Npas3 regulates stemness maintenance of radial glial cells and neuronal migration in the developing mouse cerebral cortex

Abstract

The neuronal PAS domain 3 (NPAS3) is a member of the basic helix-loop-helix (bHLH) PAS family of transcription factors and is implicated in psychiatric and neurodevelopmental disorders. NPAS3 is robustly expressed in the cortical ventricle zone (VZ), a transient proliferative zone containing progenitor cells, mainly radial glial cells, destined to give rise to cortical excitatory neurons. However, the role of NPAS3 in corticogenesis remains largely unknown. In this study, we knocked down Npas3 expression in the neural progenitor cells residing in the cortical VZ to investigate the role of Npas3 in cerebral cortical development in mice. We demonstrated that Npas3 knockdown profoundly impaired neuronal radial migration and changed the laminar cell fate of the cells detained in the deep cortical layers. Furthermore, the downregulation of Npas3 led to the stemness maintenance of radial glial cells and increased the proliferation rate of neural progenitor cells residing in the VZ/subventricular zone (SVZ). These findings underline the function of Npas3 in the development of the cerebral cortex and may shed light on the etiology of NPAS3-related disorders.

Keywords: NPAS3; cerebral cortex; neuronal migration; radial glial cells; stemness maintenance.

Figure 1

The expression of Npas3 in the developing mouse cerebral cortex. (A) Quantification for Npas3 mRNA levels in the developing cerebral cortex at the indicated stages obtained by RT-PCR. Data are presented as mean ± SEM (n = 3). One-way ANOVA test, **p < 0.01, ****p < 0.0001. (B) Western blot analysis of NPAS3 protein levels in the developing cerebral cortex at the indicated stages. GAPDH was used as a loading control.

Figure 2

Npas3 downregulation impaired neuronal radial migration in vivo. (A) Non-transfected HEK293T cells (mock) or cells transfected with an expression plasmid encoding mouse NPAS3-GFP protein together with Npas3 shRNAs or control pSuper-basic plasmid were lysed 48 h after transfection and processed to Western blot analysis with anti-GFP and anti-GAPDH antibodies. Quantification of the relative protein levels is shown in the right panel. Data are presented as mean ± SEM (n = 3). One-way ANOVA test, ****p < 0.0001. GAPDH was used as a loading control. (B) and (C) Positioning defects of Npas3-knockdown cells. E14.5 mouse embryos electroporated with Npas3 shRNAs or control pSuper-basic plasmid together with a GFP expression plasmid were allowed to develop until P0 (B) and P3 (C). Electroporated cells are in green, and nuclei are in blue. Quantification of cell position is shown on the right panel. Data are presented as mean ± SEM (n = 3–5). Two-way ANOVA test, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ucp, upper cortical plate; Lcp, lower cortical plate; IZ, intermediate zone; SVZ, subventricular zone; Cortical layers are labeled I–VI. Scale bar, 100 μm.

Figure 3

Npas3 knockdown affected the laminar fate of cells detained in deep cortical layers. (A,B) Coronal sections of P3 (A) and P7 (B) brains electroporated at E14.5 were subjected to immunostaining with the anti-GFP antibody together with anti-Cux1 or anti-Ctip2 antibodies. Representative images with higher magnifications of Npas3 knockdown cells in layers II–IV, V and VI are shown in the lower panels. Note that most Npas3 knockdown cells in upper layers II–IV are Cux1 positive (arrowheads), while the majority of those in deep layers V and VI are Cux1 negative (arrows). Electroporated cells are in green, and nuclei are in blue. Cortical layers are labeled I–VI. Quantification of Cux1 positive cells from (A) and (B) is shown in (C) and (D) as % of total electroporated cells, ±SEM (n = 3–5). One-way ANOVA test, *p < 0.05.

Figure 4

Npas3 knockdown promoted the stemness maintenance of radial glial cells in the developing mouse cerebral cortex. Npas3 shRNAs or pSuper-basic control plasmid together with a GFP expression plasmid were electroporated into mouse embryos at E14.5. The brains of mouse pups were harvested at P3 or P7 and processed for immunostaining with anti-GFP antibody together with antibodies against one of the following markers: NeuroD2 (A), BLBP (C), and Pax6 (F, G). Slices were counterstained with Hoechst (blue) to indicate lamination. High-magnification images of Npas3 knockdown cells layers II-IV, V and VI and WM were presented at the right or lower panel. Note that most Npas3 knockdown cells in layers II-IV were Neurod2 positive (arrowheads) and BLBP negative (arrows). In contrast, the majority of Npas3 knockdown cells in WM were NeuroD2 negative (arrows), but BLBP and Pax6 positive (arrowheads). Quantification of NeuroD2 positive cells from (A), BLBP positive cells from (C), and Pax6 positive cells from (F) are shown in (B), (D), and (E), respectively, as % of total electroporated cells, ±SEM (n = 3–5). One-way ANOVA test, **p < 0.01, ***p < 0.001. WM, white matter; Cortical layers are labeled I–VI.

Figure 5

Npas3 knockdown resulted in an increased proliferation rate of progenitors residing in VZ and SVZ. BrdU was injected into E16.5 pregnant mice 2 days after in utero electroporation. Mouse brains were collected 2 h later after BrdU injection, and the brain sections were processed for immunostaining with antibodies against BrdU (A), Ki67 (B), and Sox2/Tbr2 (C). Quantification of BrdU positive cells from (A), Ki67 positive cells from (B), and Sox2⁺Tbr2⁺ (C) is shown in (D), (E), and (F), respectively, as % of total electroporated cells, ±SEM (n = 3–5). The asterisks indicate the statistically significant difference with respect to control mice (one-way ANOVA test, *p < 0.05, **p < 0.01). VZ, ventricular zone; SVZ, subventricular zone.