Polycomb Ezh2 controls the fate of GABAergic neurons in the embryonic cerebellum

Abstract

Although the genetic interactions between signaling pathways and transcription factors have been largely decoded, much remains to be learned about the epigenetic regulation of cerebellar development. Here, we report that cerebellar deletion of Ezh2, the methyltransferase subunit of the PRC2 complex, results in reduced H3K27me3 and profound transcriptional dysregulation, including that of a set of transcription factors directly involved in cerebellar neuronal cell-type specification and differentiation. Such transcriptional changes lead to increased GABAergic interneurons and decreased Purkinje cells. Transcriptional changes also inhibit the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately results in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays crucial roles in regulating neurogenesis from both cerebellar germinal zones.

Keywords: Cerebellum; Ezh2; Neurogenesis; PRC2; Polycomb; Purkinje cells.

Fig. 1.

Ezh2 and Pax7 are abundantly expressed in the cerebellar primordium. Ezh2 and Pax7 immunofluorescence staining in both Ezh2^cKO and littermate control cerebella at early embryonic stages E10.5 (top panels) and E12.5 (bottom panels). Mid, midbrain; Cb, cerebellum. for all panels, ventral (V) to the left, dorsal (D) to the right. Arrow indicates Cb region. Scale bar: 200 µm.

Fig. 2.

Cerebellar gene ablation of Ezh2 induces decreased H3K27me3 and developmental defects. (A,B) Embryonic brain sagittal sections of Ezh2^cKO and littermate control embryos at E12.5 (A) and E14.5 (B) were immunostained with an anti-H3K27me3 antibody and counterstained with DAPI to highlight the nuclei. Arrows in B indicate brain ventral regions. (C) H&E histology staining of E15.5 (top) and E17.5 (bottom) cerebellar para-sagittal sections from control and Ezh2^cKO cerebella. Arrows indicate the EGL. (D) Dorsal views of P8 control and Ezh2^cKO cerebella (top panel). H&E histology staining of para-sagittal sections through the hemisphere of P8 control and Ezh2^cKO cerebella (lower panel). Mid, midbrain; Cb, cerebellum; V, vermis; H, hemisphere. For all panels, ventral to the left, dorsal to the right. Scale bars: 200 µm in A-C; 800 µm in D, lower panel.

Fig. 3.

H3K27me3 genome-wide distribution. (A) Genomic H3K27me3 peak distribution in E13.5 control cerebellum (left panel) and average (global) H3K27me3 tag density in control (blue line) and Ezh2^cKO (red line) cerebella (right panel). H3K27me3 ChIP-seq was repeated with cerebella of two control and two Ezh2^cKO embryos (n=2). (B) Gene ontology analysis of genes corresponding to H3K27me3 promoter regions. (C) Genome browser views of H3K27me3 ChIP-seq traces on representative genes Pft1a, Atoh1 (left panels) Pax2 and En1 (right panels) involved in cerebellar neurogenesis in control and Ezh2^cKO cerebella.

Fig. 4.

Transcriptional dysregulation in Ezh2^cKO embryos. (A) Gene expression changes in E13.5 control and Ezh2^cKO cerebella. A list of selected up- and downregulated transcripts is indicated. RNA-seq was repeated with cerebella of four control and four Ezh2^cKO cerebella (n=4). (B) Venn diagram of H3K27me3 promoter regions in control and upregulated genes in Ezh2^cKO cerebella. (C) Gene ontology terms of 481 upregulated genes in E13.5 Ezh2^cKO cerebella.

Fig. 5.

Pax2⁺ interneurons are increased whereas Rorα⁺ Purkinje cells are decreased in Ezh2^cKO cerebella. (A) E13.5 para-sagittal sections were immunostained with anti-Pax2 (red) antibody and counter-stained with DAPI (blue) showing ectopic Pax2 expression in control and Ezh2^cKO cerebella. (B) E13.5 para-sagittal sections were immunostained with anti-Rorα antibody (green) and counter-stained with DAPI (blue), showing the Rorα⁺ cells were distributed in a more compact manner in control than in Ezh2^cKO cerebella. (C,D) Similar phenotypes were observed in E14.5 para-sagittal sections stained for Pax2 (C) and Rorα (D). The number of Pax2- and Rorα-positive nuclei on each section was quanitified and graphed on the right side of each image (n=3), P-value was calculated by paired t-test: A, P≈0.003; B, P≈0.006; C, P≈0.003; D, P≈0.002. Error bars represent mean±s.e.m. Anterior to the left, posterior to the right in all images. Scale bars: 200 µm.

Fig. 6.

Derepression of the Ink4B-Arf-Ink4A tumor suppressor locus and cell proliferation defects in Ezh2^cKO embryos. (A) RNA-seq (left panels) and H3K27me3 ChIP-seq (right panels) UCSC genome browser tracks for cdkn2a, -b and -c in Ezh2^cKO and littermate control embryos. (B) Relative expression (qPCR, top panel) and H3K27me3 promoter enrichment (ChIP-qPCR, bottom panel) for the Cdkn2a gene through embryonic stages in Ezh2^cKO and littermate control embryos. Experiments were performed with three biological replicates (n=3). Error bars represent mean±s.e.m. (C) Cerebellar sagittal sections at E14.5 (top panels) and E15.5 (bottom panels) of Ezh2^cKO and littermate control embryos were immunostained with anti-Pax6 antibody (green) counterstained with DAPI (blue). Pax6⁺ cells were quantified and graphed below the images (n=3), P-value was calculated by paired t-test: E14.5, P≈0.09; E15.5, P≈0.003. Dotted lines indicate the pial surface, the areas correspond to the areas in Fig. 7. Scale bar: 100 µm.

Fig. 7.

Reduced proliferation of granule precursor cells in the EGL of Ezh2^cKO embryos. (A,B) EdU was administrated by intraperitoneal injection on pregnant mice at E13.5 (A) and at E14.5 (B). The embryos were collected 24 h after the treatment. EdU (red) and Ki67 (green) immunofluorescence staining was analyzed on para-saggital sections. Each section was counterstained with DAPI (blue). EdU-positive nuclei (EdU⁺) and EdU/Ki67-double-positive nuclei (EdU⁺/Ki67⁺) in the EGL region were quantified and graphed (n=3). Error bars represent mean±s.e.m. The P-value of each pair was calculated by t-test: A, P≈0.07 (left graph), P≈0.02 (right graph); B, P≈0.004 (left graph),  P≤0.0001 (right graph). The number of EdU⁺ nuclei reflects the proliferation state of EGL cells at the time point of EdU treatment. The Edu⁺/Ki67⁺ ratio reflects the proliferation state of EGL cells after one day of development. Dotted lines indicate the pial surface and areas marked in Fig. 6. Arrows indicate representitives of EdU-positive but Ki67-negative nuclei (Edu⁺/Ki67⁻). Scale bar=50 µm.