Roles of Tsukushi proteoglycan and Fibroblast Growth Factor 2 in the regulation of neocortical astrogliogenesis

TSK is an extracellular proteoglycan belonging to the small leucine-rich protein (SLRP) family, which is expressed in the cerebral cortex of the developing mouse brain. It is known to be involved in different signaling pathways, including Wnt and TGFβ. TSK knockout (TSK-KO) mice show an excessive enlargement of the lateral ventricles (LVs) of the brain, a phenotype known as communicating hydrocephalus. The molecular mechanisms involved in the etiology of this phenotype are not fully understood, although TSK has been related to the regulation of neural stem/progenitor cell (NSPC) behaviour. To better characterize the role of TSK in the regulation of NSPCs and their progeny, we assessed the expression levels of TSK in proliferating and differentiated NSPCs using an in vitro cellular model. Given the enriched TSK expression in the astroglial NSPC progeny, we overexpressed the TSK transgene, or a control GFP transgene, in two cortical TSK-KO NSPC lines and evaluated its role in TSK-KO/GFP and TSK-KO/TSK-HA astrocytes by RNA-sequencing analysis. Surprisingly, our in vitro model led us to the characterization of two different sub-types of astrocytes, which differ in morphology and molecular features. By exploiting our transcriptomic dataset to profile these two cell states, we found a key role of FGF2, when acting in collaboration with BMP4, in steering differentiating cortical astrocytes to a more mature and less reactive phenotype, as compared to astroglial differentiation driven only by BMP4. Moreover, we compared the molecular signatures of TSK-expressing and TSK deficient astrocytes, shedding light on the role of TSK in specific astroglial features. Furthermore, single-cell transcriptomic analysis of the developing mouse cerebral cortex allowed us to analyze the role of TSK in the regulation of cortical neurogenesis and gliogenesis in vivo, unveiling a new role of TSK in properly balancing these processes during corticaldevelopment.