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. 2016 Sep 9;3(1):e1232679.
doi: 10.1080/23262133.2016.1232679. eCollection 2016.

Prenatal deletion of DNA methyltransferase 1 in neural stem cells impairs neurogenesis and causes anxiety-like behavior in adulthood

Hirofumi Noguchi  1 , Ayaka Kimura  1 , Naoya Murao  1 , Masakazu Namihira  2 , Kinichi Nakashima  1
Affiliations

Prenatal deletion of DNA methyltransferase 1 in neural stem cells impairs neurogenesis and causes anxiety-like behavior in adulthood

Hirofumi Noguchi et al. Neurogenesis (Austin). .

Abstract

Despite recent advances in our understanding of epigenetic regulation of central nervous system development, little is known regarding the effects of epigenetic dysregulation on neurogenesis and brain function in adulthood. In the present study, we show that prenatal deletion of DNA methyltransferase 1 (Dnmt1) in neural stem cells results in impaired neurogenesis as well as increases in inflammatory features (e.g., elevated glial fibrillary acidic protein [GFAP] expression in astrocytes and increased numbers of microglia) in the adult mouse brain. Moreover, these mice exhibited anxiety-like behavior during an open-field test. These findings suggest that Dnmt1 plays a critical role in regulating neurogenesis and behavior in the developing brain and into adulthood.

Keywords: Dnmt1; anxiety; dentate gyrus; hippocampus; neurogenesis.

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Figures

Figure 1.
Figure 1.
Prenatal deletion of Dnmt1 impairs cell proliferation and neurogenesis in the adult brain. (A) Diagram of the Dnmt1 conditional knockout strategy in NSCs. Dnmt1flox/flox mice were crossed with Nestin-CreERT2 mice, yielding progeny in which tamoxifen administration results in the deletion of Dnmt1 (exons 4 and 5) in Nestin-expressing NSCs. (B) Experimental scheme for assessing the effect of Dnmt1 deletion on cortical development and neurogenesis. Pregnant mice were injected with tamoxifen (2 mg) at E15 to induce deletion of Dnmt1 in NSCs, and the brains of the offspring were analyzed at 8-week old. (C) Representative immunofluorescence images of DCX (red) in sagittal adult (8-week old) brain sections from control and cKO mice. White open rectangles indicating the RMS and DG are shown in higher magnification images, respectively. (D) DCX immunostaining images (white) of representative newborn neurons in the DG of control and cKO mice. (E) DCX immunostaining images (red) of representative SVZ neurons in coronal adult brain sections from control and cKO mice. White open rectangles in the left panel images are shown in higher magnification in the right images. (F) DCX+ cells in (E) were quantified along the lateral wall of the lateral ventricle (mm) (Control = 3, cKO = 3). (G) Ki67 immunostaining (green) images of representative SVZ neurons in coronal adult brain sections from control and cKO mice. The nucleus was stained with Hoechst (Blue). (H) Ki67+ cells in (G) were quantified along the lateral wall of the lateral ventricle (mm) (Control = 3, cKO = 3). Scale bars are indicated in each figure. Values represent mean ± SEM; *P < 0.05, ***P < 0.001. Student's t-test. NSC: neuronal stem cells; Dnmt1: DNA methyltransferase 1; DCX: doublecortin; RMS: rostral migratory stream; DG: dentate gyrus; SVZ: subventricular zone.
Figure 2.
Figure 2.
Prenatal deletion of Dnmt1 increases expression of GFAP in astrocytes (A) Representative immunofluorescence images of GFAP (red) in the cortex of coronal brain sections from control and cKO mice at various time points. (B) GFAP immunostaining images (red, left panels) of representative cortical neurons in coronal adult brain sections from control and cKO mice. The nucleus was stained with Hoechst (gray, right panels). Ctx: cortex. (C) GFAP immunostaining images (red) of representative regions including the SVZ in coronal adult brain sections from control and cKO mice. (D) Representative GFAP immunofluorescence images (red) of cortical neurons in coronal brain sections from control and cKO mice at various time points. (E) GFAP (red) and S100β (green) immunostaining images of representative cortical astrocytes in coronal brain sections from adult control and cKO mice. (F) Representative image of staining for S100β (green) in the cortex of coronal brain sections from adult control and cKO mice. (G) Quantification of S100β+ cells in the cortex in (F). (Control = 3, cKO = 3). Scale bars are indicated in each figure. Values represent mean ± SEM; n.s > 0.05. Student's t-test. GFAP: glial fibrillary acidic protein.
Figure 3.
Figure 3.
Prenatal deletion of Dnmt1 increases microglia in the adult brain. (A) Representative Iba1 immunofluorescence images (green) in the cortex of coronal brain sections from control and cKO mice at various time points. (B) Quantification of IbaI+ cells in the cortex at P35. (Control = 3, cKO = 3). (C) Iba1 immunostaining image (green) of representative DG neurons in coronal brain sections from control and cKO mice at various time points. The nucleus was stained with Hoechst (blue). (D) Quantification of IbaI+ cells in the DG at P35. (Control = 3, cKO = 3) (E) Representative immunofluorescence images of Iba1 (green) and CD68 (red) in the DG of coronal adult brain sections from control and cKO mice at indicated time point. The nucleus was stained with Hoechst (blue). Scale bars are indicated in each figure. Values represent mean ± SEM; *P < 0.05, **P < 0.01. Student's t-test.
Figure 4.
Figure 4.
Prenatal deletion of Dnmt1 induces anxiety-like behavior in adult offspring. (A-C) Exploratory behavior in the open-field test. Histograms show the total distance moved in the test (A) and proportion of time spent in the center area of arena (B) (Control = 6, cKO = 6). (C) Digital tracking of mice exposed to the open-field test. Representative traces of mice activity (gray) obtained from video tracking. Blue box indicates the center area of arena. Values represent mean ± SEM; n.s > 0.05, *P < 0.05. Student's t-test.
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