Deciphering molecular pathways and cellular events involved in uveal coloboma

What is Coloboma?
Coloboma refers to an eye disorder that affects approximately 1:10,000 children. As the eye develops before birth it will normally close forming a sphere. In the case of coloboma the spherical eye does not fully form leaving small or bigger gaps in its structure, usually in the bottom part of the eye. These can result in the child being blind or partially sighted.
Aetiology of Coloboma
The causes of coloboma are not fully understood, but it is known that both environmental and genetic factors contribute to it. However, most cases of coloboma seem to be caused during embryonic development by errors, known as mutations, in our hereditary components known as genes. However, there are still many cases of coloboma for which the causative gene has not yet been identified and great effort is being invested in identifying additional genes involved in this condition. As coloboma originates during foetal life, investigating how the cells interact and what genes participate in the closure of the eye during embryonic development can help us understand the causes of this disorder.
Research Aim
The main aim of this research proposal is to identify new genes and cellular events that may be responsible for coloboma in humans.
To achieve this aim we will study eye development using the mouse as our model organism. The mouse is a good model for studying human genetic disorders as it shares many common genes with humans. In many cases, these shared genes contain errors (mutations) in both human and mouse which result in similar defects. This is also the case for some of the genes associated with coloboma in both human and mouse.
Objectives
- Our first objective is to isolate different sets of genes from the bottom part of the eye of mouse embryos. These genes are potentially crucial for eye closure. Our strategy will compare genes expressed in samples of normal embryos and embryos that are mutant for the Foxg1 gene, which have severe coloboma. This approach will produce a lot of coloboma-candidate genes which we will need to prioritise. Several steps will be followed for narrowing down genes of interest.
1. With help from Bioinformaticians from the University of Edinburgh our results will be ranked based on significant differences between our two groups of study.
2. We will compare our results with those generated from a screening of patients with coloboma. This will be achieved in collaboration with Prof FitzPatrick, an expert in coloboma at the MRC-Human Genetics Unit in Edinburgh.
3. We will investigate in which areas of the developing mouse eye our best candidate genes are found.
4. We will then investigate what are the effects on the mouse of blocking the gene function or increasing the dose of these candidate genes on cultures of mouse developing eyes.
5. The genes that will arise from this study as plausible candidates for coloboma will be further tested for mutations on human samples of patients with coloboma. This, again, will be achieved in collaboration with Prof FitzPatrick's group.
- We have recently observed that in the Foxg1 mutant mouse we are able to ameliorate the coloboma defect when we subtract another gene known as Wnt8b. This indicates that Wnt8b is involved in coloboma formation and reducing the amount of this gene in mammals may recover the defect. Our second objective is to understand how the Wnt8b gene acts during eye development, its relationship with Foxg1 and with other genes that cause coloboma in mice and human, such as the Pax2 gene.
Benefits from the study
We anticipate to understand the basic biological events behind coloboma and to identify novel genes responsible for human coloboma. Such a discovery will be of great importance to the wider scientific community. Our basic research will generate data that in the future will help ophthalmologists, paediatricians and geneticists to provide better information and support to families with children with coloboma.

In this research proposal we plan to perform an expression analysis using RNA sequencing to identify genes that are differentially expressed in the anterior optic fissure (OF) in wild-type and Foxg1-/- null embryos and to investigate the role of Wnt8b in OF closure.
For our expression analysis experiment, we will dissect tissue from wild-type and Foxg1-/- null mutant mice using laser capture microdissection (available at Roslin Institute) at two different developmental time points (before and just when the two tips of the OF meet). The sequencing and statistical analysis of results will take place at GenePool (University of Edinburgh). Crucial to our selection of coloboma-candidate genes will be our collaboration with Prof FitzPatrick, who will provide us with data that will arise from a human exome analysis screen of patients with coloboma. Expression of genes shared by the two different studies will be examined in wild-types and Foxg1-/- nulls. Using ex-vivo electroporation we will alter expression of these genes in wild-type optic cups and the phenotype will be evaluated by qualitative and/or quantitative techniques. Our strongest candidate genes will be sequenced to screen for mutations in samples of coloboma patients in the MRC-HGU.
In parallel and based on our pilot data, we will study the functional relationship between Foxg1 and Wnt8b in the developing optic stalk and OF. To establish this, we will study:
-When Wnt8b expression becomes defective in the developing eye of Foxg1-/- nulls.
-How expression of downstream targets of the Wnt/beta-catenin and planar-cell-polarity pathway changes in our Foxg1-/- and Foxg1-/-;Wnt8b-/- mutants.
-What the OF phenotype is at later stages of development in our double mutants. We will finally generate a double mutant strain for the Wnt8b and Pax2 genes and study the eye phenotype in these embryos.
Once we publish our results, our RNA sequencing and optic fissure gene expression data will become publicly available.

The current proposal falls within the remit of basic research with close links to translational research. We propose to study the molecular and cellular mechanisms that lead to the medical entity known as coloboma. Coloboma results from incomplete closure of the inferior part of the eye, the optic fissure, during foetal life and, although environmental factors seem to have a causative role, most cases have a genetic basis. Although the incidence of coloboma may not be as high as that of other genetic disorders (1 in 10,000 children), its impact on the life of the individual can be detrimental, as 5-10% of European children with coloboma are blind. Our proposal aspires to identify molecules involved in optic fissure closure using the mouse as model system and exploring whether some of these molecules could be a causative gene for human coloboma.
Our work will significantly contribute to expanding our knowledge in the field of Developmental Neurobiology, providing novel data on mammalian eye morphogenesis.
Our research will ultimately contribute to generating a more in depth knowledge about the genetic causes of coloboma. This will assist children and their families in multiple ways:
- Parents with children with coloboma need to know as soon as possible how the disorder may affect their child's vision and what can be done to help them. In many cases, they also wish to know why it happened and whether it may happen again. Our research will ultimately provide ophthalmic or medical geneticists with valuable information for genetic counselling.
- A critical aspect in managing children with coloboma is the early identification of the disorder, which allows early evaluation of the visual acuity of the child. As coloboma can be a feature encountered in a variety of syndromes, an early full examination of the child is imperative to help identify any associated systemic abnormalities. A prompt approach helps paediatricians and ophthalmologists to adopt a proper therapeutic approach and provide support to the child and the family from early stages of life. Our basic research will provide information regarding the cause of the disorder, which in the future can be of immense value to clinicians for managing it successfully.
Our research will also aim in raising public awareness regarding coloboma as well as other eye disorders such as microphthalmia, which is frequently associated with coloboma.