Cell Type-Specific Transcriptional Control of Gsk3β in the Developing Mammalian Neocortex

Abstract

Temporal control of neurogenesis is central for the development and evolution of species-specific brain architectures. The balance between progenitor expansion and neuronal differentiation is tightly coordinated by cell-intrinsic and cell-extrinsic cues. Wnt signaling plays pivotal roles in the proliferation and differentiation of neural progenitors in a temporal manner. However, regulatory mechanisms that adjust intracellular signaling amplitudes according to cell fate progression remain to be elucidated. Here, we report the transcriptional controls of Gsk3β, a critical regulator of Wnt signaling, in the developing mouse neocortex. Gsk3β expression was higher in ventricular neural progenitors, while it gradually declined in differentiated neurons. We identified active cis-regulatory module (CRM) of Gsk3β that responded to cell type-specific transcription factors, such as Sox2, Sox9, and Neurogenin2. Furthermore, we found extensive conservation of the CRM among mammals but not in non-mammalian amniotes. Our data suggest that a mammalian-specific CRM drives the cell type-specific activity of Gsk3β to fine tune Wnt signaling, which contributes to the tight control of neurogenesis during neocortical development.

Keywords: Gsk3β; Wnt signaling; evolution; neocortex; neurogenesis; promoter.

FIGURE 1

The Gsk3β promoter region acts as a CRM in response to cell type-specific transcription factors. (A,B) Histone ChIP-seq data of mouse E14.5 forebrain tissue focusing on the Gsk3β genomic locus (ENCODE). The peaks represent H3K4me3 and H3K27ac enrichment. (C) Schematic drawings of the Gsk3β promoter and exon 1 and reporter vectors containing these genomic fragments. (D,E) Luciferase activities of reporter vectors in HEK293T cells (D) and cultured mouse neocortex (E). (F–H) Luciferase activities of reporter vectors containing the Gsk3β promoter or exon 1 in response to cell type-specific transcription factors, including Sox9 (F), Neurogenin2 [Ngn2, (G)], NeuroD and Sox2 (H). (I) Putative binding sites of Sox2, Sox9, and Ngn2 in mouse Gsk3β promoter region. All graphs represent mean + s.e.m.; n = 3 or 4 independent samples. Statistical analyses were performed by ordinary one-way ANOVA (D,F,G), unpaired t-test (E), and Kruskal-Wallis test with Dunn’s multiple comparison’s test (H). P-values are represented in each graph.

FIGURE 2

Species-specific genomic structures of Gsk3β in amniotes. (A–E) Expression patterns of Gsk3β in E15.5 mouse neocortex (A,B) and 23 dpo gecko dorsal cortex (C–E). Conservation analyses of Gsk3β genomic loci among representative vertebrate species [(F); mVISTA] and 12 amniote species [(G); UCSC conservation track]. Peaks in (F) and vertical lines in (G) represent conserved genomic regions in comparison of mouse genomic sequences (mm10). The purple box in (G) represents the region corresponding to the 2 kb promoter of mouse Gsk3β. (H) Conservation analyses of Gsk3β genomic loci among non-mammalian species. Peaks represent conserved genomic regions in comparison of anole lizard genomic sequences (Ano Car2.0.v2). CP: cortical plate; IMZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone; MC: medial cortex; DC: dorsal cortex; NL: neuronal layer. Scale bars: 100 μm (B), 50 μm (C,E).