A genome-wide analysis of Notch signalling in neural stem cells and neurons

All complex multi-cellular organisms originate from a single cell, and yet the final adult form has a multitude of different cell types performing specialised functions. As cells are generated within developing organisms, therefore, new cells need to acquire new characteristics, forms and fates, which has to happen at both the right time during development as well as in the right location. The problem of how this complex temporal and spatial patterning is achieved is the key question driving the field of developmental biology.

The processes involved in brain formation are of particular interest. The human cerebral cortex is an extraordinarily complex structure, comprising over 10 billion neurons in multiple interacting layers, all of which derive from neural stem cells. Creating the right numbers of the right types of neurons requires a delicate balance between the initial proliferation of neural stem cells and their subsequent differentiation -- should too few stem cells be produced, the necessary numbers of neurons will never be achieved, whereas an uncontrolled proliferation of stem cells will lead to tumour formation and cancer. The factors influencing the maintenance of this fine balance during brain development are the focus of this research proposal.

In order to control the stages of neural development and to maintain stem cells as self-renewing, cells use signalling pathways to communicate with each other. One of these signalling pathways is called the Notch signalling pathway, and it is used in many cell types during development. It may seem paradoxical that a single signalling mechanism could be used to generate many different cell types, and we currently do not completely understand how Notch signalling can create so many different outcomes depending on the context. What we do know, however, is that in certain cell types Notch signalling requires protein co-factors to enable it to activate gene expression. Currently, the co-factors that might confer specificity on Notch signalling in neural stem cells and neurons are unknown, and the first objectives of this grant are to determine which proteins allow Notch to be able to perform its specified roles in neural stem cells and in neurons.

Another means of controlling the activity of Notch signalling is through changing the chromatin state of a cell. Chromatin is a term used to refer to the format of a piece of DNA, which in the simplest sense can be in either an open or closed configuration. Open chromatin is, as the name suggests, conducive to being bound by many proteins that activate genes. These proteins are unable to act when the chromatin structure is closed and inaccessible. The last objectives of this proposal are thus to examine the form that chromatin is in within both neural stem cells and neurons, and determine whether this influences the binding activity of Notch.

Notch signalling is important for maintaining neural stem cells and establishing the fates of neurons. Currently little is known as to how Notch achieves specificity of action between cell types and whether interacting co-factors or more wide-scale changes in chromatin state are involved.

We have previously shown that the developing Drosophila optic lobe as similar to the mammalian cerebral cortex in the way in which neural stem cells ("neuroblasts") proliferate and differentiate. We and others have demonstrated that Notch signalling is vital to maintaining neural stem cell fate and establishing the fate of neurons. We have also developed a powerful new technique, Targeted DamID, to investigate the DNA binding profiles of proteins in specific cell types at specific times during development. We have used this technique to identify the regions of the genome bound by Notch in both neuroepithelial cell and in neuroblasts. We found that the regions of the genome bound by Notch are highly disparate between these two clonal cell types. Our proposal seeks to identify the factors that control the specificity of Notch signalling in neural stem cells and neurons.

Our proposed research will use a combination of the Targeted DamID technique in conjunction with standard Drosophila developmental biology tools and bioinformatics to identify the proteins that interact with Notch at promoters and enhancers during neural development. We will also use Targeted DamID to profile the way in which chromatin states change during neural differentiation in order to determine whether these influence the specificity of Notch binding. Finally, we will look at a potentially new form of genetic regulation, namely the role of the position of gene loci within chromosome territories in the interphase nucleus, and in particular at the proposed role of the nuclear lamina in silencing gene expression and influencing cellular competence.

Expected beneficiaries of the research detailed in this proposal include the medical and pharmaceutical industries, businesses recruiting graduate-level staff, and the general public and schools through our work in science communication.

The Notch signalling pathway is involved in many stages of development and is aberrantly activated in many forms of cancer, including brain tumours such as glioblastoma. Our proposal will identify interacting co-factors of Notch that maintain neural stem cell fate, and it is likely that these factors may contribute to de-differentiation and cancer development. The proteins and pathways that we uncover in our proposal will be potential targets for drug development and discovery. In the long-term (>10 years) our results could benefit health care and quality of life. The technology used in this grant, which we have termed Targeted DamID, may benefit human health when combined with patient-specific induced-pluripotent stem cells (iPS; time-frame 5-10 years).

The research proposal involves training that will ultimately prepare our staff for highly skilled employment in the private and public sectors. Former members of the lab have progressed to successful careers in the biotechnology industry, in consulting and in publishing, as well as in the medical, charitable and public sectors. The skills obtained in our lab are likely to produce individuals who will have a have a major impact on both the economy and the well-being of society.

Finally, our group is heavily involved in science communication and outreach activities, both in schools and to the general public. Past examples include public lectures, radio interviews, University open days, school careers fairs, and, as of this year, involvement in the newly opened Cambridge Science Centre. We will continue to promote greater awareness of science within the community, encourage more primary and secondary school students to consider science as a career. In particular, we aim to encourage young girls and women to participate in STEM subjects. We will disseminate our research goals to the widest possible audience.