Novel approaches to define tissue fusion mechanisms in embryonic development

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Disruptions to how tissues fuse together during human development is a common cause of birth defects, affecting approximately 1 in 500 people in the UK. This research has been prompted because despite our best efforts, most patients born with problems such as cleft palate, spina bifida, and heart defects still don't have the genetic cause of their disorder identified. This often impacts genetic counselling and efforts towards prevention. We also remain unsure what impacts maternal environment have on these conditions (e.g. illness, vitamin deficiency, or substance abuse). I aim to address these by performing transformative research to reveal the key genes, cell behaviours, and molecular systems required for normal tissue fusion, and provide a step-change in our knowledge of how these can be perturbed.To enhance our understanding of fusion, we require experimentally versatile and appropriate model systems. I will focus on the causes of ocular coloboma, the leading inherited cause of blindness arising from a fusion defect of optic fissure closure (OFC) in the eye in the first 7 weeks of pregnancy. This defect leaves a persistent gap in the retina and optic nerve which cannot be cured or repaired. I have established the chick eye as a powerful and tractable new model for fusion because of a unique combination of features: (i) it closely resembles OFC in humans; (ii) experiments can be performed inside the egg; and (iii) chick eyes are sufficiently large to accurately dissect fusing tissues for further experimentation. At Roslin Institute we have also developed unique transgenic chickens with fluorescent cells whose behaviours I can observe throughout OFC, and from which I can selectively isolate specific cells to reveal their unique gene expression profiles. This work will combine these new technologies with other cutting-edge techniques in DNA sequencing, live-cell imaging, gene-network analysis and cell behaviour modelling to generate the most robust and accurate framework of OFC and tissue fusion information available to date.The first aim of this project is to reveal gene expression levels and molecular signatures for the cells directly taking part in the OFC fusion process. I will also determine the changes in morphology, movement and organisation of these cells using mathematical and computer simulations, and integrate this with my molecular data. Next, by selectively ablating OFC-specific genes using gene-editing techniques, I will determine the consequence of their loss or dysregulation to learn more about the genetic drivers and modifiers for specific cell behaviours during fusion. Understanding the function of the Netrin-1 gene is a key part of our work, as I have recently shown that this is an essential factor for normal OFC in diverse species, and that it is required for other developmental fusion contexts (eye, ear and palate). I will then go on to reveal how Netrin-1 and other fusion-specific genes are regulated by genome-wide networks, allowing us to understand better the biological consequences of mutations identified in human patients, and to accurately predict new disease-causing candidate genes and the effect of non-genetic factors on the regulation of gene expression in fusion.This is a powerful combination of tools to transform fusion biology. It will provide valuable information for how specific challenges during embryogenesis can disrupt genetic and cellular programmes, directly or indirectly leading to developmental fusion defects. The information I reveal will help clinicians to uncover new causative mutations and provide evidence to support genetic counselling in affected families. In the longer term, my system will reveal how the maternal environment can influence the causes of fusion defects, and help define strategies to reduce their incidence. Lastly, my data will reveal how gene function links to cell behaviour, providing insight for a broad range of biological contexts in health and disease.