Roles of Ascl1 and Olig2 in the Transcriptional Regulation of Astrocytogenesis

Ascl1 and Olig2 are transcription factors highly expressed in certain neural progenitor cells, and are known to be involved in neurogenesis and oligodendrogenesis (OL) throughout the CNS; their role in astrocytogenesis (AS) is less well explored. Recent evidence shows that Ascl1-lineage AS clones in the spinal cord (SC) are spatially restricted to either gray matter (GM) or white matter (WM), but not both, and that Olig2 may be necessary for WM astrocytogenesis in the brain. We consider the following questions: (1) Do Ascl1+ progenitors give rise to astrocytes in the brain? (2) Do astrocyte clones, in general, display the GM/WM spatial restriction seen in Ascl1-lineage astrocytes? (3a) Is Olig2 expressed by astrocytes in the SC? (3b) If it is, is this expression required for astrocytogenesis in the SC? To address (1), we used the CreERT2 system under the Ascl1 promoter to label Ascl1+ progenitor cells in the neonatal murine brain (and their progeny) with the tdTom fluorescent reporter. Adult brains were obtained and immunohistochemically (IHC) labeled for factors specific to mature AS, OL and neural lineages; AS, OL, and neurons derived from neonatal Ascl1+ progenitors were observed in every major cortical and subcortical structure, showing that neonatal Ascl1+ progenitors do give rise to AS through the brain. To address (2), we used the CreERT2-Confetti system under the promoter for hGFAP (an astrocyte-specific marker) to give sparse labeling of astrocytes in multiple colors, so that any clone (one clone representing all the progeny of a single AS-progenitor cell) will be far from and visually distinct from other clones. Adult murine SCs were obtained, sectioned, and analyzed by fluorescence microscopy. The location, morphology and clonal identity of every labeled cell was cataloged and used to construct a clonal map of AS distribution in the spinal cord from neonatal development through adulthood, revealing the presence of "mixed" (non-GM/WM-restricted) AS clones - strong evidence for the existence of a GM/WM bipotent AS progenitor cell. To address (3a), we used the CreERT2 system under hGFAP, and IHC labeled for the presence of Olig2. The presence of Olig2+;tdTom+ double positive cells (i.e., astrocytes expressing Olig2) was quantified via fluorescence microscopy. Approximately 50% of astrocytes expressed Olig2. To address (3b), we repeated the above procedure in mice with floxed Olig2 alleles, allowing conditional knockout (CKO) of Olig2 at time of induction. 50% of Olig2-CKO spinal cords showed an almost-complete lack of astrocytes, tentatively indicating a vital role of Olig2 expression in astrocytogenesis in both the GM and the WM.