MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism‐like behaviors
2021; Springer Nature; Volume: 22; Issue: 7 Linguagem: Inglês
10.15252/embr.202052150
ISSN1469-3178
AutoresHongyan Ma, Libo Su, Wenlong Xia, Wenwen Wang, Guohe Tan, Jianwei Jiao,
Tópico(s)Family and Disability Support Research
ResumoArticle27 May 2021free access MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors Hongyan Ma Hongyan Ma State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Libo Su Libo Su State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Wenlong Xia Wenlong Xia State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Wenwen Wang Wenwen Wang State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China School of Life Sciences, University of Science and Technology of China, Hefei, China Search for more papers by this author Guohe Tan Corresponding Author Guohe Tan [email protected] Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education, Guangxi Key Laboratory of Regenerative Medicine, School of Basic Medical Sciences and Center for Translational Medicine, Guangxi Medical University, Nanning, Guangxi, China Search for more papers by this author Jianwei Jiao Corresponding Author Jianwei Jiao [email protected] orcid.org/0000-0002-7893-0721 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Hongyan Ma Hongyan Ma State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Libo Su Libo Su State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Wenlong Xia Wenlong Xia State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Wenwen Wang Wenwen Wang State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China School of Life Sciences, University of Science and Technology of China, Hefei, China Search for more papers by this author Guohe Tan Corresponding Author Guohe Tan [email protected] Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education, Guangxi Key Laboratory of Regenerative Medicine, School of Basic Medical Sciences and Center for Translational Medicine, Guangxi Medical University, Nanning, Guangxi, China Search for more papers by this author Jianwei Jiao Corresponding Author Jianwei Jiao [email protected] orcid.org/0000-0002-7893-0721 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Author Information Hongyan Ma1, Libo Su1,2, Wenlong Xia1,2, Wenwen Wang1,3, Guohe Tan *,4 and Jianwei Jiao *,1,2,5,6 1State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 2University of Chinese Academy of Sciences, Beijing, China 3School of Life Sciences, University of Science and Technology of China, Hefei, China 4Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education, Guangxi Key Laboratory of Regenerative Medicine, School of Basic Medical Sciences and Center for Translational Medicine, Guangxi Medical University, Nanning, Guangxi, China 5Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China 6Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, China *Corresponding author. Tel: +86 771 5606035; E-mail: [email protected] *Corresponding author. Tel: +86 010 64806335; E-mail: [email protected] EMBO Reports (2021)22:e52150https://doi.org/10.15252/embr.202052150 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2-deficient mice exhibit autism-like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders. SYNOPSIS The histone variant mH2A1.2 is critical for mouse embryonic neurogenesis. It promotes the expression of NKX2.2, which affects the differentiation and proliferation of neural progenitor cells during embryonic development. mH2A1.2 promotes the differentiation and inhibits the proliferation of neural progenitor cells during mouse embryonic development. mH2A1.2, possibly together with Brd4, promotes NKX2.2 transcription. Loss of mH2A1.2 leads to developmental defects with autistic-like behaviors in mice. Introduction During the formation of the mammalian brain, neural progenitor cells give rise to neurons and astrocytes through symmetrical and asymmetrical divisions (Kriegstein & Alvarez-Buylla, 2009; Ming & Song, 2011; Florio & Huttner, 2014). The function of the brain is precisely regulated by many genes and epigenetic regulators (Haubensak et al, 2003; Noctor et al, 2004; Fuentealba et al, 2015). During development, deletion of certain genes may cause