Parallel notch activation mechanisms provide robustness to developmental patterning in Drosophila
Lead Research Organisation:
University of Manchester
Department Name: Life Sciences
Abstract
Multicellular organisms like humans and fruit flies, develop from the proliferation of a single cell. An important problem in biology is to understand how it is that the millions of cells present in the adult body arise. These cells have to perform many different functions in diverse tissues and therefore they become differently specialised, for example into heart cells or brain cells. It is an important challenge in biology to understand, not only how this specialisation occurs, but also how each specialised cell arises in the correct place at the correct time of development. This is not only important from the point of view of adding to our fundamental biological knowledge, but it has important implications for our understanding of diseases like cancer, heart disease and dementia. This is because the signals that control the development of the organism are often signals that go wrong when we are suffering such medical problems. If we can understand how these signals function normally in development then this will provide essential insight into how to correct defects in these signals when they go wrong. For example sometimes cancer is caused when a signal is switched on permanently by a mutation, rather being regulated on and off. If we can find a way to block such signals without harming its normal functions, then it might be possible to cure particular cancers. It is obviously difficult both practically and ethically, to find out the answers to these problems by experimenting on humans. However, because all living organisms today have evolved from a common ancestor, it is possible to understand problems related to human biology and medicine, by studying other organisms. The fruit fly is one powerful model system that is often used to understand problems of signalling and development. The major signals that control fruit fly development are conserved with humans, including many of the signals that are associated with human diseases such as cancer. It is a wonderful model organism because it is so easy to manipulate by making mutations in its genes. This means we can study signals in intact tissue rather than trying to deduce their function by studying isolated cells in a dish. The latter approach is also very useful but can give misleading answers unless it can be combined with the investigation of signalling in real tissues. Studying fruit flies has resulted in the identification of many signals that are misregulated in human cancers. Our aim is to investigate a critical signal that is mediated by a protein called Notch. This protein is used many times in development and in many different tissues. One of the most well known uses is during the development of the brain. The function of Notch is to ensure that correctly specialised cells arise in the proper places in the body. My research recently found out that our understanding of how this receptor works is incomplete. This is very important to know because this protein is used many times in development and it is misregulated in many types of cancer. We have now discovered that there is a way for Notch to signal that was not known before. This programme aims to understand this new signal by identifying its required components and finding out what they do during development of the fruit fly brain, and other tissues. The work has an enormous potential to help find ways to cure diseases like cancer. It may also help find ways to solve problems associated with aging and help us to lead healthier lives for longer. This is because Notch regulates stem cells and these contribute to keeping our tissues and organs healthy over our lifespan. It also functions in the brain cells to help us remember events for a long time. A decline in the ability to form memories is a problem often associated with age-related conditions such as dementia. Understanding how Notch works may help lead to solutions to these challenging problems in the future.
Technical Summary
A critical and conserved developmental signal is mediated by the Notch receptor which is utilised in numerous key decisions and patterning processes. The Notch signal refines, to a single cell resolution, more broad patterns laid down by long-range signals. DSL-domain ligands activate Notch by causing proteolytic release of the Notch intracellular domain. The latter translocates to the nucleus to regulate gene expression. However, recent work from my group, using the Drosophila model organism, shows a proportion of Notch signalling depends on a DSL ligand-independent signal, that regulates the trafficking of Notch to the lysosome membrane. We propose that parallel pathways of Notch activation contribute to developmental robustness during cell fate patterning. To characterise the novel pathway further, we need to identify the remaining components and their functions. We will determine the full contribution of the alternative Notch pathway to development by identifying any redundancy, or parallel pathways by which Notch traffics to the lysosome. We will understand the impact of endocytic sorting on Notch signalling, by investigating how the pathway can be switched between positive and negative outcomes, and the relationship between the endocytosis-induced and the DSL ligand-induced signals. Our hypothesis is that these are distinct routes to activation rather than being part of the same linear pathway. However we will rigorously test this hypothesis by comparing the structure/function requirements of Notch in the two pathways, and the requirements for the pathway components. The outcomes from the above will enable us to generate more realistic models of the Notch signalling network during pattern formation, exploiting an existing collaboration to generate predictive computer simulations. This more comprehensive understanding will inevitably be informative for the ability to manipulate Notch signal activity for therapeutic purposes.
