Combining structure and genetic data to probe cis and trans regulation of Notch by ligands.

The well being of the organism depends on the proper development of the tissues and organs on which adult health depends. Understanding the regulation of the signals that contribute to developmental complexity will have considerable impact within the BBSRC priority area of "healthy aging across the lifecourse". This is not in the least because the same developmental signals continue to be used in the adult to ensure proper homeostasis, renewal and repair of organs and tissues. Understanding how these processes are controlled will also help advance the field of tissue engineering and exploitation of stem cells to improve the health and life of the aging population because it will facilitate ways to manipulate cell fates. The evolutionary conserved Notch receptor mediates arguably one of the most important developmental signals because it plays numerous critical roles in many aspects of development and adult tissue homeostasis, and hence is involved in regulating many cell types. These critical functions are revealed by mutations that affect the structure/function of the Notch receptor and which are associated with many different developmental disorders and with cancer. Only in a small number of cases do we fully understand how these missense mutations in different parts of Notch lead to up or down-regulation of its activity that is linked to different outcomes affecting health. This is because the regulation of Notch is complex involving many different extracellular and intracellular interactions in different parts of the receptor. Notch is localised to the cell membrane and activated there by one of two classes of membrane bound ligand. Ligands on adjacent cells (trans-presented ligands) activate the Notch signal. However, if the ligand is expressed in the same cell as Notch (cis-ligand) it instead blocks signalling. How these different outcomes of ligands interacting with Notch arise, and how these opposing regulatory mechanisms map onto the structure and function of Notch are important questions that will be addressed in this project. We hypothesise based on previous pilot studies that different mutants will selectively interfere with different modes of regulatory interactions and hence have different outcomes on activity. Understanding the genotype to phenotype connection is important because understanding the different ways in which Notch mutants lead to loss or gain of function will advance our knowledge of how this important signal is normally regulated, and it will inform new strategies to allow compensation for these defects which in the longer term will ultimately provide new routes to therapies or interventions to improve health and well being.
Our approach to this question will exploit Drosophila Notch as a model system. Guided by recent published structures of Notch ligand complexes, we will compare the structure/function/phenotype relationships of cis and trans-interactions of Notch with two different classes of ligand, the Serrate/Jagged class and the Delta class, using cell culture and in vivo assays. Drosophila has a strong track record of delivering fundamental and conserved new insights into cell signalling regulation. The fly excellent is an excellent model system for our study since Notch sequence, structure and signalling mechanisms are highly conserved between flies and humans and there is no organism in which Notch signalling is better understood and the ease by which novel mutants can be introduced into the fly genome makes probing structure/function in vivo a viable approach. Using the fruitfly Drosophila as a model organism contributes to the BBSRC priority area of "replacement, refinement and reduction in research using animals". This is because doing similar research with vertebrate models would require the use of a large number of animals particular because of the fact that mammalian genomes contain four copies of Notch, making experimental results much harder to control and interpret.

Notch is a cell surface receptor that mediates a crucial developmental signal that is precisely regulated for proper tissue development, homeostasis, and renewal. There is a complex interplay between Notch/ligand and Notch/Notch interactions that regulates its activity. Trans-presented ligands on adjacent cells activate the receptor while cis-interacting ligand in the same cell inhibit signalling by competing with trans ligands, by altering Notch trafficking and possibly by promoting receptor dimerisation. These negative interactions are important to reinforce the directionality of developmental pattern formation and also to suppress ectopic activation that would otherwise occur independently of trans-presented ligands. Genomic studies of a variety of disease states have uncovered hundreds of Notch mutations that affect function but the genotype/phenotype linkages are mostly unknown. Attempting to understand these linkages is now timely because there is now a large database of function-perturbing mutations from disease studies and crystal structures giving unprecedented structural detail regarding Notch/ligand complexes. We will use a mutational approach in cell culture and in vivo in Drosophila to map the structural requirements of Notch for different regulatory outcomes arising from different modes of ligand binding and of Notch/Notch dimer contacts, making use of the strong conservation between the fly and human Notch sequences. We will examine the variety of different phenotypic consequences in vivo arising from disruption of different ligand/Notch and Notch/Notch interface sites. Understanding in detail, at the structure/function level, how Notch is regulated will ultimately be beneficial for developing ways to manipulating cell fates by up or down regulating Notch during tissue engineering and for developing treatments for a variety of pathologies.