A comprehensive spatial analysis of active gene regulatory elements in the neural border during early development

The "BIG" PICTURE
For a fertilised egg to develop into a multicellular organism such as a tadpole or human many changes have to happen. These changes have to occur at defined times in development and in specific places such that, for example, cells that will form the brain will do so at the right time and in the right place. For these changes to occur the cells in the embryo have to be able to know where and when to begin to specialize into that particular cell type. We are studying how cells can do this. We are looking at this question using an important cell type in the embryo called the Neural Crest and the placodes. Neural Crest (NC) cells contribute to the formation of many organs in the body such as parts of the nervous system, the cartilage and muscle of the face, the heart and the pigment cells in the skin. They are therefore of importance for normal development and errors in their development are the cause of many birth defects ie. Treacher-Collins syndrome. Additionally, NC cell types are also implicated in cancer. The placodes give rise to the sensory organs in our head such as the eye, nose and ears

Enhancers are areas of DNA that control the expression of genes over large distances. Increasingly focus is not just on gene function with respect to development and differentiation but on the active DNA regions and enhancers regulating gene expression.

THE QUESTION:
What regulatory regions are required for the correct spatial expression of the early NC and Placode specific genes during development. We will determine this at the level of the whole genome to identify all active regions and enhancers controlling NC and placode development as well as enhancers involved in defining the epidermis and neural ectoderm.

WHY IS THIS IMPORTANT:
Understanding how cells become different from each other during development is a fundamental question which is important for researchers working on development, cancer, regeneration, evolution and stem cell biology. In the long term the insights gained by these studies could be applied to human stem cells and potentially be used therapeutically.

EXPERIMENTAL MODEL SYSTEM
Xenopus embryos are a classic, accessible model system for experimental embryology. They are easy to manipulate to assess gene function and for tissue dissection and genomics analysis. With resepect to these studies on early development Xenopus has advantages over zebrafish and chick as the early NC and placodes are more clearly defined and characterised and so are easier to dissect and process quickly.

As the embryo develops the ectoderm becomes subdivided into the ectoderm, neuroectoderm and neural border which further subdivides into the pre-placodal ectoderm (PPE) and the neural crest (NC). The gene regulatory networks that lead to the differentiation of these tissues are increasingly being defined. However, the regulatory landscape that defines these tissues spatially and leads to correct development has not been characterised. New technologies such as ATAC-seq are making the identification of cis-regulatory elements (CREs) over the whole genome possible. In this project we will use Xenopus as a model system to carry out ATAC-seq in order to spatially identify the regulatory landscape and thus potential CREs across the different ectodermal tissues. In pilot experiments we have carried out ATAC-seq of animal cap tissue induced to form NC by injection of Wnt/noggin or dexamethasone inducible Pax3GR and ZIc1GR constructs. Computational analysis will identify important CREs associated with the NC. Next, we will carry out ATAC-seq and correlate this with expressed genes. This will be done on animal cap tissue induced to form PPE and on dissected embryonic tissue corresponding to the ectoderm, PPE and NC of the neural border and neuroectoderm. Bioinformatic analysis of active regions across these tissues including differential comparisons with the AC data will identify important sequences in the genome that will be tested for their spatio-temporal activity using transgenic technology. CRISPR/Cas9 approaches will examine selected endogenous CREs. Our data will be shared with the scientific community by incorporating it into EctoMap which has previously identified the genes expressed in these tissues by RNA transcriptomics.

INTRODUCTION:
Determination of cell lineages and identifying simple effective ways to generate specific cells types is a major area of research. This is important with respect to regenerative medicine, organ replacement and gene therapy.

In recent years identification of active enhancers and promoters can be done at the whole genome level. Using such techniques we can identify crucial sequences in the genome associated with specific gene activation leading to specific tissue formation. Using these technologies we will overlay active enhancers and promoters to a molecular atlas currently showing temporal and spatial transcriptomes in the ectoderm during neurulation.

BASIC SCIENCE:
This is a basic science project; it addresses fundamental questions about cell lineage and cell specification. The results will be important for stem cell biology and therefore findings will be relevant to human and animal health. The project is most likely to have longer-term impacts in the biomedical and health science areas.

IMPACT FOR HUMAN (AND ANIMAL) HEALTH AND APPLIED TRANSLATIONAL RESEARCH:
The correct temporal and spatial specification of the NC and other tissues of the ectoderm during neurulation is of fundamental importance for cellular differentiation programmes in humans and animals. Deregulation of this process contributes to developmental abnormalities and leads to diseases in the adult. Understanding the molecular mechanisms that regulate the specificity of the transcriptional and cellular response in different cells will underpin the development of strategies aimed at the use of stem cell-based therapies in regenerative medicine and in developing therapies for specific cancers.

IMPACT ON GENERATION OF A SCIENTIFICALLY LITERATE WORKFORCE:
This project will help train the next generation of biomedical researchers by supporting the research career of a postdoctoral researcher, by training a technical assistant and by involving a bioinformatician in cutting edge genomics techniques. Indirect benefits will come from their contributions to a research-led environment for teaching of postgraduate and undergraduate students. The PIs laboratory has an excellent training record and researchers have successfully obtained positions in academia or been appointed in industry (e.g. University of Toronto, IMB Mainz, Oxford).

IMPACT ON WIDER PUBLIC:
Members of the public are interested in scientific progress, particularly when relevant to health. Increased understanding of gene regulatory mechanisms during neural crest development will be important and relevant for regenerative medicine and stem cell science. New information gathered in this project will be disseminated to a general audience in an accessible form using web based tools.

IMPACT ON PHARMA AND BIOTECH INDUSTRY:
The project will increase our knowledge base especially with respect to neurocristopathies. This is a prerequisite to design more sophisticated drugs targeting specific pathways in specific contexts.

CONCLUSION:
This study will directly and indirectly contribute to both improved health and economic wealth.