Organisations
- University of Manchester (Collaboration, Lead Research Organisation)
- UNIVERSITY OF OXFORD (Collaboration)
- University of Sheffield (Collaboration)
- Osaka University (Collaboration)
- Heinrich Heine University Düsseldorf (Collaboration)
- Agency for Science, Technology and Research (A*STAR) (Collaboration)
Publications
Acar A
(2021)
Inhibition of Wnt signalling by Notch via two distinct mechanisms.
in Scientific reports
Baron M
(2012)
Endocytic routes to Notch activation.
in Seminars in cell & developmental biology
Bonfini A
(2015)
Reversible regulation of stem cell niche size associated with dietary control of Notch signalling.
in BMC developmental biology
Djiane A
(2011)
Su(dx) E3 ubiquitin ligase-dependent and -independent functions of polychaetoid, the Drosophila ZO-1 homologue.
in The Journal of cell biology
Shimizu H
(2024)
Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.
in The Journal of cell biology
Shimizu H
(2017)
The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation.
in Open biology
Whiteman P
(2013)
Molecular basis for Jagged-1/Serrate ligand recognition by the Notch receptor.
in The Journal of biological chemistry
Yamada K
(2011)
Roles of Drosophila deltex in Notch receptor endocytic trafficking and activation.
in Genes to cells : devoted to molecular & cellular mechanisms
Description | Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals. |
Exploitation Route | The work provides the basis for understanding the impact of numerous Notch regulators in the framework of a regulatory endocytic network and a basis for understanding the complex genotype phenotype relationship of the Notch genetic locus with implications for the study of cancer associated mutations |
Sectors | Education Pharmaceuticals and Medical Biotechnology |
URL | http://www.sciencedirect.com/science/article/pii/S0092867414004802 |
Description | My lab has devised primary school teaching package illustrating the use of Drosophila in research and demonstrating aspects of the national curriculum associated with heredity and evolution |
First Year Of Impact | 2015 |
Sector | Education |
Impact Types | Societal |
Description | Exploiting Notch regulation to probe alternative mechanisms of TSC signalling. Tuberous Sclerosis Alliance |
Amount | £189,737 (GBP) |
Funding ID | 2016-01 |
Organisation | Tuberous Sclerosis Association |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 12/2016 |
End | 05/2020 |
Description | Synthetic lethal targeting of Notch-driven breast cancer |
Amount | £24,924 (GBP) |
Organisation | University of Manchester |
Sector | Academic/University |
Country | United Kingdom |
Start | 11/2022 |
End | 11/2023 |
Description | Molecular basis for jagged-1/serrate ligand recognition by the notch receptor. |
Organisation | University of Oxford |
Department | Department of Plant Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Research collaboration established with Department of Biochemistry, University of Oxford bringing together physical studies on the structure and function of Notch with our functional analysis and genetic dissection of Notch activities. |
Start Year | 2010 |
Description | Notch ubiquitination and trafficking |
Organisation | Heinrich Heine University Düsseldorf |
Country | Germany |
Sector | Academic/University |
PI Contribution | We have supplied expertise in ubiquitination and trafficking assays, supervision and some lab consumables |
Collaborator Contribution | The klein lab has constructed Notch receptor expression construct with a lysine to arginine replacement for every lysine in the intracellular domain. They have sent a lab member to my lab for a few weeks to perform some protein ubiquitination and trafficking assays using the expertise we have developed in this area. They have also contributed the researcher's travel, accommodation and subsistence expenses to this visit. |
Impact | Ubiquitination and trafficking data and new mutant Notch expression constructs. |
Start Year | 2018 |
Description | Regulation of Notch by Pecanex |
Organisation | Osaka University |
Department | Department of Biological Sciences |
Country | Japan |
Sector | Academic/University |
PI Contribution | We have contributed expertise in cell sorting by FACS, and Notch signalling assays in S2 cells, supervision and training |
Collaborator Contribution | Our collaborative partner Dr. Tomoko Yamakawa has contributed Drosophila mutants of the Pecanex gene and has characterised the developmental links between this gene and Notch during embryo nervous system development. She has raised funds to pay expenses for travel accommodation and subsistence for a two week research visit to my lab to perform experiments in which we have expertise not available in Osaka. |
Impact | This is a multidisciplinary collaboration involving Drosophila developmental biology and genetics , cell biology and biochemistry. |
Start Year | 2018 |
Description | Regulation of Notch by numb |
Organisation | Osaka University |
Department | Department of Biological Sciences |
Country | Japan |
Sector | Academic/University |
PI Contribution | We have provided expertise in Notch signalling assays in S2 cells, supervision and some consumables |
