A novel pathway of cell cycle activation in root formative divisions

We all learn at school that reproduction is a fundamental property of life, and the same is true of the cells from which organisms are built. All cells arise from a parent cell by division. Usually a cell divides to make two cells of the same type, increasing the population size. We refer to this as a "proliferative division", and is easy to think about in a tumour where there are many identical cells. However, when a complex organism is developing, for example as an embryo grows from a single initial fertilised egg cell, new cell types have to be produced. These arise from a special kind of division, known as a "formative division". In this case one daughter cell (or sometimes both daughter cells), are different from their parent cell and have a new identity. This process of formative division is also essential in the maintenance and function of stem cells- these are cells in the body that can undergo formative divisions to generate another stem cell and a new cell of different identity.

We know a lot about the molecules that regulate the processes of proliferative cell divisions, in part because of their importance in cancer. The molecular and cellular aspects of the process of cell division are known as the "cell cycle". However we know very little about what different mechanisms operate in the cell cycle of formative divisions or indeed even if there are different mechanisms. We have been studying a particular type of formative division in the root of a plant called Arabidopsis. We study plants because the cells do not move around, and the identity of a cell is easy to establish because it is determined by its position in the root. We use Arabidopsis because it grows rapidly, there is a great wealth of earlier studies to draw on, and there are a lot of resources that make the research fast and relatively cheaper. The roots are also thin and transparent so we can study living roots using a confocal microscope that allows us to visualise the action of proteins and genes as cells divide. The root consist of concentric layers of cells, each layer with a different identity, wrapped around a central core that conducts water and nutrients.

The particular division we have been studying involves the formation of two of these layers, the cortex and endodermis from a single layer of root ground tissue. The endodermis is a crucial tissue because it forms an impermeable layer controlling the movement of water and ions into the central conducting tissue. Without the endodermis the root cannot grow and function properly. This is exactly what happens in mutants of a gene called SHORT-ROOT. In this mutant, the formative divisions do not take place. In collaboration with a leading US group, we showed in a paper published last year in the journal "Nature" that SHORT-ROOT directly controls expression of a cell cycle regulating gene called cyclin D6, which is only switched on in cells carrying out the formative division. If cyclin D6 is missing, the formative division is not properly controlled. Cyclins work together with a partner protein called a cyclin-dependent kinase. This has now been identified, and mutants in this gene also have a defect in the formative division, confirming it is also involved. These two proteins do not normally work together, so we believe that we have identified a new mechanism by which the cell cycle is switched on in formative divisions, which also involves a third candidate we have identified. In this project, we will analyse this new mechanism in detail. We think it involves three feedback loops that all activate each other, so to understand them, we will use mathematical modelling to predict the effect of making changes to the system and test these predictions. We will carry out the work in collaboration with world-leading groups in the US, Holland and the UK, bringing exceptional expertise to bear on this problem.

Cell divisions fall into two categories. Proliferative divisions increase the number of cells of a given type, but formative divisions result in the production of two cells with new identities. Whilst we know a lot about control of the cell cycle in general, we know very little about the mechanisms that underpin these different types of division, or indeed whether there are differences in the cell cycle mechanisms that operate. These processes are easy to study in plants due to the relationship between spatial position and cell identity.

Here we present evidence of new cell cycle regulatory mechanisms in formative divisions of the Arabidopsis root ground tissue and propose to analyse and model this novel mechanism for cell cycle control. These formative divisions that create the cortex and endodermis depend on regulation by the SHORTROOT (SHR)/ SCARECROW (SCR) transcription factors. We propose here that the novel cell cycle activation involves three interlocking positive feedback loops converging on phosphoryation of the RETINOBLASTOMA-protein homologue RBR via novel cyclin-CDK complexes. We have previously shown will Philip Benfey's group that a unique D-type cyclin CYCD6;1 acts in the formative division downstream of SHR (Nature 466:128-32; 2010) and controls the spatio-temporal pattern of division. Further work by the Scheres group has shown that RBR itself also binds and regulates SCR, limiting its activity and forming the first positive feedback loop. The applicant's lab has also identified a phenotype associated with the CDK bound by CYCD6;1 and a further candidate cyclin also involved. The project proposes to analyse these regulatory mechanisms combining in vitro and in vivo approaches with mathematical modelling of the system allowing predictions to be made and tested. The work will be in collaboration with the Scheres, Benfey and Bennett groups, three of the world-leaders in studying root development.

This project will define a new pathway controlling cell division that operates specifically when new cell types are being formed. This new knowledge will have a broad impact across the biological sciences, improving our understanding of how biological processes operate in general and how pattern is generated during the development of organisms. It will also show for the first time that different types of regulatory mechanism operate in different types of cell division.
Immediate beneficiaries will be academic biologists worldwide who seek to understand how cell division is controlled and how complex organisms develop, broad questions of fundamental and general interest. The significance of the work is therefore likely to generate further interest in the media and wider public. Precursor work led to publicity and media interviews for the applicant illustrating its wider impact. The general public will therefore benefit through an enrichment of their understanding of how biological processes function. Education may well benefit since this mechanism is likely to become included in undergraduate textbooks and courses as an illustration of formative divisions.

In the longer term, business and industry will benefit from an increased understanding of the processes that are important in generating biological form. Such knowledge will be required in order to realise long term goals of engineering crops for improved yield, biomass and resistance to stress (for example through improvements in root structure or architecture). This type of knowledge will help to underpin the knowledge based bioeconomy (KBBE), goals for both UK government and the European Commission. Furthermore, this project places the UK at the centre of a collaboration involving top European and US labs, enhancing UK global prestige and scientific standing and indirectly contributing to the UK position as a leading country for research and development (R&D) and hence helping to attract associated investment.

The project will involve the researchers in state-of-the-art techniques, leading to highly skilled and trained individuals. Since the project integrates both biology and systems modeling approaches, the biologists will benefit from practical knowledge of how mathematics can be used in biological research and the mathematician in understanding practical applications of modeling. These types of integrated skill sets acquired by the researchers are essential for a highly trained and flexible workforce that will be required to deliver the KBBE and contributing to future economic development and associated societal benefits. Such individuals also enhance the skill and knowledge base available to academic and business research and further contribute to the UK's attractiveness for outside investment in R&D.