Function and mechanisms specifying the heterogeneity of forebrain astrocytes

在之前的资助阶段，我们发现大脑皮质、灰质和间脑的实质星形胶质细胞之间存在有趣的差异。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，因为它们形成了多潜能和自我更新的神经球。因此，我们在成人脑中发现了一种新的神经干细胞生态位，并提出了体内区域特异性星形胶质细胞发生的新概念。此外，我们还确定了间脑星形胶质细胞这些区域特异性标志的关键调节因子Smad4。星形胶质细胞中可诱导的Smad4缺失表明间脑中星形胶质细胞的数量选择性减少，但大脑皮层没有，并且间脑星形胶质细胞的神经球形成能力严重下降。在为即将到来的资助期提出的项目的第一部分中，我们想要检查和了解正在进行的星形细胞形成在成人间脑中的作用。这将通过克隆分析来确定星形胶质细胞的增加或更替的程度，以及间脑中增殖的星形胶质细胞的电生理记录，以确定它们的功能特性和对网络特性的潜在影响。我们还将对Smad4突变小鼠进行行为分析，以此为切入点了解间脑星形细胞生成减少的影响。在项目的第二部分，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，即外部因素，或者是内在决定的。为此，我们将把大脑皮质的星形胶质细胞移植到间脑，以确定间脑环境是否足以诱导大脑皮质星形胶质细胞的增殖和神经球的形成。由于Smad4是间脑星形胶质细胞特征的外在或内在机制的介体，我们的目标是通过RNA-SEQ和CHIP SEQ确定其介导间脑星形胶质细胞这些特征的转录靶点。最后，我们的目标是将WM星形胶质细胞与间脑星形胶质细胞进行比较，因为最近的RNA-seq数据显示，它们也表达增殖基因，实际上，第一次实验表明它们的增殖和神经球的形成。我们现在的目标是确定WM星形胶质细胞的这些特性是否由间脑中不同的作用机制决定。综上所述，这些方法可以解决成年小鼠大脑中区域特异性星形细胞发生的新概念。

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.