Collaborator Contribution | Our Collaborative partner, Kenji Matsuno has contributed a post doc to the collaboration who has characterised a role of numb in early development to restrain Not5ch activity and hence assure proper control of mitosis and morphological changes in the embryo. The researcher has visited my lab for three weeks to perform some biochemical experiments and signalling assays to discern the mechanism of numb regulation of Notch. An in kind contribution of travel accommodation and subsistence expenses for the researcher to visit my lab has been made. |
Impact | Conference presentation: "A novel role of Numb prevents embryo from twisting though the inhibition of Notch signaling" The Notch meeting, Athens 2017. |
Start Year | 2017 |
Description | Regulation of Notch4 trafficking and signalling |
Organisation | University of Manchester |
Department | Manchester Cancer Research Centre |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | Co-superverision of MCRC funded PhD student providing expertise in Notch signalling and trafficking |
Collaborator Contribution | Rob Clarke of the MCRC is providing joint supervision of a MCRC funded student and providing use of facilities at the MCRC to pursue the project which will investigate how Notch4 trafficking and signalling compares to Drosophila Notch. |
Impact | Too early for outputs |
Start Year | 2017 |
Description | Role of Notch trafficking regulation in early Drosophila embryo patterning |
Organisation | Osaka University |
Country | Japan |
Sector | Academic/University |
PI Contribution | Co-supervision of a post doc appointed in collaborators laboratory including 2 visits to Osaka per year |
Collaborator Contribution | Hosting jointly supervised post doc and providing research infrastructure for the project |
Impact | Post doc has been identified and appointed. First research visit has been completed |
Start Year | 2014 |
Description | Understanding the role of Deltex in stem cell regulation |
Organisation | Agency for Science, Technology and Research (A*STAR) |
Country | Singapore |
Sector | Public |
PI Contribution | Co-supervision, provision of materials and resources, generation of new mutant zebrafish lines |
Collaborator Contribution | Co-supervisions, expertise reagents and resources in Zebrafish developmental biology, maintenaice and generation of new mutant lines |
Impact | The Notch pathway is involved in many developmental processes and its dysfunction leads to disorders such as cancer. Notch can be activated by cell surface ligands, or ligand-independently by the E3 ubiquitin ligase Deltex. The latter regulates Notch endocytosis and its subsequent activation in the lysosomal membrane. In the Drosophila ovary, deltex mutation causes egg chamber packaging defects, with reduced interfollicular stalks, a Notch loss of function phenotype. Moreover, Deltex is involved in the regenerative response of the adult intestine after dextran sulfate sodium-induced damage and stem cell proliferation. Compared to wild type, deltex mutants present with an initial increase in enteroblasts, which are precursors to the absorptive enterocyte cells, but these subsequently showed delayed terminal differentiation, with and associated decreased viability. Without DSS treatment, we observed that deltex mutant intestines had reduced Notch signalling and a suppression of enteroblast differentiation in aged flies, with enteroblasts remaining adjacent to the intestinal stem cells, instead of migrating away. Zebrafish Deltex2 mutants also display abnormal gut morphology and goblet cell overpopulation, indicative of reduced Notch activity. To explore conservation of Deltex function between flies and vertebrates further, mutant zebrafish lines have been established in the Deltex 4a and 4b genes using CRISPR. |
Start Year | 2019 |
Description | mathematical modelling of Notch signal regulation |
Organisation | University of Sheffield |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We provided experiemental data to create and validate the mathematical model |
Collaborator Contribution | Partner supervised and provided intellectual inout into the creation and use of the modelling by a PhD student who worked on the project in Manchester |
Impact | Resulted in Cell paper Multidisciplinary Biological sciences and mathematics |
Start Year | 2012 |
Description | Science outreach |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | The brain box.. science fair in Town Hall Manchester, part of European City of Science celebrations |
Year(s) Of Engagement Activity | 2016 |
Description | school visit.. |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | visited school parents evening where `i am a governor and advertised and promoted Manchester Science week out reach activities Increased participation of parents/pupils teachers in visiting inscience week events science week promoted in school assemble |
Year(s) Of Engagement Activity | 2014,2015,2016 |
Description | science spectacular outreach activity |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Demonstrations of uses of Fruit flies in research with hands on practical experiments Enagagement with public in the role of the fly in research including as an alternative to animal models |
Year(s) Of Engagement Activity | 2014 |