PAR polarity proteins and Neurogenesis

大脑是由胚胎中的一层薄薄的细胞发育而来的。在发育到成体的过程中，它变成了一个非常复杂的结构，有许多层和许多不同的细胞类型。产生不同细胞类型的方法之一是细胞不对称分裂，即一个细胞变成两个彼此不同的子细胞。一般来说，一个子细胞就像它的母细胞（即可以以同样的方式再次分裂的干细胞），而另一个则是更特化的细胞，如神经元。众所周知，这种分裂在哺乳动物大脑发育的许多阶段都会发生，因此，它们是如何被调节的，对于总体理解大脑发育非常重要。在发育中的大脑中，不对称细胞分裂是如何发生的？我们一直在研究果蝇和蠕虫体内的一组蛋白质，这些蛋白质在非神经组织发育的几个阶段中对某些不对称细胞分裂是必需的。为了做到这一点，我们使用爪蟾作为实验系统，因为它和人类一样，是一种脊椎动物，但与人类或实验室小鼠不同，它的胚胎很大，可以很容易地观察到从产卵到蝌蚪阶段，为期四天。我们决定研究神经组织中的这些蛋白质。我们发现，当我们耗尽这两种蛋白质中的任何一种，即Par-1和Par-4，早期神经元不再形成。当我们在错误的地方引入额外的Par-1蛋白时，我们可以得到额外的神经元。这告诉我们这些Par蛋白很重要，所以我们想要发现它们是如何工作的。我们认为，我们所看到的神经元的损失和增加都是因为形成大脑的细胞薄层中最早的不对称细胞分裂现在还不够不对称。为了验证这一观点，我们将直接观察当Par-1或Par-4发生改变时，早期分裂的子细胞是否会继续成为相同类型的细胞。这包括要么早期手工分离细胞，要么就地追踪它们，然后使用特定的细胞染色试剂来观察它们是什么细胞类型。在另一组实验中，我们还将发现Par-1和Par-4的作用在其他不对称分裂中是否相似，特别是在早期皮肤和后期产生神经元的细胞分裂中。我们还将研究小鼠大脑中的细胞，这些细胞已知是不对称分裂的，但Par-1和Par-4在其中的作用是完全未知的。最后，在第三个项目中，我们将研究par1和par4的生物化学作用。它们都是将磷酸基团添加到其他蛋白质上的酶（激酶）。这是一种常见的方式，变化从细胞的一部分传递到另一部分，有数百种不同的激酶，每一种都传递不同的信号。我们知道一些Par激酶可以调节的蛋白质。它们是否在神经元发育过程中这样做，我们不知道/但可以发现。我们有特殊的试剂（“磷酸化特异性抗体”），可以用来染色特定的候选蛋白，只有当它们被磷酸化时。精密的显微镜和图像处理技术将向我们准确地展示磷酸化发生的地点和时间。了解这些磷酸化事件在发育中的神经系统中的发生地点和时间，将使我们能够在驱动发育的巨大电路图中绘制出一些更重要的联系。最终，上述所有研究不仅将帮助我们了解大脑是如何形成的，而且还将有助于了解制造能够修复患者大脑和脊髓的细胞所需的知识。

Par polarity proteins in building the nervous system The brain develops from a thin sheet of cells in the embryo. During development to the adult, it becomes an extraordinarily complex structure with many layers and many different cell types. One of the ways of generating different cell types is by cells dividing asymmetrically, that is a cell becoming two daughter cells that are different from one another. Typically, one daughter cell is like its mother (i.e. a stem cell that can divide in the same way again) while the other is a more specialised cell such as a neuron. Such divisions are known to take place in the developing mammalian brain at many stages, and so how they are regulated is very important in understanding brain development in general. How do asymmetrical cell divisions happen in the developing brain? We have been studying a group of proteins that in flies and worms are needed for certain asymmetric cell divisions at several stages of development in non-neural tissues. To do this, we have used the Xenopus frog as an experimental system because, like humans, it is a vertebrate, but unlike humans or laboratory mice, its embryos are large and can be watched easily from spawning to tadpole stages, a period of four days. We decided to look at these proteins in the neural tissue. We found that when we deplete either of two of these proteins, known as Par-1 and Par-4, early neurons are no longer formed. When we introduce extra Par-1 protein in the wrong place, we can get extra neurons. This tells us that these Par proteins are important and so we want to discover how they work. We believe that both the loss and the gain of neurons we see happen because the earliest asymmetrical cell divisions in the thin sheet of cells that will make the brain are now not sufficiently asymmetrical. To test this idea, we will observe directly whether the daughter cells of the early divisions go on to become the same type of cell when Par-1 or Par-4 is altered. This involves either manually separating the cells early or tracking them in place and then using specific cell staining reagents to see what cell types they are. In a separate set of experiments, we will also find out whether the role of Par-1 and Par-4 is similar in other asymmetric divisions, specifically in the early skin and in the later cell divisions that give rise to neurons. We will also look at cells from mouse brains that are known to divide asymmetrically, but for which the role of Par-1 and Par-4 is completely unknown. Finally, in a third project, we will examine the biochemistry of what Par-1 and Par-4 are doing. They are both enzymes ('kinases') that add phosphate groups to other proteins. This is a common way that changes are transmitted from one part of a cell to another and there are hundreds of different kinases that each relays a different signal. We know some of the proteins that the Par kinases can regulate. Whether they do this during neuron development, we do not know / but can find out. We have special reagents ('phospho-specific-antibodies') that can be used to stain the specific candidate proteins only when they are phosphorylated. Sophisticated microscopes and image processing will show us exactly where and when the phosphorylation evens take place. Seeing where and when these phosphorylation events take place in the developing nervous system will enable us to draw a few more important linkages in the vast circuit diagram that drives development. Ultimately, all of the above studies will not only help us understand how brains are made, but it will also contribute to the knowledge that is needed to make cells that can repair the brains and spinal cords of patients.