neurodevelopmental disorders such as autism and depression (Taliaz et al, 2010; Ming & Song, 2011; Li et al, 2019). During brain development, epigenetic mechanisms have pivotal roles to regulating gene expression at different stages of neurogenesis (Hirabayashi & Gotoh, 2010; Yao et al, 2016). Different combinations of epigenetic molecules and transcription factors can modify the primary DNA sequence and result in differential gene expression in different cell types (Ma et al, 2010; Albert et al, 2017). Epigenetics mainly involves the heritable expression of cell phenotypes in which changes in gene expression are caused by mechanisms such as histone modification, DNA methylation, and chromatin remodeling (Yao et al, 2016). Histone variants, the products of epigenetic modifications, affect the activation or silencing of gene transcription by regulating the structure of chromatin (Henikoff & Smith, 2015). Of all histones, only the mH2A variant has an evolutionarily conserved 25 kDa carboxy-terminal globule at the C-terminus, called the macrodomain, which interacts with metabolites and histone modifiers (Pehrson & Fried, 1992; Ladurner, 2003; Chakravarthy & Luger, 2006; Gamble & Kraus, 2010; Hussey et al, 2014). Transforming the classic H2A protein into an inhibitory mH2A variant is one of the most obvious epigenetic chromatin changes that occur at the nucleosome level (Buschbeck & Di Croce, 2010; Chen et al, 2014). mH2A not only inactivates the X chromosome, but also positively or negatively regulates the transcription of specific genes (Kapoor et al, 2010; Kim et al, 2013; Dell'Orso et al, 2016). mH2A has two isoforms, mH2A1 and mH2A2, which are encoded by the H2Afy1 and H2Afy2 genes, respectively. MacroH2A1 transcripts are alternately spliced to produce two macroH2A1 variants, macroH2A1.1 and macroH2A1.2, which differ in an ∼30 amino acid regions of the macrodomain (Gamble & Kraus, 2010; Creppe et al, 2012). MACROH2A1 was identified as a candidate gene in a previous genome-wide linkage analysis for autism (GWAS) (Philippi et al, 2005; Karine et al, 2007). Autism spectrum disorder (ASD) is a neurodevelopmental disorder in which various sensorimotor processes are affected, leading to significant qualitative difficulties in behavior, social interaction, and communication with others (Tang et al, 2014; Lin et al, 2016; Trutzer et al, 2019). The presence of mH2A in the monkey brain is mentioned in the article as having an important role for neurons, and its absence may lead to similar neurological disorders such as autism or Rett syndrome (Liu et al, 2016). In addition to this, studies of macroH2A in zebrafish embryonic development in previous reports have shown that macroH2A variants constitute an important epigenetic mark involved in the concerted regulation of gene expression programs during cellular differentiation and development (Buschbeck et al, 2009; Gonzalez-Munoz et al, 2019). It is therefore important to elucidate the mechanisms by which mH2A affects neurogenesis at the genetic level during embryonic development. Although it is generally accepted that mH2A has an important regulatory role in gene expression, it is unclear whether mH2A affects neurogenesis during embryonic development and leads to autism-like symptoms. We therefore explored the functions and mechanisms of mH2A in terms of neurogenesis and animal behavior. To this end, we knocked down mH2A1.2 in embryonic neural stem cells (NSCs) by in utero electroporation (IUE) and knocked out the H2Afy1 gene encoding mH2A1 in mice. The entire MACROH2A1 gene and the expression of both splice variants downregulated. We found that silencing mH2A1.2 promoted the proliferation of mouse progenitor cells and inhibited differentiation. We discovered that downregulation of mH2A1.2 expression or deletion of the entire MACROH2A1 gene resulted in abnormal cortical development and abnormal neuronal dendrite morphology. Mechanistically, histone variants have multiple covalent modifications, including methylation and acetylation. Among these modifications, acetylation at lysine 27 of histone H3 (H3K27ac) around the TSS is an indicator of the transcriptional program of embryonic stem cells (Vermunt et al, 2016). We found that mH2A1.2 interacts with