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

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The brain contains two major classes of neuron: (1) excitatory neurons, which transmit signals between cells by activating 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. As well as doing different things, excitatory and inhibitory neurons look different and have different chemistries. These differences are created because, early in their existence, each type activates its own distinct set of genes that specify the way in which it develops. We have evidence implicating a protein called Pax6 in the high-level control of this specification process. Pax6 is a transcription factor, meaning that it binds to DNA at specific sites and controls many other genes. 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 turn cells into inhibitory neurons and promote the activation of genes that would turn cells into 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. That is, 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? We aim to provide answers to these questions. To address the first question, we shall remove Pax6 from specifically the cortex in the embryo, using genetic methods, and examine the consequences at progressively older ages through to adulthood. At each age, we shall look at what genes are activated, whether the mutated cells contain proteins associated with inhibitory or excitatory neurons, what the cells look like and what their electrical properties are. To address the second question, we shall test a hypothesis supported by previous evidence suggesting that removal of Pax6 might promoted 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, we suggest that Gsx2 might mediate many of Pax6's actions. To test this hypothesis, we shall simultaneously remove Pax6 and prevent Gsx2 activation and then examine the consequences for inhibitory vs excitatory neuron development in the cortex. We shall assess the effects on neuronal type using methods mentioned above.In summary, we aim to provide new knowledge about the ways in which the cerebral cortex develops a correct balance of inhibitory vs excitatory neurons and also about the capacity of cortical neurons to alter how they develop when perturbed genetically.