Investigating cellular plasticity in the avian primitive streak

THE 'BIG' PICTURE: A fascinating question in biology asks how multicellular organisms arise from a single cell: the fertilized egg. During embryogenesis naïve and still plastic progenitor cells interact with each other, first to form different embryonic layers and to organize the main body axes, and later to build specialized organs with highly differentiated cell types and functions. The cells in an embryo coordinate these complex events by communicating with each other using molecular signalling mechanisms. Cell-cell communication is important throughout the life of an organism, for example for repair after injury or for growth and remodelling. A good example for this is skeletal muscle, a tissue that can regenerate and rebuild itself (after injury or after exercise). This is possible, because there are specialized cells in muscle, but also in other tissues, which can divide when stimulated and make more differentiated cells when needed. These specialized cells are tissue-resident stem cells and they respond to the same communication signals that act in the embryo. We need to understand the detailed intricacies of these signals, as they can have different effects depending on the context in which they act. Defects in signalling mechanisms can be detrimental to an embryo, but they can also lead to diseases in adults such as cancer, when cells "mis-behave" and ignore the signals or respond incorrectly and grow uncontrollably as a result.

EXPERIMENTAL MODEL SYSTEM: We have used the early chick embryo, which is very similar in its morphology to early human embryos, and investigated the effects of two important cell-cell signaling pathways. From these studies we know that BMP and WNT signals have different effects in different types of early progenitor cells, depending on the stage of development. We also know that these pathways act via a common transcriptional regulator (SMAD), which can switch other genes 'on' or 'off'. In a "HH stage 3" embryo the signals change how cells migrate, but in a "HH stage 4" embryo the SAME signals have no effect on migration but instead alter the fate of the cells; thus at this later stage the cells respond by changing what they will become. We will use state-of-the-art molecular protocols to identify the genes that are switched on or off by the SMAD-switch in these cell populations. We have experience with these methods in the chick embryo, an accessible experimental system and we have assembled a highly skilled team of researchers to execute this programme of research.

WHY IS THIS IMPORTANT? BMP and Wnt signaling are widely used cell communication signals that act in many tissues and organs. We know many of the components of the pathways, but we still do not understand why cellular responses to the (apparently) same trigger vary depending on the context. We now have a well-defined system where the cellular response to the same signals has been characterized and is quite divergent: EITHER cell migration is affected OR cell specification is affected. This gives us the unique opportunity to identify which genes have been switched on or off in the responding cells and provide a deeper mechanistic insight at the molecular level. This is needed in order to fully understand how specialised cells form and how they build functional organs. This is not only relevant in developing embryos, if things go wrong the embryo will not survive or become malformed, but is also relevant for stem cell science and tissue engineering, emerging fields of increasing importance and with significant future potential for medicine and health.

SPECIFIC OBJECTIVES: We will capitalize on our recent observations and use our well established model system to (1) identify genes that are differentially expressed in response to BMP and Wnt signaling, and to (2) test the expression and function of these genes in both cell migration and cell specification/differentiation.

In amniote embryos, including chick and mouse, mesoderm progenitor cells ingress at the primitive streak during gastrulation. The cells subsequently migrate on defined trajectories towards their final destination, for example the cranial, cardiac, paraxial or lateral mesoderm. The type of mesoderm that is generated depends on the stage and on the location of ingression along the primitive streak. Experiments in vertebrate embryos showed that early mesoderm cells are multi-potent and primitive streak cells are plastic and able to adapt to a new environment where they respond to extrinsic cues.

We identified the BMP/Smad and Wnt/GSK3b signals as extrinsic cues that control the migration of prospective cardiac cells. We showed that BMP and Wnt pathways converge on a common effector: the transcription factor Smad1. BMP mediates the activation of Smad1 and Wnt facilitates its stabilisation by inhibiting GSK3b kinase (Song, McColl et al., PNAS 2014). In mesoderm progenitors, emerging at HH3 from the primitive streak, prolonged Smad1 activity alters cell migration behaviour causing prospective cardiac cells to change their exit trajectory and to migrate in a wider, abnormal migration pattern. We also found that paraxial mesoderm progenitors, which ingress through the primitive streak slightly later at mid-gastrulation (HH4), respond differently to BMP signaling. They do not alter their migration behaviour, but their cell fate is affected and they become lateralized.

We now need to use a systematic approach to determine the context dependent targets of BMP/Smad1 and Wnt/GSK3b signalling, in order to reveal mechanisms by which the same signals induce distinct responses in different populations of mesoderm cells. Deep sequencing will identify differentially expressed genes, which will be validated and their function examined in accessible chick embryos. This will uncover their roles in controlling cell behaviour, in particular cell migration and cell fate choice.

INTRODUCTION: This is a basic science project; it addresses fundamental questions about molecular signaling mechanisms that control embryonic development. Similar mechanisms will be important for stem cells and discoveries made are therefore relevant for human and animal health. The project is most likely to have longer-term impacts in the biomedical and health science areas.

HUMAN (AND ANIMAL) HEALTH AND APPLIED RESEARCH: BMP and Wnt signaling pathways are major biological mechanism for cell-to-cell communication in humans and animals. Deregulated signaling contributes to developmental abnormalities, for example cardia bifida, and diseases in the adult such as colorectal cancer. Understanding the molecular mechanisms that regulate the specificity of the transcriptional and cellular response in different cells is of fundamental importance in order to develop strategies aimed at the use of stem cell-based therapies in regenerative medicine. This includes, but is not limited to cardiomyocyte (heart muscle) regeneration.

GENERATION OF A SCIENTIFICALLY LITERATE WORKFORCE: This project will train the next generation of biomedical researchers by directly supporting the academic research career of Dr McColl and by training an RA in chick embryology. Indirect benefits will come from the team's contributions to a research-led environment for teaching of postgraduate and undergraduate students, who enter many science related careers.

THE WIDER PUBLIC: Members of the public are interested in scientific progress and embryo development is a fascinating topic that people can easily relate to. Understanding how genes regulate and drive this process has become easier to tackle with the recent advances in genomics technologies. This project will contribute discoveries towards this intriguing issue by focusing on two highly relevant signaling pathways that govern discrete cellular responses in the very early embryo.

PHARMA AND BIOTECH INDUSTRY: Context-specific signaling mechanisms are important for drug development and longer-term beneficiaries will be biotech and pharmaceutical industry. The project will increase our knowledge base, a prerequisite to design more sophisticated drugs targeting specific pathways in specific contexts. Detailed insights into the control of cellular behaviour will also benefit regenerative medicine and tissue engineering.

OVERALL this study will contribute to health improvements and to economic wealth generation in the UK and beyond, both directly and indirectly.