The Generation of Positional Identity in Mesendoderm; Mechanism(s) of Lineage Specification in Vertebrate Development

During early embryonic development, the cells of the future gut move along the midline of the embryo signalling to the nervous system. To understand the molecular instructions driving the differentiation of these cells it is necessary to isolate them from the complex and changing environments in the embryo. Being able to break down this in vivo process of lineage specification is also essential for the generation of gut associated cell types and organs from human ES cells. In this proposal, we utilise genetically modified mouse ES cells that report on differentiation towards the embryonic gut in real time. We have used this system to generate monolayer conditions for ES cell differentiation toward embryonic gut. Our ability to differentiate these cells attached to plastic means that we can easily change media components as well as following differentiation visually. This proposal exploits this system alongside embryonic models to tease apart the stages of differentiation. It contains a program of work that is both focused on identifying new determinants and understanding how these determinants drive differentiation. This proposal will provide basic mechanistic insight into developmental biology and define conditions for the efficient differentiation of ES cells towards organs like the liver and pancreas.

At the beginning of gastrulation the major embryonic organising centre generates mesendoderm that migrates along the midline to pattern the anterior-posterior neural axis and form the gut-associated organs. Segregating cell migration and the complex local microenvironments it generates from specific molecular mechanism(s) is difficult. The intimate relationship between mesendoderm migration and differentiation also complicates embryonic stem (ES) differentiation to foregut-derived organs such as liver and pancreas, with the majority of these protocols relying on inconvenient suspension culture systems. In this proposal I address the underlying molecular basis for lineage specification in the anterior mesendoderm (anterior definitive endoderm (ADE)) using a novel in vitro system. This system makes use of genetically modified murine ES cells, including a fluorescent reporter of ADE, HexRS (a dsred reporter driven by the Hex locus). We have used this reporter to identify intermediates in the process of foregut specification and established a defined serum free system in which we can monitor ADE specification in adherent monolayer culture. In the process of establishing this in vitro model we also uncovered novel requirements for FGF signalling in the specification of ADE. In this fellowship I exploit sophisticated genetic technologies in mouse ES cells alongside our defined differentiation protocols to determine the molecular basis for anterior specification within the mesendoderm. We will further characterise ES cell derived ADE and explore transcription factor and signalling requirements for it‘s induction. We will investigate the means by which FGF signalling drives ADE specification and potential interactions with the core pathways required for mesendoderm; Nodal and Wnt. We will also follow up our recent findings suggesting that the homeodomain proteins Oct4 and Hex regulate Nodal signalling and that the Groucho related/TLE co-repressors are key regulatory nodes for mesendoderm specification. We will explore the mechanism by which these transcription factors and their downstream networks regulate lineage specification and signalling in our defined system. We will also use our in vitro system to identify novel regulators of ADE and then return to embryonic models to validate function. This work builds on our already existing transcriptional profile for these ES derived populations and exploits our ability to move rapidly between mouse ES cells, Xenopus and mouse models. In this way we hope to rapidly advance our understanding of the molecular basis for mammalian development and provide markers and protocols that will aid in the efficient differentiation of human ES cells.