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

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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".