Earlier studies show that Hes1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of Hes1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of Hes1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA-9 (miR-9) is complementary to the 3′-UTR sequence of Hes1 mRNA. MiR-9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over-expression of miR-9 negatively regulates the Hes1 protein expression by interacting with the 3′-UTR of Hes1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR-9 inhibits neuronal differentiation. Furthermore, knockdown of miR-9 inhibits the oscillatory expression of Hes1 mRNA in neural stem cells. These results indicate that miR-9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes1 expression in the developing brain.

SUMMARYIn the developing brain, neural progenitor cells switch differentiation competency by changing gene expression profiles that are governed partly by epigenetic control, such as histone modification, although the precise mechanism is unknown. Here we found that ESET (Setdb1), a histone H3 Lys9 (H3K9) methyltransferase, is highly expressed at early stages of mouse brain development but downregulated over time, and that ablation of ESET leads to decreased H3K9 trimethylation and the misregulation of genes, resulting in severe brain defects and early lethality. In the mutant brain, endogenous retrotransposons were derepressed and nonneural gene expression was activated. Furthermore, early neurogenesis was severely impaired, whereas astrocyte formation was enhanced. We conclude that there is an epigenetic role of ESET in the temporal and tissue-specific gene expression that results in proper control of brain development. RESEARCH ARTICLE ESET in neural development MATERIALS AND METHODS Mouse linesESET flox (f) (Matsui et al., 2010), Emx2-Cre (Kimura et al., 2005) and Nes-Cre (Isaka et al., 1999) mice were described previously. Emx2-Cre;ESET(f/+) or Nes-Cre;ESET(f/+) male mice were crossed with ESET(f/f) female mice, and the day when a vaginal plug was observed was scored as embryonic day (E) 0.5. In situ hybridization, immunohistochemistry and antibodiesEmbryos were dissected, fixed in 4% paraformaldehyde (PFA)/PBS overnight and cryoprotected with 20% sucrose for at least 10 hours. On the following day, embryos were embedded in OCT compound (Tissue-Tek) and frozen at -80°C. Fixed samples were sectioned at 16 m with a cryostat and dried for 2 hours. In situ hybridization was then performed as previously described (Imayoshi et al., 2008). Primers used to produce in situ hybridization probes are listed in supplementary material Table S1. For immunohistochemistry, embryos at different ages were fixed in 4% PFA for 2 hours, equilibrated and sectioned as described above. Sections were blocked with 5% normal goat serum or donkey serum in 0.1% Triton X-100/PBS and incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: