans of multicellular organisms arise from so few developmental signals that are highly conserved during evolution. Understanding how this complexity arises is an important research goal and will have considerable impact particularly within the BBSRC priority area of "healthy aging across the lifecourse". This is because the same developmental signals continue to be used in the adult to ensure proper renewal and repair of organs and tissues. It is of vital importance therefore to understand the normal mechanisms, by which such signals are controlled to affect cellular behaviour. The Notch receptor mediates one such key developmental signal that plays numerous and critical roles in many aspects of development and is a key player in numerous diseases affecting healthy aging including cancer. There is therefore considerable and wide ranging interest in the ways in which Notch signalling is utilised and regulated in development. Notch is activated by membrane bound ligands on adjacent cells and thus it normally mediates short-range signals. We have identified an alternative means of Notch activation, which does not require the Notch ligands but instead depends on Notch internalisation from the cell surface, and activation within internal organelles. We now wish to use Drosophila to investigate in what physiological contexts this new form of signal activation may be utilised during development and how it functions together with the ligand-dependent Notch activity to regulate tissue formation. To pursue this aim we have identified a requirement for both forms of Notch signal for the development of specialised cells that comprise the niche for germline stem cells for in the adult ovary. These are required to maintain continuous egg production in the adult. The ovary stem cell niche exemplifies the importance of proper developmental regulation to the adult because it is stem cells that are required to renew tissues and maintain homeostasis in the adult. The niche is a specialised environment that provides anchorage and signals that regulate stem cell population size, maintenance and differentiation and the proper development of these niches is paramount to adult well-being. The ovary niche is an ideal developmental model because it can be dissected as living tissue at different stages of its development, allowing us to perform assays of Notch endocytic uptake in vivo. The different cell populations that comprise the developing niche can be easily distinguished by staining for different markers. The developing ovary is also easily manipulated genetically to remove specific gene functions from different cells at different stages and to allow us to lineage trace the cells that will go on to form adult niche and associated structures. We will therefore be in a position to examine the requirement for different forms of Notch signalling in the different cells that comprise the developing ovary and examine the contribution of each signal to niche formation. Furthermore we have identified a cell junctional protein ZO-1 as a regulator of ovary niche size and stem cell capacity and from cell culture data we hypothesise that ZO-1 acts as a specific down-regulator of the ligand-independent form of Notch signalling. We will use the ovary developmental model to test our models of ZO-1 action on Notch. ZO-1 is the first identified upstream regulator of the alternative mode of Notch activation which potentially links tissue morphogenesis with cell fate regulation. This work will therefore be of wide interest and impact. Because our research utilises the fruitfly Drosophila as a model organism and thus contributes to the BBSRC priority area of "replacement, refinement and reduction in research using animals".