Genes, Environment and Evolution in Eye Development
and Malformations: PAX6, SOX2, OTX2

 

 

Much of this work uses reporter transgenic analysis in mouse and zebrafish models, but with enhancer analysis with differentiating stem cells is also planned. Network building is also in progress using bioinformatics approaches, particularly through predicting downstream targets and upstream regulators of PAX6 and SOX2, followed by experimental validation, predominantly using zebrafish.

 

Further studies (NIH collaborative funding) in collaboration with Dr Rob Grainger (University of Virginia, USA) will use comparative analysis in Xenopus tropicalis and mouse and zebrafish.

 

We are also beginning to explore the role of regulatory mutations in disease and some of this work will be carried out in collaboration with an EU FP7 funded group coordinated by Dr Thomas Becker (Norway and Australia).

 

How stress response pathways modulate the effect of mutations

 

Analysis of phenotypic variation: Mediating Environmental Factors

Interestingly, AMC rarely shows a clear-cut mode of inheritance (ie no regular Mendelian pattern of segregation in families), suggesting the possible involvement of several genes and/or interaction with environmental factors which may modulate the exact outcome of the underlying mutation - hence the variability in phenotype. We are exploring the underlying mechanisms using zebrafish as models. We have shown that reducing levels of the stress-responsive chaperone protein HSP90, which plays a role in helping other proteins to fold correctly, we can modify the phenotypic outcome of some mutations. This knowledge may help us to develop strategies to reduce or prevent the incidence of severe birth abnormalities.

 

Zebrafish revealing anophthalmia and microphthalmia phenotypes following partial inhibition of HSP90 activity

 

We are currently exploring in more detail some of the myriad molecular interactions of HSP90. This work is providing new insights into cellular maintenance and function.

 

 

 

Our work is currently focused on three key areas

1. Genetics of developmental eye malformations
2. How gene expression is regulated
   (this page)
3. How stress response pathways modulate the effect of mutations (this page)

• Key Publications
• Collaborations and Laboratory Members

 

 

How gene expression is regulated

 

Molecular analysis of gene function and interactions: Long Range Control of Gene Expression

PAX6, SOX2 and OTX2 are all DNA-binding transcription factors with major roles in control of development. They themselves require complex regulation to fulfil their tissue-specific functions. We are dissecting the way this regulation takes place, through conserved elements that may lie upstream and/or downstream of the transcribed gene, as well as within its introns. We now know that these regulatory elements are generally highly conserved through evolution among vertebrates and we have shown for PAX6 that these elements can function as enhancers that drive reporter gene expression in a partial PAX6 pattern. We have shown that PAX6 regulates itself at more than one site and others have shown that it does this partly by associating with SOX2 protein. Conversely, SOX2 is co-regulated by PAX6 and SOX2 binding together to specific sites. There is therefore a clear physical basis for suggesting that these genes belong to an interacting network, whose complexity is only just emerging.

Partially overlapping expression of PAX6 (red) and SOX2 (green) protein in developing mouse eye

 

We are pursuing the mechanisms by which all the conserved regulatory elements associated with PAX6 work together. We have used DNase hypersensitivity studies to reveal an open chromatin conformation in expressing cells and tissues and we are working on chromosome conformation capture methods to reveal DNA interactions across the wider PAX6 genomic domain. Additional insight into regulatory element function is emerging from comparison of the two duplicated and diverged copies of the zebrafish pax6a and pax6b genes.