Function and mechanisms specifying the heterogeneity of forebrain astrocytes

在前一个资助期，我们发现了大脑皮层灰质和间脑的实质星形胶质细胞之间有趣的差异。最重要的是，我们发现间脑星形胶质细胞在体内增殖，并在体外具有神经干细胞的特征，因为它们形成多能和自我更新的神经球。因此，我们已经确定了一个新的神经干细胞龛在成年人的大脑，并提出了一个新的概念，区域特异性星形胶质细胞在体内。此外，我们确定了间脑星形胶质细胞的这些区域特异性标志的关键调节因子Smad 4。星形胶质细胞中的可诱导Smad 4缺失显示间脑中星形胶质细胞数量的选择性减少，但大脑皮层中没有，以及间脑星形胶质细胞的神经球形成能力的严重减少。在为下一个资助期提出的项目的第一部分中，我们想检查和了解成人间脑中正在进行的星形细胞发生的作用。这将通过克隆分析来完成，以确定星形胶质细胞添加或周转的程度，以及间脑中增殖星形胶质细胞的电生理记录，以确定其功能特性和对网络特性的潜在影响。我们还将对Smad 4突变小鼠进行行为分析，作为了解间脑星形细胞发生减少的影响的切入点。在该项目的第二部分，我们的目标是了解间脑和皮质星形胶质细胞的区域特异性差异在多大程度上取决于它们的位置，即外在因素，或内在决定。为此，我们将移植星形胶质细胞从大脑皮层到间脑，以确定间脑的环境是否足以引起增殖和神经球形成的星形胶质细胞从大脑皮层。由于Smad 4是介导间脑星形胶质细胞标志的外在或内在机制，我们的目标是通过RNA-seq和ChIP seq鉴定其介导间脑星形胶质细胞标志的转录靶点。最后，我们的目标是检查WM星形胶质细胞与间脑星形胶质细胞的比较，因为最近的RNA-seq数据显示它们也表达增殖基因，并且确实第一次实验显示它们的增殖和神经球形成。我们现在的目标是确定是否WM星形胶质细胞的这些特性是由间脑中相同或不同的机制决定的。两者合计，这些方法允许处理的新概念的区域特异性星形胶质细胞在成年小鼠大脑。

In the previous funding period we identified intriguing differences between parenchymal astrocytes from the cerebral cortex grey matter and the diencephalon. Most importantly we showed that diencephalic astrocytes proliferate in vivo and have neural stem cell hallmarks in vitro as they form multipotent and self-renewing neurospheres. Thus we have identified a novel neural stem cell niche in the adult brain and propose a novel concept of region-specific astrocytogenesis in vivo. In addition we identified a key regulator of these region-specific hallmarks of diencephalic astrocytes, Smad4. Inducible Smad4 deletion in astrocytes shows a selective reduction in astrocyte number in the diencephalon, but not the cerebral cortex, as well as a severe reduction in the neurosphere forming capacity of diencephalic astrocytes. In the first part of the project proposed for the coming funding period we would like to examine and understand the role of the ongoing astrocytogenesis in the adult diencephalon. This will be done by clonal analysis to determine the extent of astrocyte addition or turnover, as well electrophysiological recordings of proliferating astrocytes in the diencephalon to determine their functional properties and potential influences on the network properties. We will also perform behavior analysis of the Smad4 mutant mice as an entry point to understand the effects of reduced astrocytogenesis in the diencephalon. In the second part of the project we aim to understand to which extent the region-specific differences of diencephalic and cortical astrocytes depend on their location, i.e. extrinsic factors, or are intrinsically determined. Towards this end, we will transplant astrocytes from the cerebral cortex into the diencephalon to determine if the diencephalon environment is sufficient to elicit proliferation and neurosphere formation in astrocytes from the cerebral cortex. As Smad4 is a mediator of extrinsic or intrinsic mechanisms specifying diencephalic astrocyte hallmarks, we aim to identify its transcriptional targets that mediate these hallmarks of diencephalic astrocytes by RNA-seq and ChIP seq. Lastly we aim to examine WM astrocytes in comparison to diencephalic astrocytes, as recent RNA-seq data show that they also express proliferative genes and indeed first experiments show their proliferation and neurosphere formation. We now aim to determine if these properties of WM astrocytes are determined by the same of different mechanisms acting in the diencephalon. Taken together, these approaches allow tackling the novel concept of region-specific astrocytogenesis in the adult murine brain.