The role of transcription factor Pax6 in determining glutamatergic versus GABAergic cell fate in developing mammalian cerebral cortex

The brain contains two major classes of neuron: (1) excitatory neurons, which activate the neurons they connect to; (2) inhibitory neurons, which help keep the excitatory neurons in check by dampening down their activity. Both types are needed in the correct numbers for the brain to work normally. There is evidence that an imbalance in numbers contributes to cognitive disorders such as autism and major psychoses such as schizophrenia. The differences between excitatory and inhibitory neurons are created early in embryogenesis. Each type activates its own distinct program of gene expression that specifies the way in which it develops. We have evidence implicating the Pax6 protein in the high-level control of this process. Pax6 is a transcription factor, binding to DNA at specific sites and controlling many downstream genes (1,2). We have new data indicating that one of Pax6’s most important functions during early embryonic development of the cerebral cortex is to prevent the activation of genes that would generate inhibitory neurons and promote the activation of genes that would produce excitatory neurons.

In mouse, 70-80% of neurons in mature cerebral cortex are excitatory. They are generated within the cortex from cells that contain high levels of Pax6. The other 20-30% are inhibitory. In normal development, they are generated outside the cortex by cells that do not express Pax6 and they then migrate into the cortex. The mouse cortex does not normally make its own inhibitory neurons. We have discovered that if Pax6 is removed specifically from cortical cells in the embryo, when they are just starting to make cortical neurons, these cells undergo a highly abnormal, rapid and powerful activation of genes that would be expected to promote the development of inhibitory neurons. In other words, they respond to Pax6 removal by altering their program of gene activation from a pro-excitatory to a pro-inhibitory one.

Our finding raises two important questions. (1) What happens to cortical cells that undergo this early reprogramming? Do they go on to form mature inhibitory neurons, or do they later revert to an excitatory type, or do they make cells with a mixture of properties? (2) How does Pax6 normally prevent a pro-inhibitory and promote a pro-excitatory program of gene activation in the embryonic cortex? This project will address these questions.

Question (1) will be addressed by removing Pax6 from specifically the cortex in the embryo, using conditional transgenesis, and examining the consequences at progressively older ages through to adulthood. At each age, next-generating sequencing would be used to study the transcriptome. Histological and electrophysiological methods would be used to discover the morphologies and functional properties of the mutant cells. Question (2) will be addressed by testing a hypothesis supported by previous evidence suggesting that removal of Pax6 might promote inhibitory neuron development because it allows abnormal cortical activation of another transcription factor, called Gsx2, that is not normally present in cortex and which can itself activate a pro-inhibitory program. In other words, Gsx2 might mediate many of Pax6’s actions. This will involve simultaneously removing Pax6 and preventing Gsx2 activation and then examining the consequences for inhibitory vs excitatory neuron development in the cortex. The effects on neuronal type would be assessed using high throughput sequencing, histological and electrophysiological methods mentioned above.

In summary, the project will generate new knowledge about the mechanisms of development of a correct inhibitory vs excitatory neuronal balance in the cerebral cortex and about the capacity of cortical neurons to alter how they develop when perturbed genetically. The project will offer cross-disciplinary training in genetics and transgenics, high-throughput sequencing, bioinformatics and computer modelling, morphological, cellular and electrophysiological analyses.

1 Manuel MN, Mi D, Mason JO, Price DJ. Regulation of cerebral cortical neurogenesis by the Pax6 transcription factor. Front Cell Neurosci. 2015, 9:70.

2 Mi D, Carr CB, Georgala PA, Huang YT, Manuel MN, Jeanes E, Niisato E, Sansom SN, Livesey FJ, Theil T, Hasenpusch-Theil K, Simpson TI, Mason JO, Price DJ. Pax6 exerts regional control of cortical progenitor proliferation via direct repression of Cdk6 and hypophosphorylation of pRb. Neuron. 2013, 78:269-84.