Resolving the basis of phenotypically variable hereditary abnormalities of eye formation

Our eyes start out as outpocketings of brain tissue during early embryonic development. The cells destined to form the eyes originate within the neural plate, the precursor of the central nervous system. As the neural plate folds up to form the brain, the eye-forming cells bulge out laterally forming optic cups, the structures that later differentiate as eyes. Each optic cup undergoes shape changes and tissue fusion closes a gap (the optic fissure) present on one side of the cup, leading to formation of the intact globe shaped eye. The complex orchestration of cell movements that form the eyes is an example of morphogenesis - the process by which embryonic cells form into tissues and organs.

Many of the genes that regulate the formation of the eye have yet to be identified. One reason that this lack of knowledge needs to be addressed is that congenital malformations of the eye, such as anophthalmia (lack of eyes), microphthalmia (small eyes) and coloboma (a failure in optic fissure fusion), being compatible with life and reproduction, are relatively common in the human population. As these are congenital defects, this means that eye problems are present from birth. While anophthalmic patients are blind, microphthalmic and coloboma patients can have severe visual impairment. For instance, colobomas are a common cause of visual problems, can cause retinal detachment and cataracts, and often lead to blindness.

In this project, we will use zebrafish embryos to identify genes and genetic interactions important for eye formation. Zebrafish embryos are small, transparent and develop externally, facilitating the study of normal development and disease in the intact animal. Together with their amenability to genetic analysis, these features make fish embryos an excellent model system to study eye formation in normal and pathological conditions. Indeed, we can visualise all of the cells in the developing eye in living embryos both in healthy fish and in fish carrying one or more genetic mutations that compromise eye formation. Consequently, we can use research in fish both to identify those genes needed for eye formation and to understand the mechanisms by which such genes build functional eyes.

Although some congenital abnormalities of eye formation are due to mutations in single genes, we suspect that in many cases, such defects are due to disruption of two or more genes. Consequently, in this project, we will use novel, powerful approaches that allow us to systematically analyse the consequences of simultaneous disrupted function of two or more genes that are candidates for causing eye defects when non-functional. To facilitate this research, our current MRC funding has enabled us to develop lines of fish carrying mutations that make the fish more likely to show eye phenoytpes when additional genes are disrupted. We will remove function of one or more additional genes in these "sensitised" fish lines to identify new genes and genetic interactions important for eye formation. We will also study the function of several genes that, when disrupted, give very similar eye defects both in fish and in humans as although we know these genes to be important, we do not understand how they function. Finally, we will study why individuals carrying the same genetic mutations can show quite different eye phenotypes. To facilitate this, we have lines of fish in which we can perform genetic or environmental perturbations that affect the severity of the eye defects.

Overall, our research will help to bridge the gap between the highest quality research in model systems and human disease phenotypes. We will improve our understanding of normal eye development and will use new zebrafish models of human eye diseases to gain further insights into the causes of hereditary ocular malformations. Our research also has the potential to be of great value in the diagnosis of congenital abnormalities of eye formation.

Although abnormalities of eye formation are a major cause of blindness, the genetic bases of such phenotypes are poorly understood. The list of genes implicated in microphthalmia, anophthalmia and coloboma (MAC) is growing but only accounts for a minority of cases. This proposal will identify new genes involved in eye formation and will elucidate why MAC phenotypes show variable penetrance. Our proposal builds on ongoing MRC Programme Grant research which has enabled us to establish novel zebrafish models in which to explore phenotypically variable MAC phenotypes.
We will identify novel genetic interactions contributing to MAC phenotypes. Due to the robustness of eye formation, MAC phenotypes may only be revealed when embryos carry more than one genetic mutation. Consequently, novel geneticially sensitised zebrafish lines showing low penetrance phenotypes will be subject to Crispr/Cas9 gene targetting to assess the consequences of systematically disrupting hundreds of potential genetic interactions upon the penetrance and expressivity of MAC phenotypes. We also aim to understand the molecular and tissue level functions of selected genes that are critical in the expression of MAC phenotypes. We will clarify the conserved roles for Yap and Mab21l1/2 proteins in eye morphogenesis using genome wide screens in worms to complement novel transgenic approaches in zebrafish. Finally, we will elucidate the epistatic interactions which affect severity of MAC phenotypes. We will exploit novel fish models in which we can vary the penetrance of MAC phenotypes to study why genetic lesions can result in different outcomes between individuals and even between left and right eyes.

This research will both inform and be informed by large-scale human genomics studies, and will deliver a comprehensive analysis of genetic interactions that build the eyes and will provide a wealth of information to inform diagnosis and understanding of debilitating abnormalities of eye formation.

Who will benefit from this research? Our direct academic and clinical collaborators and wider research communities will benefit from our study of the genetic basis of eye development in normal and disease conditions. Our work also addresses fundamental cell-biological processes of tissue organisation as well as genetic and developmental compensatory mechanisms, thus, reaching communities beyond our direct field of research. Furthermore, the public sector (NHS), including human geneticists, consultant ophthalmologists and genetic councillors and their patients benefit from the identification of new genetic markers to help diagnose patient cohorts. Our work is of relevance to charities working with families affected by congenital eye disorders (e.g. Fight for Sight and EURODIS). The general public benefits from our outreach into the community through web-postings, public talks, school visits and the hosting of school-age children.

How will they benefit from this research? Our characterisation of genetic, molecular and cell-biological processes underlying eye development in normal and abnormal conditions will generate a wealth of information and tools that we will share with academic and clinical researchers. Genes that, when disrupted, cause harmful eye phenotypes in zebrafish will be identified in our novel genetic screens and this data can be used to predict which genes are causative of similar phenotypes in humans. Consequently, human geneticists, clinical researchers and practitioners will use our data to inform identification of genetic lesions in their patient cohorts. In accordance with MRC's strategic objective on Genetics and Disease, specific genes will be linked with disease allowing for better diagnosis and targeting of therapies to individuals. In support of MRC's strategic objective on translation of research, our novel genetic and transgenic models of human eye pathologies may be of benefit to the pharmaceutical industry in order to design and perform targeted drug screens, potentially bringing the health impacts of fundamental research to society more quickly. Charities such as Fight for Sight, with which we work closely and from which we have received funding, will benefit from our research by facilitating identification of areas of research priorities. Others, such as EURORDIS, will gain an understanding of the genetic mechanisms underlying the eye disorders that affect the patients and their families within their remit. We regularly host A-level student placements in the lab, school visits and give presentations to University students from diverse disciplines. In our experience, school children and students are fascinated when introduced to scientific research and such exposure can direct career decisions [http://zebrafishucl.org/#outreach]. We share resources [http://zebrafishucl.org/#resources] and generate many beautiful images through our work [http://zebrafishucl.org/#images], often used for publicity and in museums [https://www.ucl.ac.uk/culture/#previous]. The public benefits from a better understanding of science and scientific terms and public engagement allows us to explain the crucial role animal models play in advancing our understanding of the causes of diseases and the development of treatments. Our laboratory attracts national and international researchers to undertake further training. We train our researchers to highest standards and equip them with transferrable skills always according to the RCUK Policy on Code of Conduct. In accordance with MRC's strategic objective on Capacity, we strengthen and sustain a skilled workforce and develop world-class research leaders. With this project we will maintain the UK at the forefront of research in the field, and by sharing knowledge and resources with our network of colleagues in Europe we contribute to the development of the ERA.