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

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".

The proper development of tissues and organs is vital to the healthy maintenance of the adult organism. My group uses Drosophila as a model to investigate the regulation of key signals mediated by the Notch receptor that are vitally important to many aspects of development. We uncovered a novel ligand-independent mechanism of Notch activation and are seeking to understand how this mode is integrated with the canonical ligand-induced Notch signal in tissue development. We identified the Drosophila ovary and germline stem cell niche formation as a model in which the two forms of Notch signalling are required and their contributions genetically distinguished by removal of specific components. We will test a working model that the two modes of Notch signalling act in different pools of cells through different target genes each providing a subset of niche precursors. We hypothesise that the cell junction associated scaffold protein ZO-1 regulates niche recruitment by suppressing ligand-independent Notch activation as the niche assembles. We propose that ZO-1 regulates Notch endocytosis and its endosomal sorting by Deltex, a key intracellular activator of ligand-independent Notch signalling. To test these ideas we will use cell lineage tracing to identify the origin of cells in the niche, along with genetic manipulations to identify the requirements for different mechanisms of Notch activation in different cell populations. We will use established Notch endocytosis assays to identify the role of ZO-1 in regulating Notch endocytosis and sorting of Notch between the endosomal membrane and lumen, which we propose is a key regulatory node in the ligand-independent activation mechanism. This project will bring significant advances to understand Notch signal regulation and will identify new concepts of how niches are formed and regulated. Given the strong track record of Drosophila in identifying evolutionary conserved paradigms, we expect our work to have wide impact.

Notch signalling plays a crucial role in development. The endosomal trafficking system, which plays a central role in regulating Notch activity, also plays a central role in cellular physiology. In this project we investigate how Notch signalling is regulated in the Drosophila developmental model which comprises a stem cell niche. Altered regulation of Notch during normal aging is also associated with declining ability to replace and repair tissues contributing to age-related changes affecting health and well being. Numerous medical and age related conditions have been linked to Notch signalling including cancer and Notch plays a key role in stem cell regulation. By bringing together both of these aspects in a single programme this project leads to a number of different routes to impact with a wide range of beneficiaries.
1:- Medical research
I have established a collaboration with Dr. Keith Brennan of the Manchester breast centre who is already translating our findings from Drosophila into human cancer-research. A similar impact pathway yielded a patented IP relating to prognostic uses of expression of human Su(dx)-related protein in breast cancer patients. I have now established similar links to clinicians at the University of Liverpool who are interested in Notch activation in head and neck cancers. In addition, in collaboration with the Genetics department of St. Mary's hospital in Manchester, we have identified in flies, novel links between certain genes linked to neurodegenerative disorders and defective Notch endocytosis and activation. Our work on regulation of Notch may have an impact on understanding the mechanisms of these debilitating diseases. My close links to the genetics department at St. Mary's hospital thus provides a clear route to transfer findings such as this into clinical research. These collaborations will be managed through joint research meetings and an annual review which will assess how work arising from our programme can be best progressed towards clinically utility.
2: Public education
Key goals of this programme are:- to educate the public regarding how regulation of normal cell behaviour is vital to the body systems that normally function to keep us healthy. We further wish to educate the public as to how understanding these normal processes underpins our understanding and hope of medical intervention in pathological situations whereby normal means of control are corrupted, and may contribute to new medical approaches like tissue engineering and regenerative medicine. A further goal is to promote the importance of model organisms such as Drosophila as an alternative to animal models, as part of the NC3Rs replacement, refinement and reduction policy. I will regularly review teaching opportunities to communicate my scientific goals and outcomes to our undergraduate students who will go on to fulfil many diverse roles in society. I will utilise numerous opportunities to communicate with the public and schools using informative displays during annual outreach activities comprising of "meet a scientist" school visit days, Faculty of Life Science Open days, the Body Experience exhibition at the Manchester Museum, and the Manchester Science festival. I will further promote use in schools of simple practical experiments we have devised for teachers to demonstrate genetics and the utility of the fruit fly as a model organism. My recent election as parent governor to a local primary school will provide an excellent means to enhance contact with the teaching profession, understand how science teaching is performed in schools and to provide input and feedback on the design of the new science curriculum which emphasises ways of working scientifically.