Morphogenesis and growth of the eye in health and disease

Although our eyes have a very different appearance to our brain, they originate as outpocketings of brain tissue during early embryonic development. The neuroepithelial tissue destined to form the eyes is initially a coherent group of cells within the neural plate, the precursor of the central nervous system. As the neural plate folds up to form the brain and spinal cord, the eyefield cells bulge out laterally to the left and right to form the two eyes. Each eye undergoes dramatic shape changes as it forms. These include a tissue fusion that closes a fissure present on one side of the eye (the choroid fissure), and leads to the formation of the intact globe of the eye. The complex orchestration of cell movements required to form the eyes is an example of tissue morphogenesis - the process by which embryonic cells form into tissues and organs.

The genes that regulate the specification and morphogenesis of the eye and the cell and tissue behaviours that accompany eye formation are poorly understood. One of the reasons that this lack of knowledge needs to be addressed is that congenital malformations of the eye, such as anophthalmia, microphthalmia and coloboma, are relatively common in humans and can cause severe visual impairment or more severe craniofacial phenotypes. For instance the failure of the choroid fissure to close results in eye colobomas that encompass a group of common eye defects affecting people of all ages, but especially young children. Colobomas are usually congenital conditions diagnosed by detection of a notch, gap, hole or fissure in any of the structures of the eye, including the cornea, retina and optic nerve. These pathologies are a common cause of visual problems, can cause retinal detachment and cataracts and often lead to blindness in affected patients.

In this project, we will use zebrafish embryos to identify genes important for eye formation and to characterise the cell behaviours that accompany various stages of this process. 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, highly sophisticated imaging techniques allow us to visualise all of the cells in the developing eye in the living zebrafish embryo. Coupled with this, we have generated a novel collection of families of fish that give rise to progeny exhibiting a variety of defects including absence of the eyes, abnormal morphogenesis and coloboma.

We will identify the genetic mutations in the families of fish with eye defects and this will give us insights into the genes and genetic pathways that regulate normal eye formation. The human versions of these genes are candidates for causing congenital eye abnormalities when mutated and so, with collaborators, we will screen patients with eye defects for mutations in the genes. As eye defects often arise as a consequence of defective function of more than one gene, one of our aims will be to look at the genetic interactions between pairs of genes implicated in eye formation. In parallel to the gene identification, we will characterise the cell and tissue movements that accompany eye formation to gain an understanding of the mechanistic bases of this complex process. As our understanding increases, we will then be able to use the acquired knowledge to resolve why the congenital eye defects arise when certain genes are not functioning correctly.

Overall, our research will help to bridge the gap between the highest quality basic research in model systems and human disease phenotypes. These analyses will enable us to establish new models for human eye diseases and will allow us to gain further insight into normal eye development and into the causes of hereditary ocular malformations.

This programme of research will elucidate the genetic pathways, cellular behaviours and developmental mechanisms underlying the specification and morphogenesis of the vertebrate eye. Our studies will help reveal the mechanistic bases of ocular phenotypes such as anophthalmia and coloboma. The knowledge we acquire from genetic analyses of mutant zebrafish will be used to screen for comparable genetic defects in patients with ocular malformations.

One goal will be to identify the genetic lesions in a novel collection of fish lines carrying mutations that alone, or in combination lead to defective eye specification or morphogenesis. We will use next generation sequencing approaches to identify the genetic lesions underlying the phenotypes. Once the mutated genes are identified, we will use a transcript counting approach which involves 3' capture of mRNAs in mutants to identify the changes in gene expression that precede the appearance of morphological phenotypes and/or sensitise embryos to eye defects.

We will resolve the cellular behaviours and developmental mechanisms that underlie optic vesicle evagination and choroid fissure closure in normal conditions and in embryos carrying genetic lesions. This will be achieved through the use of transgenes that label eye cells or subcellular structures in vivo, coupled with high-resolution multi-photon imaging of wildtype and mutant eyes.

Finally, we will use results from the studies above to help to identify genetic lesions that contribute to human ocular phenotypes. We will use an amplicon based approach to sequence exons from candidate genes in patients but may switch to exome sequencing should costs reduce significantly. In parallel, we will expand our collection of lines of fish that carry mutations in genes that render carriers susceptible to ocular phenotypes. These genetically sensitised lines will be used to assess the consequence of mutations in genes that are candidates for causing human disease.

Who will benefit from this research?

As well as scientists in the field, in the short to medium term, clinical researchers will benefit by using our data to screen patient cohorts for genetic mutations. Establishing novel fish models of human eye pathologies will potentially benefit pharmaceutical industry researchers who need suitable disease models for drug screening. Our work will be of relevance to charities such as Fight for Sight and EURORDIS working with families affected by congenital eye disorders. Our research has an impact on school students, university students from different disciplines and the general public. We are established as leaders in the field and thus contribute to maintain the UK at the forefront of this area of research. Through our collaborations with EU scientists, we contribute to the development of the European Research Area (ERA).

How will they benefit from this research?

Our work is predominantly fundamental research aimed at understanding developmental processes in normal and genetically abnormal conditions. Our research is cross-cutting, combining genetic and transgenic approaches with high resolution imaging and cell biology techniques. Clinical researchers will use our data to identify genetic mutations in cohorts of patients. Thus, and 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 will 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. Our proposed programme of research will contribute knowledge of genes that are candidates for human congenital eye diseases, a better understanding of the processes that these genes control, and novel animal models of human eye phenotypes. Charities such as Fight for Sight, with which we have established channels of communication, will benefit from our research in order to identify 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 students for placements in the lab, school visits and give presentations to students from diverse disciplines. In our experience, school children and students in non-scientific fields are fascinated when introduced to scientific research. Such exposure can direct career decisions and can inspire creativity in the students' own areas of research [1]. We write summaries of our findings for the public [2], and we generate many beautiful images through our work [3], which are used often for publicity and in museums [4]. The public benefits from a better understanding of science and scientific terms and a more intangible appreciation of the beauty of the developing embryo [5].

Our laboratory attracts national and international researchers to undertake further training with us. We train our researchers to high 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.

[1] www.ucl.ac.uk/zebrafish-group/outreach/
[2] www.ucl.ac.uk/zebrafish-group/outreach/summaries/index.php
[3] www.ucl.ac.uk/zebrafish-group/researchImages.php
[4] www.ucl.ac.uk/museums/zoology/exhibitions/#previous
[5] www.pimedia.org.uk/zebrafish-glowing-results