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

大脑包含两大类神经元：(1)兴奋性神经元，通过激活与之相连的神经元在细胞间传递信号；(2)抑制性神经元，它通过抑制兴奋性神经元的活动来帮助控制它们。这两种类型都需要正确的数量才能使大脑正常工作。除了做不同的事情，兴奋性和抑制性神经元看起来也不同，化学成分也不同。这些差异之所以产生，是因为在它们存在的早期，每种类型都激活了自己独特的一组基因，这些基因指定了它们的发育方式。我们有证据表明，一种名为Pax6的蛋白质参与了这一规范过程的高层控制。Pax6是一种转录因子，这意味着它在特定位点与DNA结合，并控制许多其他基因。我们有新的数据表明，在大脑皮层早期胚胎发育过程中，Pax6最重要的功能之一是阻止将细胞转化为抑制性神经元的基因的激活，并促进将细胞转化为兴奋性神经元的基因的激活。小鼠成熟大脑皮层70-80%的神经元具有兴奋性。它们是在皮层内由含有高水平Pax6的细胞产生的。另外20-30%是抑制性的。在正常发育中，它们是由不表达Pax6的细胞在皮层外产生的，然后迁移到皮层中。也就是说，老鼠的大脑皮层通常不会产生自己的抑制性神经元。我们发现，如果在胚胎皮层细胞刚开始制造皮层神经元时，将Pax6特异性地从皮层细胞中移除，这些细胞就会经历一种高度异常的、快速而有力的基因激活，这种基因激活有望促进抑制性神经元的发育。换句话说，它们对Pax6去除的反应是通过改变它们的基因激活程序，从促兴奋变为促抑制。我们的发现提出了两个重要的问题。经历这种早期重编程的皮质细胞发生了什么？它们是继续形成成熟的抑制性神经元，还是后来恢复为兴奋性神经元，还是形成具有多种特性的细胞？(2) Pax6通常如何阻止胚胎皮质基因激活的前抑制性和促进前兴奋性程序？我们的目标是为这些问题提供答案。为了解决第一个问题，我们将使用遗传方法从胚胎时期的皮层中移除Pax6，并检查从年龄逐渐增长到成年期的后果。在每个年龄段，我们将观察哪些基因被激活，突变细胞是否含有与抑制性或兴奋性神经元相关的蛋白质，细胞的样子以及它们的电特性。为了解决第二个问题，我们将检验先前证据支持的一个假设，该假设表明，去除Pax6可能促进抑制性神经元的发育，因为它允许另一种转录因子的异常激活，称为Gsx2，这种转录因子通常不存在于皮层中，它本身可以激活促抑制性程序。换句话说，我们认为Gsx2可能介导Pax6的许多作用。为了验证这一假设，我们将同时去除Pax6和阻止Gsx2的激活，然后检查皮层中抑制性和兴奋性神经元发育的后果。我们将使用上述方法评估对神经元类型的影响。总之，我们的目标是提供关于大脑皮层如何发展出抑制性和兴奋性神经元的正确平衡的新知识，以及皮层神经元在基因干扰时改变其发育方式的能力。

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.