Browsing by Subject "Astrocytes"
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Item Novel Astrocyte-Specific Transgenic Mice Identify Distinct Populations of Transient Amplifying Progenitor Cells And Long-Lived Neural Stem Cells In the Subgranular Zone of the Adult Mouse Brain(2011-02-01) Boies, Lori Nicole Loomis; Bachoo, RobertA lack of previous interest in the astrocyte biology field has resulted in a meager number of astrocyte specific molecular markers and practical protocols to study their biology and function. The most common and accepted astrocyte marker is glial fibrillary acidic protein (GFAP), but in normal, healthy adult brains, GFAP expression is limited to the neurogenic regions, white matter tracks, and pia. This limited expression is not a true representation of astrocyte distribution patterns and does not account for astrocyte heterogeneity. Through a careful screen, cystatin C (CstC) and phospholipase A2 Group VII (PLA2G7) were identified as astrocyte specific molecular markers. To study the role of these genes in astrocyte biology, inducible CreERT2 mice were generated. Using the CstC-CreERT2 and PLA2G7-CreERT2 mouse lines, in concert with the Rosa26YFP reporter line, cell fate tracking experiments were performed in both embryonic and adult time points in tandem with extensive immunohistochemistry. Both astrocyte-specific CreERT2 lines demonstrated limited expression in embryos as early as embryonic day 12.5 and extensive expression throughout the entire central nervous system in all adult time points analyzed. In adult animals, both CstC-CreERT2 and PLA2G7-CreERT2 Rosa26YFP positive cells had strong expression in both neurogenic niches. Using careful quantification and immunohistochemistry colocalization, the dynamics of these transgenic lines were meticulously researched in the subgranular zone (SGZ) of the dentate gyrus. The CstC-CreERT2/Rosa26YFP cells represent a transient amplifying compartment in the SGZ while PLA2G7-CreERT2/Rosa26YFP positive cells comprise a long-lived neural stem cell compartment. Discovery of these astrocyte dynamics in the SGZ complements the literature indicating astrocytes are the true neural stem cells and adds knowledge of new viable markers and cell populations to the field. Results of these studies reveal not only new molecular markers for astrocytes in both embryonic and adult scenarios, but also help to identify the underlying heterogeneity of astrocyte populations, as well as adult neural stem cells. Future utilization of these inducible transgenic mouse models could provide the potential to study both astrocyte and neural stem cell pathophysiologies.Item The Role of Ascl1 in NG2 Cells in the Spinal Cord(2015-01-26) Kelenis, Demetra; Johnson, Jane; Vue, Tou YiaNG2 cells, one of the major glial cell populations within the central nervous system (CNS), are highly proliferative cells identified by the expression of the NG2 proteoglycan. Throughout life, NG2 cells can differentiate into oligodendrocytes to myelinate axon fibers, or they can be maintained in a proliferative or quiescent state as NG2 cells indefinitely. A recent study showed that deletion of tumor suppressor genes specifically within NG2 cells was sufficient to produce brain tumors in a mouse model, indicating that NG2 cells may serve as a potential cell of origin for gliomas. At present, however, understanding of how NG2 cells are regulated to proliferate or differentiate in the CNS remains incomplete. Interestingly, Ascl1, a proneural basic-helix-loop-helix (bHLH) transcription factor that is highly expressed in neural progenitor cells, is also expressed in NG2 cells. Although previous studies have shown that the loss of Ascl1 affects the initial specification and differentiation of NG2 cells, the specific role of Ascl1 in NG2 cells during embryonic and postnatal development remains unknown. In this study, we investigated the direct requirement of Ascl1 in NG2 cells during embryonic and postnatal development in the grey matter (GM) and white matter (WM) of the spinal cord. More specifically, we conditionally deleted Ascl1 specifically within NG2 cells (Ascl1-CKO) at E14.5 or at P30 using an NG2-CreERT2 mouse strain in which the tdTomato (tdTom) fluorescence reporter was also incorporated to allow direct visualization of the development of NG2-labeled (tdTom+) cells. We found that Ascl1-CKO at E14.5 resulted in a decrease in the number of tdTom+ cells in the GM, but an increased number of tdTom+ cells in the WM. In contrast, Ascl1-CKO at P30 resulted in a significant reduction in the number of tdTom+ cells in both the GM and WM. Quantification of the percentage of tdTOM-labeled cells that had differentiated to mature oligodendrocytes revealed that Ascl1-CKO at E14.5 does not affect NG2 cell differentiation, while Ascl1-CKO at P30 accelerated NG2 cell differentiation. Taken together, these findings indicate that Ascl1 is differentially required to regulate the number of NG2 cells in the GM and WM during embryonic development, whereas Ascl1 is essential for regulating both the differentiation and number of NG2 cells in the adult CNS.Item Roles of Ascl1 and Olig2 in the Transcriptional Regulation of Astrocytogenesis(2016-01-19) Combiths, Adam; Vue, Tou Yia; Johnson, Jane E.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.