Diverse gene expression of diencephalon astrocytes reveals proliferation regulated by Smad4 - scRNA-seq II

Astrocytes are crucial for neural network function throughout the brain and also show region-specific gene expression differences. To explore this at the single cell level, we isolated cells by ACSA2 MACS from the diencephalon. Single cell RNA-seq revealed two super clusters of cells expressing astrocyte markers, one of which expressed many ependymal markers. Fluorescent labelling of ependymal cells allowed removing them during dissection. The remaining astrocytes and astrocyte-like cells show considerable heterogeneity with some subsets or gene expression states showing more wide-spread and others more restricted mapping of gene expression throughout the forebrain. Surprisingly, diencephalic astrocytes express proliferation regulating genes. Immunostaining for proiliferation markers, DNA-base incorporation and clonal analysis revealed ongoing low level astrocytogenesis even in 8 months old mice. Comparing gene expression of astrocytes in diencephalon and cortex grey matter identified Smad4 as a key regulator of astrocyte in vivo proliferation and in vitro neurosphere formation. Thus, astrocyte diversity is seemingly partitioned in wide-spread and region-specific subsets and reveals the novel concept of adult astrocytogenesis in the diencephalon in a Smad4-dependent manner. Diencephalic astrocytes from 3 months old Aldh1l1-eGFP mice were ACSA2 MACS sorted and processed using Single Cell Reagent Kits V2 from 10x Genomics (scRNA-seq).