Interferon-gamma inhibits the neuronal differentiation of neural progenitor cells by inhibiting the expression of Neurogenin2 via the JAK/STAT1 pathway.

Interferon-gamma (IFN-γ) is one of the critical cytokines released by host immune cells upon infection. Despite the important role(s) of IFN-γ in host immune responses, there has been no in vivo study regarding the effects of IFN-γ on brain development, and the results from many in vitro studies are controversial. In this study, the effects of IFN-γ on embryonic neurogenesis were investigated. Treatment of E14.5 mouse neural progenitor cells (NPCs) with IFN-γ resulted in a decrease in the percentage of TuJ1-positive immature neurons but an increase in the percentage of Nestin-positive NPCs. Similar results were obtained in vivo. Treatment of NPCs with a JAK inhibitor or the knockdown of STAT1 expression abrogated the IFN-γ-mediated inhibition of neurogenesis. Interestingly, the expression of one of proneural genes, Neurogenin2 (Neurog2) was dramatically inhibited upon IFN-γ treatment, and cells overexpressing Neurog2 did not respond to IFN-γ. Taken together, our results demonstrate that IFN-γ inhibits neuronal differentiation of NPCs by negatively regulating the expression of Neurog2 via the JAK/STAT1 pathway. Our findings may provide an insight into the role of IFN-γ in the development of embryonic brain.

Ultrasound backscatter microscopy image-guided intraventricular gene delivery at murine embryonic age 9.5 and 10.5 produces distinct transgene expression patterns at the adult stage.

In utero injection of a retroviral vector into the embryonic telencephalon aided by ultrasound backscatter microscopy permits introduction of a gene of interest at an early stage of development. In this study, we compared the tissue distribution of gene expression in adult mice injected with retroviral vectors at different embryonic ages in utero. Following ultrasound image-guided gene delivery (UIGD) into the embryonic telencephalon, adult mice were subjected to whole-body luciferase imaging and immunohistochemical analysis at 6 weeks and 1 year postinjection. Luciferase activity was observed in a wide range of tissues in animals injected at embryonic age 9.5 (E9.5), whereas animals injected at E10.5 showed brain-localized reporter gene expression. These results suggest that mouse embryonic brain creates a closed and impermeable structure around E10. Therefore, by injecting a transgene before or after E10, transgene expression can be manipulated to be local or systemic. Our results also provide information that widens the applicability of UIGD beyond neuroscience studies.

GSK3ß, but not GSK3α, inhibits the neuronal differentiation of neural progenitor cells as a downstream target of mammalian target of rapamycin complex1.

Glycogen synthase kinase 3 (GSK3) acts as an important regulator during the proliferation and differentiation of neural progenitor cells (NPCs), but the roles of the isoforms of this molecule (GSK3α and GSK3ß) have not been clearly defined. In this study, we investigated the functions of GSK3α and GSK3ß in the context of neuronal differentiation of murine NPCs. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3ß expression increased the percentage of TuJ1-positive cells, while knock-down of GSK3α seemed to have no effect. When primary NPCs were engineered to stably express either isoform of GSK3 using retroviral vectors, GSK3ß, but not GSK3α, inhibited neuronal differentiation and helped the cells to maintain the characteristics of NPCs. Mutant GSK3ß (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3ß is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3ß was delivered to E9.5 mouse brains using the ultrasound image-guided gene delivery technique. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, our results demonstrate that GSK3ß, but not GSK3α, negatively controls the neuronal differentiation of progenitor cells and that GSK3ß may act downstream of the mammalian target of rapamycin complex1 signaling pathway.

Notch intracellular domain deficiency in nuclear localization activity retains the ability to enhance neural stem cell character and block neurogenesis in mammalian brain development.

Notch has a broad range of regulatory functions in many developmental processes, including hematopoiesis, neurogenesis, and angiogenesis. Notch has several key functional regions such as the RBP-Jκ/CBF1 association module (RAM) domain, nuclear localization signals (NLS), and ankyrin (ANK) repeats. However, previous reports assessing the level of importance of these domains in the Notch signaling pathway are controversial. In this study, we have assessed the level of contribution of each Notch domain to the regulation of mammalian neural stem cells in vivo as well as in vitro. Reporter assays and real-time polymerase chain reactions show that the ANK repeats and RAM domain are indispensable to the transactivation of Notch target genes, whereas a nuclear export signal (NES)-fused Notch intracellular domain (NICD) mutant defective in nuclear localization exerts a level of activity comparable to unmodified NICD. Transactivational ability appears to be tightly coupled to Notch functions during brain development. Unlike ANK repeats and RAM domain deletion mutants, NES-NICD recapitulates NICD features such as promotion of astrogenesis at the expense of neurogenesis in vitro and enhancement of neural stem cell character in vivo. Our data support the previous observation that intranuclear localization is not essential to the oncogenesis of Notch1 in certain types of cells and imply the importance of the noncanonical Notch signaling pathway in the regulation of mammalian neural stem cells.