Brd4 to regulate the expression of the downstream molecule NKX2.2 molecule, thereby promoting gene transcription activated by H3K27ac. Brd4 is recruited to the activated enhancer by the acetylation of H3K27, facilitating the initiation and activation of gene transcription during brain embryo development (Rahnamoun et al, 2018). Based on behavioral tests, a significant decrease in exploratory and social skills was observed in the mH2A1.2 KO mice, and ultrasound experiments also showed a tendency toward autism. Taken together, we showed that loss of mH2A1.2 can affect neurogenesis during brain embryogenesis and that KO mice exhibit autism-like behavior. Results mH2A1.2 is expressed in the developing brain The histone variant mH2A has two isoforms, mH2A1 and mH2A2, and mH2A1 transcripts are alternately spliced to produce two macroH2A1 variants, macroH2A1.1 and macroH2A1.2 (Hurtado-Bagès et al, 2020). To investigate the function of mH2A1.2 during the development of the nervous system, we quantified the relative abundance at the mRNA level and found that the mouse brain contained mainly mH2A1, and mH2A1.2 was the predominant component of mH2A1 (Fig EV1A and B). Since these data suggest that the macroH2A1.2 form constitutes more than 80% of all cellular macroH2A in the mice brain, we decided to focus on the functional role of this isoform. Next, we used Western blotting to determine the expression pattern and found that mH2A1.2 expression was dynamic during brain development. The results showed that mH2A1.2 was expressed at E13(embryonic 13), and as development progressed, the expression level gradually decreased (Fig 1A and C). The expression pattern was similar to that of PAX6, which is a marker of neural progenitor cells. Furthermore, RT–PCR (qRT–PCR) was used to explore several time points in the developmental process, and the results were consistent with the Western blotting results. The temporal expression pattern of mH2A1.1 in the developing cortex was similar to that of mH2A1.2 (Fig EV1C), In addition, we used immunohistochemistry (IHC) to determine the location of mH2A1.2 during brain development and found that mH2A1.2 was expressed in the VZ/SVZ and CP layers at all stages of development and localized in the nucleus (Fig 1B). We also isolated neural progenitor cells from E12(embryonic 12) mice to detect mH2A1.2 in vitro. The results showed that mH2A1.2 was also expressed in neural progenitor cells in vitro and costained with SOX2, PAX6, and NESTIN (Fig 1D). mH2A1.2 was also coexpressed with the TUJ1 neuronal marker (Fig EV1D). These experimental data show that mH2A1.2 is expressed in neural progenitor cells and neurons during embryonic development. Click here to expand this figure. Figure EV1. mH2A1.2 is predominantly expression in the developing brain and neural progenitor cells qRT–PCR analysis shows that exogenous expression of mH2A1 with mH2A2. n = 3 mice, independent replicates. qRT–PCR analysis shows that exogenous expression of mH2A1.1 with mH2A1.2 n = 3 mice, independent replicates. The temporal expression (E13, E15, E17, and P0) of mH2A1.1 and mH2A1.2 mRNAs by qRT–PCR in the developing cortex. mH2A1.2 is co-labeled with PAX6 and TUJ1 in neural progenitor cells and neurons cultured in vitro. Scale bar represents 50 μm. Western blot analysis shows that exogenous Flag-mH2A is efficiently reduced in mH2A1.2-shRNA-transfected 293 cells. Graph shows that the amount of mH2A is obviously decreased in mH2A1.2-shRNA-transfected 293 cells. n = 5 samples, independent replicates. Immunostaining images show that endogenous mH2A1.2 is obviously reduced in mH2A1.2-shRNA-electroporated cells. Hatched lines are cell boundaries. Date information: Representative images from at least three independent experiments. Error bars represent means ± S.E.M.; two-tailed unpaired t-test, **P < 0.01 or ***P < 0.001. n.s., not significant. Download figure Download PowerPoint Figure 1. mH2A1.2 is expressed in the developing brain mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0). Immunofluorescence staining for mH2A1.2 in the cerebral cortex at E13, E15, and P0. Scale bar represents 100 μm. A scatter diagram shows the changing trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates. mH2A1.2 is expressed in neural progenitor cells and neurons cultured in vitro. Scale bar represents 50 μm. Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm. Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ, and CP. n = 7 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± SEM; two-tailed unpaired t-test, **P < 0.01 or ***P < 0.001. n.s., not significant. Download figure Download PowerPoint To explore the function of mH2A1.2 in development, we designed mH2A1.2 shRNA to knockdown the expression of mH2A1.2. Western blotting showed that mH2A1.2 was obviously decreased in 293T cells infected with the mH2A1.2-shRNA lentivirus (Fig EV1E and F). Next, we transferred mH2A1.2-shRNA and control plasmids into the brains of E13.5 mice using the in utero electroporation method and harvested the brains at E16.5. mH2A1.2 was also decreased in shRNA-electroporated cells in vivo (Fig EV1G). The coronal sections were subjected to immunofluorescence staining (Fig 1E). The number of GFP+ cells in the VZ/SVZ region was significantly increased, and the number of GFP+ cells in the IZ region was greater than that in the control group, but the number of GFP+ cells in the cortical plate (CP) layer was significantly reduced compared with that in the control group (Fig 1F). These results indicate that mH2A1.2 is abundantly expressed during development and that mH2A1.2 knockdown causes abnormal cell distribution in the developing neocortex, also indicating that macroH2A1.2 can affect cortical neurogenesis. mH2A1.2 knockout promotes neural progenitor cell proliferation To further investigate the functional role of mH2A1.2 in brain development, we constructed mH2A1 knockout mice (Fig 2A). We designed a gRNA to target the MACROH2A1 encoded by H2Afy1. gRNA leads to total MACROH2A1 KO which mainly affects splice isoform macroH2A1.2. Guide RNA mediated the Cas9 nuclease cleavage of the gene sequences in the first and eighth exons of the H2Afy gene. We used Western blotting and RT–PCR to examine the knockout efficiency in the macroH2A1 KO mice. Compared with WT mice, KO mice barely expressed endogenous mH2A1.2 (Fig 2B and C and Fig EV2B and C). In addition, mH2A1.2 was scarcely expressed in the brains of KO mice compared with WT mice by IHC (Fig EV2D). To study the role of mH2A1.2 during brain development, the embryonic brain at E13.5 was transformed with GFP plasmid by the IUE method and harvested at E16.5. Quantitative staining showed that GFP+ cells had an abnormal distribution compared with WT mice. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ cells in the CP layer increased significantly (Fig 2D and E). This result is consistent with the results of mH2A1.2 knockdown. We also counted the number of GFP+ cells in the same-sized area after harvesting the brains of E16 mice and found no significant differences (Fig EV2A). Figure 2. Loss of mH2A1.2 increases proliferation of neural progenitor cell Schematic of the mH2A1 knockout mouse construction strategy. The H2Afy1 gene-encoded mH2A1 was knocked out by the CRISPR-Cas9 method. Western blot analysis of mH2A1 expression levels in WT and mH2A1 KO cortices at E13.5. Graph shows the normalized density of mH2A1.2 versus control. n = 6 mice, independent replicates. Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ, and CP. n = 5 mice, independent replicates. Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm. Statistics of pH3+ cells of the cortex. n = 6 mice, independent replicates. Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. Quantification of GFP and Pax6 double-positive cells in the VZ/SVZ. n = 6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± SEM; two-tailed unpaired t-test, *P < 0.05, **P < 0.01 or ***P < 0.001. n.s., not significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. mH2A1.2 regulates the proliferation of neural progenitor cells Counted the number of GFP+ cells in the same size area after E16 harvesting of the brain and found no significant differences. n = 6 mice, independent replicates. Expression of mH2A1.2 at the WT and KO mRNA levels was analyzed by qRT–PCR. n = 5 mice, independent replicates. Expression of mH2A1.1 and mH2A1.2 at the WT and KO mRNA levels was analyzed by qRT–PCR. n = 5 mice, independent replicates. Immunostaining