An evolutionarily restricted role for purinergic signalling in the maintenance of retinal stem cells

在胚胎发育期间，眼睛是由一种高度组织和刻板印象的事件模式形成的，其中视网膜前体细胞在退出细胞周期之前进行多次分裂(增殖)，并在一个称为分化的过程中形成不同类型的成年视网膜神经细胞。哺乳动物的眼睛一旦形成，就被认为不能再生。因此，任何视网膜细胞，包括光敏感感光细胞的损失，无论是由于疾病或生命中的损伤，都是永久性的。相比之下，鱼类和青蛙等低等脊椎动物的眼睛在动物的整个生命过程中都会继续生长。新的视网膜神经元，包括光感受器，是由一群干细胞增殖产生的，这些干细胞位于视网膜最外围的睫状缘区域(CMZ)。在过去的十年里，人们对人类视网膜修复的可能性产生了极大的兴趣，因为在哺乳动物眼睛的睫状体中发现了睫状上皮(CE)内的一群细胞，这是一个类似于低等脊椎动物CMZ的区域。这些细胞表现出许多干细胞样的特性，尽管目前还不清楚这些细胞是否可以继续形成所有类型的视网膜细胞。更重要的是，这些细胞只有在培养皿中培养时才会增殖-它们在眼睛本身就处于休眠状态。这就提出了这样一个问题：这些细胞是否代表了一群干细胞，这些干细胞与在青蛙和鱼中发现的干细胞相似，但在进化过程中失去了增殖和修复能力。此外，可以想象这些细胞可以被重新激活以产生新的视网膜神经元。调控低等脊椎动物干细胞增殖和哺乳动物干细胞样群明显休眠的机制还知之甚少。最近，一种被称为嘌呤能信号的信号通路被证明对启动一些眼睛形成所需的基因是必不可少的。我们还表明，在胚胎眼睛发育的早期阶段，这种信号机制对于控制视网膜前体细胞的增殖非常重要。在这里，我们的目标是确定嘌呤能信号是否代表控制睫状缘视网膜干细胞增殖的基本调节机制。此外，我们希望确定这个信号系统在进化过程中是否已经丢失或受到限制，从而为哺乳动物CE的干细胞不再增殖提供一个解释。我们已经发现，在培养皿中，嘌呤能信号参与哺乳动物视网膜干细胞的增殖。我们还发现，一种对大脑其他地方的嘌呤能介导的增殖至关重要的特定类型的受体不存在于哺乳动物CE中，但当它们在培养皿中培养时，在这些细胞中发现了这种受体。这意味着有可能诱导哺乳动物眼睛正常休眠的干细胞增殖，并在眼睛中形成新的视网膜神经元。因此，我们的目标是首先在成人眼和培养皿中检测嘌呤能信号在控制视网膜干细胞增殖中的作用。我们还将确定导致这种增殖的嘌呤能信号的下游途径。我们将比较进化上不同的物种，以检查这些动物视网膜中嘌呤能信号的作用，最后我们将确定是否有可能通过嘌呤能信号重新激活哺乳动物CE的干细胞。虽然在这一点上只是理论上的，但最后一个目标可能为修复因疾病或损伤而受损的视网膜提供信息。

During embryogenesis the eye is formed by a highly organized and stereotyped pattern of events, in which retinal progenitor cells divide (proliferate) many times before exiting the cell cycle and forming the different neuronal cell types of the adult retina in a process called differentiation. Once formed, the mammalian eye is thought to be incapable of regeneration. Thus, any loss of retinal cells, including the light-sensitive photoreceptors, either by disease or injury during life is permanent. In contrast, the eye of lower vertebrates, such as fish and frogs, continues to grow throughout the life of the animal. New retinal neurons, including photoreceptors, are produced by the proliferation of a population of stem cells that reside in a region called the ciliary marginal zone (CMZ), at the very periphery of the retina. Over the past decade, there has been significant interest in the possibility of retinal repair in humans following the discovery of a population of cells within the ciliary epithelium (CE), found within the ciliary body of the mammalian eye, a region analogous to the lower vertebrate CMZ. These cells exhibit a number of stem cell-like properties, although it is not yet clear if these cells can go on to form all the cell types of the retina. More importantly, these cells only proliferate when cultured in a dish - they are dormant in the eye itself. This raises the question of whether these cells represent a population of stem cells that are similar to those found in frogs and fish but that have lost the capacity for proliferation and repair during evolution. Furthermore, it is conceivable that these cells could be reactivated to generate new retinal neurons. The mechanisms that regulate both the proliferation of the stem cells in the lower vertebrate and the apparent dormancy of this stem cell-like population in mammals are poorly understood. Recently, one signalling pathway, called purinergic signalling, has been shown to be essential for turning on a number of genes required for eye formation. We have also shown that this signalling mechanism is very important for controlling the proliferation of retinal progenitor cells in the early stages of eye development in the embryo. Here, we aim to determine whether or not purinergic signalling represents a fundamental regulatory mechanism that controls the proliferation of retinal stem cells at the ciliary margin. Moreover, we wish to determine whether or not this signalling system has become lost or restricted during evolution and so provide an explanation for why the stem cells of the mammalian CE no longer proliferate. We have already found that purinergic signalling is involved in the proliferation of mammalian retinal stem cells in the culture dish. We have also found that a particular type of receptor that is critical for purinergic-mediated proliferation elsewhere in the brain is not present in the mammalian CE, but is found in these cells when they are cultured in the dish. This means it may be possible to induce the normally dormant stem cells of the mammalian eye to proliferate and form new retinal neurons in the eye. Therefore, we aim to firstly examine the role of purinergic signalling in controlling retinal stem cell proliferation both in the adult eye and in the culture dish. We will also determine the downstream pathway of purinergic signalling that leads to this proliferation. We will compare evolutionarily distinct species to examine the role of purinergic signalling in the retina of these animals and finally we will determine whether or not it is possible to reactivate the stem cells of the mammalian CE by purinergic signalling. Although only theoretical at this point, it is possible that the last objective may provide information for the repair of the retina damaged through disease or injury.