Crosstalk between genetic and epigenetic components plays a crucial role in determining cell fate of neural stem cells during cortical neurogenesis in the developing brain, according to new research from Byoung-San Moon, Wange Lu and colleagues.
During neurogenesis, NSCs are derived from neuroepithelial cells (NECs), which first divide symmetrically to expand the population and then undergo a series of asymmetric cell divisions to produce neural progenitor cells (NPCs), lineage-restricted precursor cells (RPCs), and mature neural cells. NSC fate determination is tightly regulated by intrinsic and extrinsic factors.
Recent findings suggest that neurodevelopmental and neurological anomalies, such as schizophrenia, autism, and depression, can emerge from abnormal specification, growth, and differentiation of NSCs.
Suppressor Of Mek Null
Suppressor of Mek null (Smek), an evolutionarily conserved protein family, consists of two isoforms, Smek1 (PP4R3A) and Smek2 (PP4R3B), first reported as playing a role in the formation of a functional phosphatase group with PP4c, PP4R1, and PP4R2 complex. Smek was initially identified in Dictyostelium discoideum as a playing a role in cell polarity, chemotaxis, and gene expression.
Smek also has several functions in lower eukaryotes, such as Caenorhabditis elegans, including roles in longevity by modulating DAF-16/FOXO3a transcriptional activity, DNA repair through dephosphorylation of phosphorylated H2AX (g-H2AX) during DNA replication, and glucose metabolism by controlling cAMP-response element binding protein (CREB)-regulated, transcriptional coactivator 2 (CRTC2)-dependent gene expression.
Notably, Smek also plays a critical role in cell-fate determination in higher eukaryotes.
Suppressor of Mek null (Smek) expression and function in mouse cortical development. (A) Fixed cyroembedded coronal sections from E12.5 or E14.5 mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), and microtubule-associated protein 2 (MAP2) (red). Nuclear staining is shown by 4′,6-diamidino-2-phenylindole (DAPI) (blue). (Right) Quantification of Fig 1A and S1D Fig. (E12.5: WT, n = 4, dKO, n = 5; E14.5: WT, n = 6, dKO, n = 5). Scale bars, 50 μm. (B) Same as Fig 1A for Pax6. (Right) Quantification of Fig 1B (E12.5: WT, n = 5, dKO, n = 4; E14.5: WT, n = 6, dKO, n = 3). Scale bar, 50 μm. (C) Immunostaining with Nestin (green) and Tuj1 (red) antibodies in WT and Smek1/2 dKO neural progenitor cells (NPCs). Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of anti-Tuj1–positive (WT-Un, n = 3; WT-2DIV, n = 6; dKO-Un, n = 3; dKO-2DIV, n = 6) and anti-Nestin–postive (WT-Un, n = 6; WT-2DIV, n = 6; dKO-Un, n = 6; dKO-2DIV, n = 6) cells in Fig 1C. Un, undifferentiation; 2Diff, differentiation 2 d in vitro (DIV). (D) Single cells of WT and Smek1/2 dKO NPCs were separated by serial dilution and sphere formation was induced for 8 d in vitro. Relative size of primary spheres grown up to 8 DIV were quantified by the ImageJ quantification software. Scale bars, blue (100 pixel), red (200 pixel). (E) Quantitative PCR (qPCR) analysis of indicated mRNAs. Values correspond to the average ± SD. Diff. (d), days in differentiation. Statistical t test analysis was performed to calculate significance (*p < 0.05, **p < 0.005, ***p < 0.0005; not significant (ns), p > 0.05). Credit: pbio.2001220
In Drosophila neuroblasts, PP4R3/Falafel (Flfl), which is an orthologous of Smek and is conserved throughout eukaryotic evolution, regulates asymmetric cell division by controlling localization of Miranda. In mice, which express orthologous Smek 1 and 2, both Smek proteins suppress brachyury expression in embryonic stem cells (ESCs), and Smek1, especially, promotes NSC neuronal differentiation by negatively regulating Par3.
Although it has been shown that the Smek isoform Smek1 promotes NSC neuronal differentiation, signaling pathways required for that activity remain unclear.
Methyl-CpG–binding domain protein 3 (Mbd3), a core component of the repressive nucleosome remodeling and deacetylase (NuRD) complex, possesses a conserved methyl-CpG–binding domain (Mbd). Unlike other family members, which recognize 5′-methyl-cytosine (5′-mC)-modified DNA, Mbd3 specifically recognizes 5′-hydroxymethyl-cytosine (5′-hmC), an epigenetic marker highly enriched in NSCs.
Mbd3 plays an important role in brain development. Mbd3 expression is reported to be predominant in cortical NECs of the embryonic forebrain. Mice lacking Mbd3 die in utero before neurogenesis is completed.
Conditional knockout of Mbd3 in neural progenitor cells leads to defects of differentiation of appropriate cell types during neurogenesis. Despite emerging evidence that Mbd3 has a critical function in the CNS, little is known about its regulatory mechanism in NSCs.
Neural Progenitor Cell Differentiation
To understand Smek protein function during mammalian CNS neurogenesis, Byoung-San Moon’s team screened for novel Smek-binding proteins that regulate NPC neuronal differentiation and identified Mbd3, a potent epigenetic regulator, as a Smek-interacting protein. They found that Mbd3 is highly expressed in NPC populations in the ventricular zone, and it was predominantly expressed in the nucleus.
Smek interacted directly with the Mbd3’s Mbd domain, destabilizing Mbd3 protein and its interaction with NuRD components, and sequentially, preventing accumulation of the Mbd3/NuRD complex on target gene loci functioning in neurogenesis. Such dissociation of Mbd3/NuRD complex promotes NPC neuronal differentiation.
Moreover, overexpression of Mbd3 significantly inhibited neuronal differentiation of wild-type NPCs, while Mbd3 depletion rescued neurogenesis defects seen in Smek knockout mice.
The work identifies a novel pathway of Smek and Mbd3/NuRD complex in brain development and could encourage discovery of novel epigenetic regulators governing neuronal differentiation.