Regulation of gene expression in developing cortex by the transcription factor Pax6

Our knowledge of how the brain controls its own development during embryogenesis is still relatively poor. What we do know is that a relatively small group of genes, encoding proteins called transcription factors, control the processes by which cells divide, move and mature in the embryo to make the brain. These transcription factors are often thought of as master regulators of development. Severe defects can result from abnormalities in the activity of any one of these genes in humans or in other animal species, indicating their fundamental importance for normal brain development. Transcription factors work by controlling the activity of other genes; but which genes, how directly are they controlled and are different genes controlled at different times in development? These questions remain largely unanswered, although we know enough to realise that the answer will be complex, with many hundreds of genes falling under the control of each individual transcription factor.

Our research is aimed at deepening understanding of the actions of one such transcription factor, Pax6. There has been sustained interest in the analysis of this particular transcription factor over many years by our group and others. Its disruption is well-known to cause severe human disease. It is a high-level regulator of the development of nerve cells in many contexts and it can programme stem cells to become nerve cells, which could be useful in the future development of new therapies based on the exploitation of stem cells. This application is timely since we have recently made and validated new transgenic mice for this work.

These new transgenic lines will allow us to circumvent some of the major problems encountered in previous studies aimed at discovering and investigating the genes that Pax6 regulates at different times during development. First, they will allow us to home in on just those cells that express the Pax6 gene, in which Pax6's direct actions will be found; we shall be able to ignore those cells that no longer express Pax6 and are, therefore, no longer under its direct control even though they might still be influenced indirectly by Pax6's actions at earlier times and/ or in other cells. Second, they will allow us to test the effects of removing Pax6 at different developmental ages on the activity of other genes, providing information on which genes are controlled by Pax6 at different ages.

The aim of the first part of the work will be to use experimental techniques followed by computational (bioinformatic) methods to generate and analyse large amounts of data on how loss of Pax6 at different ages affects the activity of other genes. The important outcome expected from this aspect includes robust identification of genes that are regulated by Pax6 at different developmental ages and the nature of the regulation, e.g. which genes are suppressed by Pax6, which are activated, how quickly are they affected by loss of Pax6, etc. The aims of the second part of the work will be to test specific hypotheses on whether Pax6 regulates particular genes directly or not and to correlate Pax6's actions on gene activity with its effects on how developing brain cells divide, move and mature.

This will be a vital step towards the challenging goal of gaining a comprehensive understanding of the molecular functions of a key transcription factor in brain development. This work is important not only because it will tell us about Pax6's mechanisms of action: more generally, progress here will inform research in related fields, for example the analysis of genetic diseases, cancer and stem cell biology.

Aim 1. To test how profiles of gene expression in a defined population of Pax6-expressing cerebral cortical progenitors vary (i) normally with developmental age and (ii) following experimenter-induced removal of Pax6 at different ages.

We shall remove Pax6 acutely at different ages using tamoxifen-induced activation of Cre recombinase from an Emx1-CreER allele to delete our new floxed Pax6 allele. Progenitor cells that would normally express the Pax6 gene (whether its sequence is mutated or not) will be isolated from cells that do not express Pax6 using our new YAC reporter transgene (DTy54), allowing comparison of equivalent progenitor populations in cortices that express Pax6 versus those that lose Pax6. For each age of tamoxifen administration, changes in gene expression profiles will be followed by RNA sequencing. Profiles will be analysed using bioinformatics.

Aim 2. To study in more detail Pax6's regulation of sets of genes involved in cortical cell proliferation, migration, specification, differentiation and axon guidance. We shall test for direct regulation by Pax6 of genes whose expression levels change when Pax6 is removed and correlate changes in expression with cellular defects.

The immediate priority, based on our existing pilot data, is to test whether Pax6 exerts control over progenitor cell proliferation by direct regulation of cell cycle genes. Further sets of genes for testing will be prioritised based on results from work in Aim 1, focussing on clusters of co-regulated genes that change expression rapidly after Pax6 loss and are associated with functions that are disrupted when Pax6 is removed. Methods to be used include chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSAs) and in vitro and in vivo reporter assays, as well as proliferation, migration and differentiation assays in vivo.

This proposal outlines a plan of fundamental scientific research that addresses a challenging biomedical question. We expect that the answer to this question, which will be very complex, will have a long-term impact on our understanding of disease in humans and will eventually contribute to the development of new therapies. In the immediate future, societal impacts are anticipated since our work is likely to provide a better understanding to patients suffering from relevant neurological and psychiatric conditions. Although we shall not be developing any new treatments for relevant diseases here, our work is an essential step towards improving opportunities for the development of new therapies for currently incurable neurological and neuropsychiatric diseases.

Most of the immediate impact of the research will be on other members of the scientific community with an interest in unravelling the molecular pathways by which transcription factors regulate brain development. In addition, the analysis of our datasets and the conclusions drawn from them will have a relatively early societal impact since it will give people suffering from defects of PAX6 and their families a much clearer understanding of the biological causes of their abnormalities. Heterozygous loss-of-function mutations of PAX6 in humans cause a syndrome called aniridia. This condition comprises eye defects including iris and retinal hypoplasia, associated with neurological and psychiatric conditions including nystagmus, impaired auditory processing and verbal working memory, autism and mental retardation. These disorders are linked to structural defects of cortex, cerebellum, corpus callosum and anterior commissure. The principal applicant of this proposal has participated in a two day meeting of scientists and aniridia patients at which it was very clear that a greater understanding of the basis of the components of the syndrome is something that many aniridia sufferers crave. It is hoped that, at future meetings of this type, we shall be able to provide patients with continually improving explanations. It is also likely that increasing understanding of the molecular and cellular defects that occur in patients with PAX6 mutations will prompt clinicians working with aniridia patients to test for defects that have so far gone unnoticed, allowing the potential for additional medical assistance where it might be possible and effective.

Whilst a pathway to curative treatment for patients with aniridia is hard to envisage at present, we do foresee an impact for a greater understanding of the molecular functions of Pax6 in stem cell and cancer biology. The therapeutic potential of stem cells is widely recognised, but the generation of cells of the appropriate type to replace damaged neurons remains a significant challenge. Pax6 is a critically important regulator of a neurodevelopmental programme in stem cells. Knowledge of the molecular pathways by which Pax6 drives neural development should provide more informed and efficient ways of developing methods for directing neurogenesis in stem cells, as an alternative to empirical approaches. In cancer biology, Pax6 has been implicated as a tumour-suppressor and understanding the mechanism of this action could help develop new therapies in the long-term. We consider, therefore, that the results of this present study will contribute to impact both on society and economically by improving the potential for new treatments.