Mouse models of forebrain defects caused by Pax6 haploinsufficiency

Lead Research Organisation: University of Edinburgh
Department Name: Centre for Discovery Brain Sciences


In the human population, ~1/50,000 live births suffer from a condition called congenital aniridia, which results from mutations that disrupt the function of a gene called PAX6. This gene, which codes for a protein that is a high-level regulator of the activity of other genes, is expressed in developing eye and brain and is critical for their normal formation. Here, we are interested in understanding the consequences of the loss of function of one copy of PAX6 for development of the most advanced part of the brain, the forebrain. If both copies of the PAX6 gene are mutated so that there is no functional PAX6 at all, the result is fatal before or shortly after birth and there are severe abnormalities of the brain. Patients with mutations causing one copy of PAX6 to lose function have normal life expectancy but show abnormalities of brain structure and function. These abnormalities include a reduced volume of the major structure that links the two hemispheres of the brain, the corpus callosum, and a range of neurological and psychiatric defects. It is well-recognized that mutant mice that have lost Pax6 gene function provide excellent models of at least some of the defects found in congenital aniridia. Heterozygous mutant mice (i.e. mutants in which just one of the two copies of Pax6 is abnormal, designated Pax6+/-) are the best mouse models for congenital aniridia, since humans surviving with the condition are heterozygous for PAX6 loss-of-function (PAX6+/-). Whereas defects of the eye, olfactory bulbs and specific hindbrain structures have been found before in Pax6+/- mice, until now forebrain defects that model those in human patients have not been identified. We now have new pilot data showing that heterozygous Pax6 mutations in mice cause specific forebrain defects. These findings are potentially very relevant for understanding human congenital aniridia. We have found: (i) A significant narrowing of the corpus callosum in adult Pax6+/- mice, a result that fits well with observations of corpus callosum abnormalities in heterozygous PAX6+/- humans with congenital aniridia. (ii) An increased density of a set of Pax6+/- cells of a specific type (cells that activate, or in their past activated, a gene called Zic4: i.e. so-called Zic4-lineage cells) in a region of the forebrain, known as the geniculate nuclei, that connects with the cerebral cortex; studies of corresponding defects in PAX6+/- humans have not yet been reported. Our objective in this proposal is to gain a better understanding of these forebrain defects and their causes in mice, with a view to improving our knowledge of the forebrain abnormalities associated with human congenital aniridia.

In the first part, we shall study callosal defects in Pax6+/- mice, their age of onset, their cellular causes and whether Pax6+/- mice show behavioural abnormalities that have been linked by previous authors with callosal abnormalities.

In the second part, we shall study the effects of mutating one copy of Pax6 on the development of Zic4-lineage cells in the geniculate nuclei. We shall first study the effects of mutating Pax6 specifically in Zic4-lineage cells on the postnatal development of these cells and their connections to the cortex. We shall then study what happens to Zic4-lineage cells in mice carrying one mutant copy of Pax6 in all cells, a condition that most closely models human congenital aniridia.

In summary, our objective is to capitalize on our recent identification of defects that are likely to be highly relevant for understanding forebrain abnormalities in congenital aniridia. Once we know the answers to the questions posed here (e.g. are defects early or late onset, are they cell autonomous or cell non-autonomous?) we shall be able to design future experiments to provide molecular explanations for our discoveries and to test ways of rescuing abnormalities that might be applicable to humans.

Technical Summary

The overall aim is to study forebrain defects resulting from Pax6+/- heterozygosity in mice. These defects have been identified in our pilot work.

Aim 1. Callosal defects:
Aim 1a. To identify the age of onset of abnormal callosal thinning in Pax6+/- mice by studying histological sections of the forebrain.
Aim 1b. To discover the cellular cause of callosal thinning in Pax6+/- mice. This will involve testing the hypothesis that a lowering of the number of contralaterally projecting cortical neurons contributes to callosal thinning, using retrograde tracers combined with specific molecular markers of callosal neurons and indicators of cell death. We shall use electron microscopy to test whether reduced axonal diameter and/ or reduced myelination contribute to callosal thinning.
Aim 1c. To test whether callosal defects are cell autonomous or cell non-autonomous by making chimeric mice comprising a mixture of Pax6+/+ and Pax6+/- cells and applying the approaches used in Aim 1b to them.
Aim 1d. To test Pax6+/- mice for behavioural abnormalities that have been linked with callosal abnormalities.

Aim 2. Diencephalic defects:
Aim 2a. To examine what happens to the perinatal defects of Pax6+/- geniculate Zic4-lineage cells during the postnatal development of Zic4Cre;Pax6fl/+ conditional mutants, using immunohistochemistry to identify Zic4-lineage cells for quantification.
Aim 2b. To study the cortex of Zic4Cre;Pax6fl/+ conditional mutants using immunohistochemistry and in vivo calcium imaging to measure the extent of the visual and other cortical areas.
Aim 2c. To test the cause of the enlarged populations of Pax6+/- geniculate Zic4-lineage cells in Zic4Cre;Pax6fl/+ conditional mutants using markers of proliferating cells and immunohistochemical methods to measure cell cycle parameters.
Aim 2d. To study the nature of defects of geniculate Zic4-lineage cells in constitutive Pax6+/- mutants, using the approaches in Aims 2a-c to identify the defects.

Planned Impact

In addition to the immediate benefit to the scientific community (see Academic Beneficiaries above), the conclusions drawn from our work 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 congenital 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, corpus callosum and anterior commissure. There are several websites providing links between suffers, for whom understanding their condition and its implications for their and their families' livelihoods and prospects is of paramount importance (,, 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. In the future, we shall seek to understand at a molecular level why Pax6 haploinsufficiency causes the abnormalities identified in the current proposal. We shall also test ways of rescuing the defects identified by this project, using approaches that might be applicable to humans.


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