Thesis title: 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 family (SLRP), expressed in the cerebral cortex of the mouse developing brain. It is known to be involved in different signaling pathways, including Wnt and TGFβ. TSK-KO mice show an excessive enlargement of the lateral ventricles of the brain (LV), 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 NSCs 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 model. Given the potential role in the astrocyte progeny, we overexpressed the TSK transgene in two cortical TSK-KO NSC lines and further 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 signature of TSK-expressing and TSK-deficient astrocytes, shedding light on the role of TSK on specific cellular processes. 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 cortical development.