Conditional Loss of BAF (mSWI/SNF) Scaffolding Subunits Affects Specification and Proliferation of Oligodendrocyte Precursors in Developing Mouse Forebrain

Abstract

Oligodendrocytes are responsible for axon myelination in the brain and spinal cord. Generation of oligodendrocytes entails highly regulated multistage neurodevelopmental events, including proliferation, differentiation and maturation. The chromatin remodeling BAF (mSWI/SNF) complex is a notable regulator of neural development. In our previous studies, we determined the indispensability of the BAF complex scaffolding subunits BAF155 and BAF170 for neurogenesis, whereas their role in gliogenesis is unknown. Here, we show that the expression of BAF155 and BAF170 is essential for the genesis of oligodendrocytes during brain development. We report that the ablation of BAF155 and BAF170 in the dorsal telencephalic (dTel) neural progenitors or in oligodendrocyte-producing progenitors in the ventral telencephalon (vTel) in double-conditional knockout (dcKO) mouse mutants, perturbed the process of oligodendrogenesis. Molecular marker and cell cycle analyses revealed impairment of oligodendrocyte precursor specification and proliferation, as well as overt depletion of oligodendrocytes pool in dcKO mutants. Our findings unveil a central role of BAF155 and BAF170 in oligodendrogenesis, and thus substantiate the involvement of the BAF complex in the production of oligodendrocytes in the forebrain.

Keywords: BAF complex; BAF155 and BAF170; OPC specification and proliferation; oligodendrocyte development; oligodendrogenesis.

FIGURE 1

Cortex-specific loss of BAF155 and BAF170 led to decreased number of OPCs in postnatal cortex. (A) Coronal brain sections of hGFAP-Cre, Rosa-tdTom transgenic mouse embryo revealing the cortex-specific hGFAP-Cre recombinase activity pattern at E15.5 (Nguyen et al., 2018). (B) Graph showing the variance distribution or clustering in a principal component (PC) analysis of the four control and four mutants P3 hGFAP-Cre dcKO cortices used for the RNA sequencing. In a principal analysis (C) Volcano plot showing the distribution of genes upregulated and downregulate in the P3 hGFAP-Cre dcKO cortices compared with control. Examples of key oligodendrogenesis-related genes downregulate in the BAF155 and BAF170 mutant cortex are indicated. (D) Immunostaining of PDGFRα (green) in coronal sections of control and dcKO_hGFAP-Cre brains at P3. (E) Quantitative analysis comparing the percentage of the PDGFRα⁺ cells per total cortical cells (DAPI⁺) in the control and hGFAP-Cre dcKO cortex at P3. Data are presented as means ± SEMs; ***p < 0.001; Experimental replicates (n) = 7. dTel, dorsal telencephalon; vTel, ventral telencephalon. Scale bars = 200 μm (A,D).

FIGURE 2

BAF155 and BAF170 expression in the oligodendroglial lineage in the ventral telencephalon. (A) Diagram illustrating the stepwise oligodendrocyte lineage progression and the expression pattern of key molecular drivers. Curved arrows denote proliferation. (B–F) Micrographs showing BAF155 and BAF170 expression (B) alongside the oligodendrocyte lineage markers Olig2, Sox10 for pan-oligodendrocyte (C,F), Sox9 for glioblasts (D), and PDGFRα for OPCs (E) in coronal sections of the mouse forebrain at E15.5 using immunohistochemistry. (G) Bar graph showing quantitative estimation of proportion of Olig2, Sox9, PDGFRα, and Sox10 positive cells with BAF155 and BAF170 expression. Inserted box shows selected area for quantification. Arrows refer to the oligodendrocytic lineage expressing BAF155 and BAF170. Experimental replicates (n) = 3. NSC, neural stem cell; OPC, oligodendrocyte precursor cell; iOL, immature oligodendrocyte, mOL, mature oligodendrocyte. Scale bars = 200 μm (B), 25 μm (C), 50 μm (D–F).

FIGURE 3

Decrease in the number of Olig2-expressing cells due to loss of BAF155 and BAF170 in ventral telencephalon. (A,B) Immunohistochmical micrograph (A) and quantitative analyses (B) indicating diminished number of Olig2⁺ cells caused by loss of BAF155 and BAF170. (C,E,G) In situ hybridization micrographs using Digoxigenin-labeled Olig2 RNA probes in the control and dcKO_Olig2-Cre E15.5 mouse telencephalon along the rostral–caudal axis. (D,F,H) Statistical analyses indicate significant depletion of Olig2⁺ cells in the rostral middle, and caudal levels of the dcKO_Olig2-Cre mutant telencephalon compared with control. Data are expressed as means ± SEMs. Experimental replicates (n) = 6; **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Scale bars = 200 μm (A), 100 μm (D).

