Integrating developmental pathways and chromatin structure during lineage specification

In order for the fertilized egg to develop into a live-borne animal, genes that control the regenerative capacity, identity and fate of cells must be switched on and off at the right time and place. Changes in the way that the DNA sequence is packaged up with proteins, to form a structure called chromatin, are important in this regulation of gene expression. However, to date this has mainly been studied in artificial cell culture systems and little is known about the changes in chromatin structure that happen at specific genes in a situation that is more relevant to the development of the embryo. We propose to use a newly developed cell culture system that enables mouse embryonic stem cells to be directed to undergo development towards cell types that usually go on to form muscle and bone (mesoderm) or gut, lung, liver and pancreas (endoderm). With this system we can produce large quantities of cells that closely resemble their equivalents in an embryo and challenge these cells with specific chemical signals that are known to be important for embryonic development. This system will be used to study how chromatin structure is changed both globally and at a particular set of genes, the Hox genes, which are key regulators of development. Our global analysis will help us to understand the way in which cells are progressively restricted to the mesoderm and endoderm lineages, while at the Hox cluster in particular we will be able to ask specific questions about how this happens. This work will help to better understand how stem cells can be used to target organs derived from these cell types in regenerative medicine.

The focus of this proposal is to investigate changes in chromatin structure during the initial differentiation events of embryonic development - gastrulation - and the development of defined cell populations therein. There are insufficient cells in the early embryo to investigate chromatin structure biochemically, therefore, we plan to exploit a system for the defined in vitro differentiation of mouse embryonic stem cells that mimics lineage specification through a primitive streak-like stage. Using this system, we will couple genome-wide analysis of expression and chromatin structure, to a focused study of a single chromosomal cluster (Hox). These studies are only made possible by bringing together a unique combination of expertise in chromatin and developmental biology. Since polycomb repressive complexes are known to be important chromatin regulators during gastrulation, we will use Chromatin Immune precipitation (ChIP) to assess changes in the histone marks laid down by polycomb complexes, on both a genome-wide scale and in more detail at Hox loci, as pluripotent cells differentiate into defined populations of primitive streak like, mesodermal and endodermal cells and in response to signals such as Nodal and FGF. Previous studies have only analysed the modulation of polycomb marks in non-physiological differentiation systems. We will test the affect of mutations and knock-downs of specific polycomb components on the ability to respond to these pathways. This will be coupled with engineering of the Hoxb locus, to allow for the isolation of pure populations of cells expressing either early or both early and late members of the cluster as material for ChIP and a resource for single-cell assays examining higher-order chromatin compaction using fluorescence in situ hybridization. This may help in the understanding of the mechanism of temporal colinearity.

Lineage specification is intricately tied to embryonic development. It is difficult to imagine generating insulin responsive Beta-cells, dopimenergic neurons or hepatocytes in the absence of the complex morphogenetic movements of embryonic development. One of the focuses of this application is the further development of technologies that attempt to recapitulate normal differentiation from ES cells to intermediate developmental stages. Here we use these technologies to take systems level approaches impossible in an embryo. As a result our work benefits the broader community of developmental and systems biologists who are looking for an experimental model that is both amenable to this level of analysis and yet still recapitulates normal developmental processes. It also has implications for regenerative medicine, where work with pluripotent stem cells has demonstrated limited success in producing differentiated functional cell types. The Brickman group has already made two major breakthroughs in the manipulation of embryo derived stem cells; the establishment of an in vitro system for controlled specification of anterior mesendoderm, endoderm and mesoderm from embryonic stem (ES) cells, and the development of a cell culture system for endodermal progenitors. Both technologies are the basis for an International Patent Application No. PCT/GB2008/0028675 and the ability to cultivate and expand endodermal progenitors in particular, received significant attention in the media (see Semb, (2008) Cell Stem Cell for comment). These technologies reduce the need for animals in research and generates the volumes of defined materials necessary for biochemical and systems level approaches to developmental biology. Moreover, coupling this system to modern genomic and deep-sequencing technologies has the potential to generate a road map for the specification of mesoderm and endoderm and different cell types within these lineages. In addition to the community at large, an obvious beneficiary of this proposal is the biotechnology sector. Both the construction of genome level maps of differentiation and regulatory interactions, alongside improvements to directed differentiation that will come as a result of this project has a clear application to the differentiation of human pluripotent cells. The initial IP generated by Dr. Brickman was patented by the University and is being developed by the Edinburgh University's technology transfer office. Dr. Brickman also participates in the development of technology for the UK industrial and medical communities through, 'Stem Cells for Safer Medicine (SC4SM).' This root exists for propagating technology with in this proposal. To communicate to the community as a whole, both Dr. Brickman and Prof. Bickmore participate in and organize a significant number of scientific meetings. Dr. Brickman works with the Scottish Stem Cell Network to organize their, 'Stem Cells in Embryos,' series with Prof. Kate Storey and co-organizes a biannual course in Latin America, focused on transferring ES cell technologies to laboratories where animal work is impossible. Prof. Bickmore is an organizer of the EMBO Nuclear Organisation biannual series of meetings. Communication and outreach to lay audiences is a priority for both the Institute for Stem Cell Research/ MRC Centre for Regenerative Medicine and the MRC Human genetics Unit/Institute of Genetics and Molecular Medicine. They engage staff in communicating and debating their work, through hands-on activities, exhibitions, print, audiovisual and new media, debates, open-door days and the Edinburgh International Science Festival. Prof Bickmore also tours Scottish schools as part of the Royal Society of Edinburgh's RSE@schools outreach programme, and on a wider scale, she is chair of the the EMBO Science and SocietyCommittee. Through these activities we target and interact with; students, teachers, the media, decision makers and the general public.