TCF 20 dysfunction leads to cortical neurogenesis defects and autistic‐like behaviors in mice
2020; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês
10.15252/embr.201949239
ISSN1469-3178
AutoresChao Feng, Jinyue Zhao, Fen Ji, Libo Su, Yihui Chen, Jianwei Jiao,
Tópico(s)Congenital heart defects research
ResumoArticle8 June 2020free access Source Data TCF20 dysfunction leads to cortical neurogenesis defects and autistic-like behaviors in mice Chao Feng State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Sino-Danish College at University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jinyue Zhao State Key Laboratory of Stem Cell and 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 Fen Ji State Key Laboratory of Stem Cell and 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 Libo Su State Key Laboratory of Stem Cell and 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 Yihui Chen Corresponding Author [email protected] Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Jianwei Jiao Corresponding Author [email protected] orcid.org/0000-0002-7893-0721 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Innovation Academy for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chao Feng State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Sino-Danish College at University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jinyue Zhao State Key Laboratory of Stem Cell and 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 Fen Ji State Key Laboratory of Stem Cell and 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 Libo Su State Key Laboratory of Stem Cell and 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 Yihui Chen Corresponding Author [email protected] Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Jianwei Jiao Corresponding Author [email protected] orcid.org/0000-0002-7893-0721 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China Innovation Academy for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Author Information Chao Feng1,2,3, Jinyue Zhao1,2, Fen Ji1,2, Libo Su1,2, Yihui Chen *,4 and Jianwei Jiao *,1,2,5 1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 2University of Chinese Academy of Sciences, Beijing, China 3Sino-Danish College at University of Chinese Academy of Sciences, Beijing, China 4Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai, China 5Innovation Academy for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China *Corresponding author. Tel: +86 021 65690520; E-mail: [email protected] *Corresponding author. Tel: +86 010 64806335; E-mail: [email protected] EMBO Rep (2020)21:e49239https://doi.org/10.15252/embr.201949239 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Recently, de novo mutations of transcription factor 20 (TCF20) were found in patients with autism by large-scale exome sequencing. However, how TCF20 modulates brain development and whether its dysfunction causes ASD remain unclear. Here, we show that TCF20 deficits impair neurogenesis in mouse. TCF20 deletion significantly reduces the number of neurons, which leads to abnormal brain functions. Furthermore, transcriptome analysis and ChIP-qPCR reveal that the DNA demethylation factor TDG is a downstream target gene of TCF20. As a nonspecific DNA demethylation factor, TDG potentially affects many genes. Combined TDG ChIP-seq and GO analysis of TCF20 RNA-Seq identifies T-cell factor 4 (TCF-4) as a common target. TDG controls the DNA methylation level in the promoter area of TCF-4, affecting TCF-4 expression and modulating neural differentiation. Overexpression of TDG or TCF-4 rescues the deficient neurogenesis of TCF20 knockdown brains. Together, our data reveal that TCF20 is essential for neurogenesis and we suggest that defects in neurogenesis caused by TCF20 loss are associated with ASD. Synopsis Transcription factor 20 (TCF20) is essential for neurogenesis. It promotes the expression of TDG, and TDG controls the DNA methylation level at the TCF-4 promoter, affecting TCF-4 expression and modulating neural differentiation. The TCF20-TDG-TCF-4 pathway has a role in neurogenesis. TCF20 knockout mice show neurogenesis defects with imbalanced differentiation and proliferation of neural progenitor cells. Deletion of TCF20 leads to autistic-like behaviors in mice. Introduction During cerebral cortex development, neuron generation from neural stem cells and progenitor cells is precisely controlled. Neural stem cells located in the ventricle zone and subventricle zone use symmetrical and asymmetrical division to self-renew and generate neuroblast cells. After terminal mitosis, newborn neurons migrate to the appropriate regions following the inside-out rule, at which point they form functional neural circuits 123. Precise control of neurogenesis is indispensable for normal brain development and function. Abnormal regulation of neurogenesis usually leads to neurodevelopmental disorders comprising ASD, ADHD, and intellectual