Emerging mechanisms underlying astrogenesis in the developing mammalian brain

Abstract

In the developing brain, the three major cell types, i.e., neurons, astrocytes and oligodendrocytes, are generated from common multipotent neural stem cells (NSCs). In particular, astrocytes eventually occupy a great fraction of the brain and play pivotal roles in the brain development and functions. However, NSCs cannot produce the three major cell types simultaneously from the beginning; e.g., it is known that neurogenesis precedes astrogenesis during brain development. How is this fate switching achieved? Many studies have revealed that extracellular cues and intracellular programs are involved in the transition of NSC fate specification. The former include growth factor- and cytokine-signaling, and the latter involve epigenetic machinery, including DNA methylation, histone modifications, and non-coding RNAs. Accumulating evidence has identified a complex array of epigenetic modifications that control the timing of astrocytic differentiation of NSCs. In this review, we introduce recent progress in identifying the molecular mechanisms of astrogenesis underlying the tight regulation of neuronal-astrocytic fate switching of NSCs.

Keywords: astrogenesis; central nerve system (CNS); epigenetics; neural stem cells (NSCs).

Figure 1.

Schematic illustration of cytokine signaling pathways related to transition from neurogenic to astrogenic competence of NSCs. ① During mid-gestation, by the activation of Wnt/β-catenin signaling, a complex consisting of β-catenin and T-cell factor (TCF) is formed in the nucleus to evoke Neurog1 expression. Then, ② Neurog1 binds to form a complex with P300/CBP, and to upregulate neuronal genes such as NeuroD. During late-gestation, ③ both the JAK-STAT3 pathway and BMP signaling cooperate and synergistically induce the target genes, in which STAT3 and Smads form a complex via P300/CBP. Neurog1 no longer deprives P300/CBP-Smad1 complex of STAT3, due to decreased Neurog1 expression in late-gestation, promoting interaction of STAT3 with P300/CBP.

Figure 2.

NFIA potentiates astrocytic differentiation of mgNSCs. A, B. NSCs derived from mouse telencephalons at embryonic day 11.5 (E11.5) were infected with retroviruses engineered to express green fluorescent protein (GFP) alone (A) or GFP together with NFIA (B), cultured for 24 hr in the presence of bFGF, and then stimulated with LIF for a further 3 days to induce astrocytic differentiation. The cells were stained with antibodies against GFP (green) and GFAP (red). Scale bar = 50 µm. C. GFAP-positive astrocytes in GFP control (GFP) and GFP-NFIA-expressing (NFIA) cells were quantified. Data are shown as means ± SD. Statistical significance was examined by Student’s t test (**p < 0.01). D. NSCs derived from E11.5 mouse telencephalons were infected with GFP control (GFP) or GFP-NFIA-expressing (NFIA) retroviruses, and cultured for 4 days with bFGF. After cell sorting based on GFP fluorescence, genomic DNA was extracted, and the methylation status of the Gfap promoter including the STAT3 binding site was examined by bisulfate sequencing. Red indicates STAT3 binding site. Filled portion of circles indicates the percentage of methylation at each CpG site. E. Methylation frequency of the CpG site within the STAT3 binding sequence in the Gfap promoter. Data are shown as means ± SD (n = 3). Statistical significance was examined by Student’s t test (*p < 0.05). F. ChIP assay with a specific antibody for DNMT1 from GFP- and GFP-NFIA-expressing retrovirus-infected NSCs, cultured as in A and B. (Reproduced with modification from Namihira et al., 2009.²⁴⁾)

Figure 3.

Schematic illustration of the alterations of epigenetic modifications in NSCs from mid- to late-gestation. ① SETDB1 associates with Gfap promoter, leading to transcriptional repression of the gene mediated by repressive methylation of H3K9. ② The neuronal-committed precursors (neuroblasts) and newly generated immature neurons expressing Notch ligands such as DLL1 activate Notch signaling in neighboring NSCs, producing cleaved Notch (NICD). Then, released NICD binds to RBP-Jκ and activates Nfia expression. NFIA dissociates DNMT1 from the Gfap promoter, resulting in demethylation at the region. ③ In a hypoxic environment, stabilized HIF1α associates with NICD and enhances the transcriptional activity of RBP-Jκ/NICD complex. ④ RAR/RXR forms a complex with transcriptional repressors, leading to formation of a closed chromatin structure. Once RA binds to RAR, the repressor complex is replaced with the activator complex, inducing a relaxed chromatin structure through H3K9ac. ⑤ miR-153 negatively regulates the expression of Nfia. ⑥ The ER stress-transducing protein OASIS induces GCM, which may contribute to active demethylation of the Gfap promoter.

Figure 4.

Chromatin accessibility is important for the GFAP expression. (A) Western blot analysis of tyrosine-phosphorylated STAT3 and total STAT3 in wild-type (WT) and triple knockout (TKO) ESCs. (B) The expression of GFAP in each cell type. WT and TKO ESCs were stained with antibodies against GFAP (blue). E14 NSCs-derived astrocytes were used as a positive control for staining. Nuclei were stained with Hoechst 33258 (white). (C) The DNA methylation status of the Gfap promoter in wild-type (WT) and triple-knockout (TKO) ESCs. Red indicates STAT3 binding site. Filled portion of circles indicates the percentage of methylation at each CpG site. (D) MNase digestion status of Gfap promoter around STAT3 binding site. Quantitative PCR against MNase-digested products from WT ESCs (WT), TKO ESCs (TKO), E11.5 NSCs, E14.5 NSCs, neurons, astrocytes, and mouse embryonic fibroblasts (MEFs) was performed. Amounts of genomic DNA remaining after MNase digestion were determined by qPCR. Each mean value was normalized by that of a linker histone H1foo gene. The H1foo gene is silent in all cells except for oocytes, and the region of its locus used here was completely resistant to MNase digestion in all the tested cell types. Error bars represent standard deviation from the mean of three independent experiments. **P < 0.01, *P < 0.05. (Reproduced with modification from Urayama et al., 2013³⁹⁾)