FIGURE 4

Specification of oligodendrocyte precursors is impaired in the dcKO_Olig2-Cre. (A,C) Immunomicrographs showing Sox9 and Sox10 in the E13.5 (A) E15.5 (C) control and dcKO_Olig2-Cre forebrain. Selected regions of the striatum are shown at higher magnification. Yellow arrows point to cells co-expressing Sox9 and Sox10. Green arrows point to cells expression only Sox10. Red arrows point to cells expressing only Sox9. DAPI counter staining is shown. (B,D) Bar plots showing statistical difference in the number of cells (co)expressing Sox9, Sox10, or Sox9/Sox10 in the E13.5 (B) E15.5 (D) control and dcKO_Olig2-Cre forebrain. Data are shown as means ± SEMs. Experimental replicates (n) = 6; *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001. Scale bars = 100 μm.

FIGURE 5

Conditional removal of BAF155 and BAF170 leads to an impaired proliferative capacity of OPCs. (A) Images showing immunostaining of PDGFRα-labeled oligodendrocyte precursor cells in the dcKO E15.5 forebrains compared with controls. (B,C) High magnification images of the outlined regions (inserted boxes) of the ventral telencephalon showing immunolabeling of PDGFRα and Ki67 (B) and PDGFRα and IdU (C) to mark proliferating oligodendrocyte precursor cells in the E15.5 control and mutant forebrain. Filled arrows show PDGFRα positive oligodendrocyte precursor cells expressing Ki67 or IdU, and empty arrows point to PDGFRα oligodendrocyte precursor cells negative for Ki67 or IdU. (D,E) Bar charts showing significant reduction of the ratio of PDGFRα⁺ and Ki67⁺ per total PDGFRα⁺ cells as well as the ratio PDGFRα⁺ and IdU⁺ per total PDGFR⁺ cells in the dcKO_Olig2-Cre ventral telencephalon compared with control. (F) High magnification images showing PDGFRα, Sox10 (violet) and Casp3 immunostaining in the E15.5 ventral telencephalon. Empty arrows point to PDGFRα⁺ and Sox10⁺ cells lacking Casp3 expression. Data are presented as means ± SEMs; ****p ≤ 0.0001; Experimental replicates (n) = 6. Scale bars = 200 μm (A), 50 μm (B,C,F).

FIGURE 6

Number of PLP⁺, MBP⁺ oligodendrocytes is reduced in dcKO_Olig2-Cre mutant forebrain at E18.5. (A–C) In situ hybridization images showing the E18.5 control and dcKO_Olig2-Cre forebrain coronal sections with riboprobed for PLP expression. (D,E) Bar charts showing quantification of PLP-expressing cells in the E18.5 control and dcKO_Olig2-Cre at the middle (D), and caudal (E) levels of the ventral telencephalon Experimental replicates (n) = 5, Scale bars = 100 μm. (F–H) Micrographs showing the rostral, middle, and caudal sections of the E18.5 mouse forebrain immunostained with MBP and with DAPI counterstaining. Inserted images are higher magnification of MBP-expressing cells indicate by white and red solid arrows. (I,J) Bar graph showing significantly diminished number of cells expression MBP in the E18.5 control striatum compared with that of dcKO_Olig2-Cre. Note that PLP⁺, MBP⁺ cells are very rare in E18.5 control and dcKO_Olig2-Cre forebrain at rostral level and are not included in statistical analysis. Data are presented as means ± SEMs; ****p ≤ 0.0001); Experimental replicates (n) = 6. Scale bars = 100 μm.

FIGURE 7

Schema summarizing the effects of loss of chromatin remodeling BAF complex activity on oligodendroglial lineage progression in BAF155/BAF170dcKO and Brg1cKO mutants. The illustration shows a comparison of the progression of oligodendrocyte development in the developing mouse forebrain between control (Wild-type, in the left panel) and BAF mutants (in the right panel), i.e., BAF155/BAF170cKO mutants (this study) and Brg1cKO mutants (Yu et al., 2013; Bischof et al., 2015). In control, multipotent neural stem cell (NSC) gives rise to oligodendrocyte precursor cells, which are able to proliferate (shown by curved arrow) and differentiate into immature oligodendrocyte (imOL). The imOL undergoes maturation to become fully myelinating mature oligodendrocyte (mOL). However, in the absence of BAF155 and BAF170, the OPC is improperly specified from the mutant NSC and undergoes reduced proliferation (this study). In addition, in the absence of Brg1, OPCs were unable to differentiate and mature properly (Yu et al., 2013; Bischof et al., 2015). The diagrams at the end of the schema depict the overall depletion of OLs in the BAF mutant brain compared with control. References to key studies that reported phenotypes due to ablation of the BAF complex are indicated. Dashed-arrows are used to indicate defective developmental processes.