Genetic and imaging studies of eye morphogenesis in development and disease

Eye colobomas encompass a group of common eye defects affecting patients of all ages, but especially young children. These pathologies are a common cause of visual problems, can cause retinal detachment and cataracts, and often induce blindness in affected patients. Colobomas are generally hereditary diseases with a highly variable clinical presentation, being diagnosed by the detection of a notch, gap, hole or fissure in any of the structures of the eye, including the cornea, retina, optic nerve or even the eyelid. The defects in the ocular structures associated with the colobomas result from a failure in the embryonic formation of the eye. During embryogenesis, the forming eye and optic nerve undergo dramatic shape changes that lead to the closure of a fissure present on one side of the eye (the choroid fissure), and eventually to the formation of the eye globe. If choroid fissure closure is disrupted, an ocular coloboma develops. Recently, some of the congenital defects responsible for certain forms of coloboma have been identified. These defects consist of mistakes or ?mutations? in certain genes that normally make proteins important during eye formation. Despite these recent advances, we know virtually nothing about how cells behave during closure of the choroid fissure and how they are affected in coloboma conditions. Understanding these issues is fundamental to understand coloboma pathologies and to find ways of treating them. For this purpose, we will use the zebrafish, a small striped fish that has been used for years as a model to study embryonic development and that recently has been successfully used to model other human diseases. Highly developed imaging techniques allow visualisation of the developing eye in the living zebrafish embryo. By using zebrafish that exhibit colobomas similar to those in humans, we will compare the process of choroid fissure closure between these coloboma disease models and healthy conditions. In addition, we will perform a number of genetic screens to search for other genes affected in coloboma. These analyses will provide us with a list of candidate genes responsible for human ocular pathologies, will lead us to identify and generate 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.

Ocular colobomas constitute a family of ocular pathogeneses that are caused by non-closure of the choroid fissure, an event that normally occurs during morphogenesis of the eye. These abnormalities are the most common hereditary ocular malformations, causing as much as 10% of childhood blindness. However, their genetic bases remain elusive. Identification and functional characterisation of genes responsible for hereditary coloboma is not easy to do in humans as the low number of affected families and wide phenotypic and locus heterogeneity, make it difficult to identify disease loci. Moreover, a thorough understanding of the cellular events leading to choroid fissure closure during morphogenesis of the eye is essential to understand the causes of these hereditary ocular malformations. In this project, we aim to gain insight into the cellular events leading to optic cup morphogenesis and choroid fissure closure and to identify molecules involved in this process. Preliminary results have prompted us to identify two well-known signalling pathways, the Wnt and the Eph/ephrin pathways, as strong candidates for participating in choroid fissure formation and closure and we will pursue the functional analysis of these pathways. Other molecules important for this process have recently been identified by ourselves and others but, with the exception of Ncadherin, all are signalling molecules and their target transcription factors. The final downstream effectors of optic cup morphogenesis activated by these signals are still largely unknown. To identify genes with roles in choroid fissure morphogenesis and closure, we will make use of both forward and reverse genetic approaches in zebrafish. We will interfere with the function of genes known to be important for optic fissure closure and will use microarray expression profiling of cells from the resulting colobomatous eyes to identify genes misregulated when colobomas occur. As a complementary forward genetic approach, we will characterise existing zebrafish mutants with coloboma phenotypes and will participate in a genetic screen to identify mutations affecting eye morphogenesis. In parallel with these approaches, we will make use of highly advanced imaging techniques to elucidate the cellular mechanisms underlying these morphogenetic processes in living fish. These analyses will identify candidate genes responsible for human ocular pathologies and will lead us to identify and generate new models for human eye hereditary diseases. Analysis of the cell biology underlying the identified genetic defects will allow us to gain further insight into the normal eye morphogenesis of the eye and the aetiologies of hereditary ocular malformations.