shows that the expression of mH2A1.2 is depleted in the E16.5 KO mice brains. Scale bar represents 50 μm. Mouse embryos were electroporated in utero with control, mH2A1.2-shRNA at E13.5 and the harvested brain sections were immune-stained with mitotic marker pH3 at E16.5. Scale bar represents 50 μm. Graph shows the percentage of pH3+GFP+ cells in VZ/SVZ. The increase in the number of pH3+ cells in the coronal sections of mH2A1.2 knockdown mice at E16.5. n = 6 mice, independent replicates. BrdU was injected intraperitoneally for 2 h of pulse labeling, and the harvested brain sections were immune-stained with anti-BrdU. Scale bar represents 50 μm. n = 8 mice, independent replicates. Percentage of GFP+BrdU+ cells among GFP+ cells in VZ/SVZ. More GFP+ BrdU+ cells were found in the brains of KO than in those of WT mice. n = 8 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent means ± S.E.M.; two-tailed unpaired t-test, *P < 0.05, **P < 0.01, or ***P < 0.001. n.s., not significant. Download figure Download PowerPoint To confirm whether the increase in GFP+ cells in the VZ/SVZ region is due to increased proliferation of neural progenitor cells, we used pH3 stain to label the mitotic activity of cells and observed that mH2A1.2 knockdown increased mitotic activity (Fig EV2E and F). The increase in the number of pH3+ cells in the coronal sections of KO mice at E16.5 also confirmed that mitotic activity was increased when mH2A1.2 was deleted (Fig 2F and G). Immunofluorescence staining revealed that the number of GFP+ and PAX6+ costained cells increased, which suggests that the number of neural progenitor cells increased (Fig 2H and I). We also labeled progenitor cells with BrdU two hours in advance, and IHC analysis revealed that there were more GFP+ BrdU+ cells in the brains of KO mice than in the brains of WT mice, suggesting that knockout of mH2A1.2 increases the amount of incorporated BrdU in the GFP+ cell population (Fig EV2G and H). Although previous reports indicated we understand that the mH2A variant in the mouse brain is predominantly mH2A1.2 (Posavec Marjanović et al, 2017), but mH2A1.1 is also expressed in mouse brain (Kozlowski et al, 2018). We explored whether the mH2A1.1 variant has a role during embryonic development. We used electroporation to overexpress the mH2A1.1 plasmid in the brains of E13.5 mice, and collected brain sections at E16 and P0 (Fig EV3A and C), to determine that the distribution of GFP+ was not significantly affected (Fig EV3B and D). These results suggest that overexpression of mH2A1.1 did not have a significant effect on the proliferation or differentiation of neural stem cells. Click here to expand this figure. Figure EV3. The role of mH2A1.1 in neural stem cell differentiation and migration The mH2A1.1 overexpressed plasmid was electroporated into mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ, and CP. The GFP-positive cells distribution no significant change. n = 5 mice, independent replicates. The mH2A1.1 overexpressed plasmid was electroporated into mice brains at E15.5, and the mice were sacrificed at P0. Scale bar represents 50 μm. Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ, and CP. The GFP-positive cell distribution no significant change. n = 5 mice, independent replicates. Representative images of E16.5 cortices electroporated with Control, mH2A1.2-shRNA and mH2A-shRNA+ mH2A1.2 into mice at E13.5, the mouse was sacrificed at E16.5. Scale bar represents 50 μm. Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ, and CP. mH2A1.2-shRNA+ mH2A1.2 restored the distribution of GFP+ cells to the normal state. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent means ± S.E.M.; two-tailed unpaired t-test, *P < 0.05, **P < 0.01. n.s., not significant. Download figure Download PowerPoint Next, we used electroporation to overexpress the mH2A1.2 plasmid in the brains of mH2A1.2 KO and WT mice. Compared with the control group, the number of GFP+ cells in the VZ/SVZ brain regions of the KO mice was rescued by overexpression of mH2A1.2. The number of GFP+ cells in the CP layer appeared to be normal (Fig EV4A and B). This suggests that the abnormal distribution of GFP+ cells observed in mH2A1.2-deficient embryos can be rescued by overexpression of the mH2A1.2 plasmid. Similar results were obtained using mH2A1.2-shRNA and mH2A1.2 overexpression by in utero electroporation (Fig EV3E and F). The above experimental results suggest that mH2A1.2 affects NPC proliferation in early embryonic development. Click here to expand this figure. Figure EV4. mH2A1.2 knockdown affects proliferation and differentiation of neural progenitor cells Representative images of E16.5 cortices electroporated with GFP into WT and KO brain and GFP+mH2A1.2 into mice at E13.5, the mouse was sacrificed at E16.5. Scale bar represents 50 μm. Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ, and CP. Overexpression of mH2A1.2 restored the distribution of GFP+ cells to the normal state. n = 5 mice, independent replicates. Western blot analysis reveals that the expression levels of the neural stem cell markers, including pH3, PCNA, PAX6, TBR2, and mH2A1.2, are increased in the mH2A knockdown NSCs versus the control. Statistics of the normalized density of pH3, PCNA, PAX6, TBR2, and mH2A1.2. n = 3 mice, independent replicates. Western blot analysis reveals that the expression levels of the neuron markers NeuN, Tuj1, and mH2A1.2 are downregulated in mH2A1.2 knockdown NSCs versus the control. Statistics of the normalized density of NeuN, TUJ1, and mH2A1.2. n = 3 mice, independent replicates. Representative images of mH2A1.2 WT and KO cortex at newborn (P0) by hematoxylin and eosin stain (H&E) staining. Reduced cortical thickness is observed in KO cortex. CP: cortical plate. Scale bar = 800 μm. Quantification of the cortical thickness in mH2A1.2 WT and KO cortex. Reduced cortical thickness is observed in KO cortex. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent means ± S.E.M.; two-tailed unpaired t-test, *P < 0.05, **P < 0.01. n.s., not significant. Download figure Download PowerPoint Deletion of mH2A1.2 inhibits neural progenitor cell differentiation Deletion of mH2A1.2 leads to abnormal distribution of GFP+ cells in the VZ/SVZ region, and radial glial cells (RGs) and intermediate progenitor cells (IPs) are the main progenitor cell types in the VZ/SVZ region. To determine whether the deletion of mH2A1.2 affects the lineage transition of progenitor cells from the VZ to the SVZ, we compared the proportion of TBR2+ cells in the VZ/SVZ region of WT and KO mice (Fig 3A) and found that the number of TBR2+ cells increased more significantly (Fig 3B). Consistently, the immunolabeling of BrdU on coronal sections from WT and KO mice showed that more BrdU-incorporating cells were detected in KO mice (Fig 3C), suggesting that progenitor cells in S phase were also increased when mH2A1.2 was ablated. To test whether the increase in the number of TBR2+ cells is due to the proliferation of IPs alone, we injected BrdU to label proliferating IPs two hours before the brain was harvested. The results confirmed that TBR2+BrdU+ cells were increased in KO mice compared with WT mice (Fig 3D). These data demonstrated that mH2A1.2 regulates brain neural progenitor cell proliferation. In addition, we injected BrdU into mice, and 24 h later, we removed the brain at E16.5 for immunofluorescence analysis of BrdU and Ki67 staining. We found that the percentage of cells in the mouse brain that exited the cell cycle decreased after mH2A1.2 deletion (BrdU+Ki67−/BrdU+Ki67+ cells; Fig 3E and F), indicating that increased cell proliferation was accompanied by decreased cell cycle exit. Figure 3. Depletion of mH2A1.2 reduces neural progenitor cell differentiation Representative images of E16.5 coronal brain sections immune-stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2 h of pulse labeling. Scale bar represents 20 μm. Statistics of TBR2+ cells per 100 μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared with WT mice. n = 6 mice, independent replicates. Statistics of BrdU+ cells per 100 μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared with WT mice. n = 6 mice, independent replicates. Percentage of TBR2+BrdU+ cells among all TBR2+ cells. TBR2+BrdU+ cells were increased in KO mice compared with WT mice. Two-tailed unpaired t-test. n = 6 mice, independent replicates. Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice
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