disability 4. Autism spectrum disorders describe a series of neurodevelopmental disorders with deficits in social communication and interaction, as well as stereotypical repetitive behavior patterns 5. Because they are neurodevelopmental disorders, ASDs are lifelong diseases that have a huge impact on not only patients’ lives but also their families and society. Since being first reported by Kanner in 1943, the worldwide prevalence of autism has significantly increased from 1/2,500 to 1/100, and it is even higher in some countries, such as 1/86 in the UK and 1/59 in the United States 678. Due to the current global state of ASD, many efforts have been made to investigate its pathogenetic mechanism. Currently, genetic factors are regarded as the most important risk factors for the development of ASDs. For parents of a child with autism, the next child has a 20–50 times higher risk of suffering ASD than the next child of parents who have a child without autism 791011. However, the causes of autism are still unclear. Studies of autism candidate genes are urgently needed. Recently, many gene mutations were identified as high-risk factors in the pathogenesis of neurodevelopmental disorders through large-scale exome sequencing technology 1213. Among these novel gene mutations, de novo mutations of TCF20 (transcription factor 20) were identified in the sequencing data of autism patients, which might imply that TCF20 dysfunction is related to the development of ASD 141516. TCF20, also called stromelysin-1 platelet-derived growth factor (PDGF)-responsive element binding protein, is a transcription factor involved in the regulation of stromelysin-1 transcription 17. TCF20 was also found to play a role as transcriptional coactivator in modulating the transcriptional activity of Sp1, c-Jun, Est1, and RNF4 181920. TCF20 is located in the chromosome 22q13.2 region, which is close to the Phelan-McDermid syndrome (PMS) candidate gene shank3 21. PMS, known as 22q13.3 deletion syndrome, involves a range of phenotypes, including global developmental delay, intellectual disability, neonatal hypotonia, autism, and autistic-like behaviors, which has caused PMS to be considered a syndromic form of ASD 222324. TCF20 mutations or microdeletions were also found in PMS patients 212325. All these studies suggest that TCF20 dysfunction could be related to ASDs. However, because of a lack of direct evidence, it is still unclear whether TCF20 dysfunction causes ASDs. In addition, the function of TCF20 in the brain development process also remains unclear. To understand the biological function of TCF20 in neurogenesis and the possible relationship between TCF20 mutations and ASD, we generated TCF20 knockout mice and found that the deletion of TCF20 results in neurogenesis defects as well as autistic-like behavioral patterns. Specific depletion of TCF20 in NSCs leads to a decrease in neuronal differentiation and an increase in NPCs proliferation, which might be responsible for abnormal behaviors. In terms of molecular mechanisms, TCF20 regulates the expression of TDG, a DNA demethylation enzyme, by binding to the TDG promoter region and promoting its transcription. Furthermore, TDG reduces the methylation level in the promoter CpG island region of the downstream neural differentiation gene T-cell factor 4 (TCF-4), which enhances its expression. Overexpression of TDG or TCF-4 could rescue the phenotype caused by TCF20 dysfunction. Together, our work reveals that TCF20 is essential for neurogenesis in the developing brain, and we provide insight into the relevance of DNA methylation of neural differentiation genes to the etiology of ASDs. Results TCF20 is expressed in the cerebral cortex of mouse embryos To investigate the function of TCF20 in neurogenesis, we first detected the expression of TCF20 in the brain of E13 and E16 mouse embryos. The immunofluorescence results showed that TCF20 was ubiquitously expressed in cortex. Co-immunostaining with the neural progenitor cell (NPC) marker PAX6, NESTIN, and SOX2 in isolated neural progenitor cells from E13 embryos also revealed the expression of TCF20 in NPCs (Fig 1A and B). Then, we used absolute quantitative RT–PCR combined with Western blotting to detect the expression level during brain development. The results showed that expression increased throughout development until it reached an expression peak at E16, and then, expression slightly decreased (Fig 1C–E). Figure 1. TCF20 is expressed in the developing cerebral cortex and neural stem cells Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Costaining with the NSC markers NESTIN, SOX2, and PAX6 reveals that TCF20 is expressed in NSCs. Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0. n = 4 independently repeated tests. Western blotting results show the changes in TCF20 protein expression level during brain development. Statistical analysis of normalized band intensity in the Western blot results. n = 4 samples for each group. In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution. These electroporated embryos were collected at E16. Statistical analysis of the distributions of GFP-positive cells in different brain regions. The percentage of GFP-positive cells in the CP (cortical plate), IZ (intermediate zone), and VZ/SVZ (ventricle zone and subventricle zone) was analyzed. n = 7 brains. Data information: Bars and error bars represent the means ± SEM. Two-tailed unpaired t-tests were used to analyze the data, n.s. (no significant difference), P < 0.01 (**), P < 0.001 (***). Scale bar in (A) e13, 50 μm, e16 100 μm; (B) 50 μm; (F) 50 μm. Source data are available online for this figure. Source Data for Figure 1 [embr201949239-sup-0006-SDataFig1.TIF] Download figure Download PowerPoint Next, we generated 2 shRNA plasmids targeting the TCF20 CDS region. First, their knockdown efficiencies were confirmed through Western blotting, RT–qPCR, and immunostaining in isolated NSCs (Fig EV1A–E). Results indicated that TCF20-sh2 had a better efficiency. We next generated an overexpression vector of TCF20 CDS region. Western blotting showed that this overexpression vector had ability to overexpress TCF20 (Fig EV1F and G). Then, TCF20 knockdown plasmids were transferred into NPCs located in the VZ through in utero electroporation (IUE) at E13.5. After 3 days, these embryos were harvested and sectioned. We found that two TCF20 knockdown plasmids both resulted in the reduction of GFP+ cells proportion in the CP and the increase of GFP+ cells proportion in the IZ and VZ/SVZ (Fig 1F and G). The abnormal cell distribution might result from three possibilities: the imbalance of NSCs proliferation and differentiation, migration defects, and cell death. The increase proportion of GFP+ cells in VZ/SVZ might be ascribed to enhanced cell proliferation, while the decrease percentage of cells in CP might come from the cell differentiation defects or migration problems or both. These three possibilities would be separately analyzed in the following experiments. Click here to expand this figure. Figure EV1. TCF20 shRNAs and overexpression vector knock down and overexpress TCF20, respectively Western blotting analysis of TCF20 shRNA efficiencies. Statistical analysis of TCF20 band intensity; n = 3 samples for each group. RT–qPCR analysis of TCF20 shRNA efficiencies in knocking down RNA levels; n = 3 samples for each group. Immunostaining of TCF20 in TCF20 shRNAs infected isolated NSCs. White dashed lines show the cells transfected with control or knockdown vectors. Scale bar 50 μm. Statistical analysis of fluorescence intensity of TCF20. n = 11 cells. Western blotting analysis of TCF20 overexpression efficiencies. Statistical analysis of FLAG band intensity; n = 3 samples for each group. Data information: Bars and error bars represent the means ± SEM. Two-tailed unpaired t-tests were used to analyze the data, n.s. (no significant difference), P < 0.01 (**), and P < 0.001 (***). Source data are available online for this figure. Download figure Download PowerPoint Then, a rescue experiment was performed through co-IUE TCF20 knockdown vectors with TCF20 overexpression vectors. This result indicated that TCF20 shRNA did not have off-target effect (Fig EV2D and E). The abnormal cell distribution phenotype indeed resulted from TCF20 dysfunction. Because TCF20-sh2 had better knockdown efficiency and a more dramatic phenotype, further explorations of cell proliferation, differentiation, and migration were mainly carried out using the TCF20-sh2 group. Click here to expand this figure. Figure EV2. TCF20 depletion resulted in a reduction in cell cycle exit and differentiation A diagram of the BrdU labeling time point in cell cycle exit experiments. Co-immunostaining of Ki67 and BrdU. White arrows represent BrdU+Ki67−GFP+ cells. Scale bar = 50 μm. Statistic analysis of ratios of BrdU GFP double-positive but Ki67-negative cells in total BrdU GFP double-positive cells, n = 3 brains. Overexpression of TCF20 rescued TCF20 knockdown phenotype of the IUE experiment in E13 to E16 brains. Scale bar = 50 μm. Statistic analysis of percentage of GFP-positive cells in different zones. n = 3 brains. Immunostaining of CUX1 revealed that CUX1+ cell differentiation was insufficient when TCF20 was depleted from E15 to P0. Scale bar = 50 μm. Statistic analysis of ratio of Cux1 and GFP double-positive cells to GFP-positive cells, n = 3 brains. Data information: Bars and error bars represent the means ± SEM. Two-tailed unpaired t-tests were used to analyze the data, n.s. (no significant difference), P < 0.05 (*), P < 0.01 (**). Download figure Download PowerPoint TCF20 knockdown promotes neural progenitor cell proliferation and inhibits differentiation After we observed the abnormal GFP distribution of TCF20 knockdown mice, 2 h of BrdU (bromodeoxyuridine) labeling experiments was conducted to label proliferating cells. BrdU is an analog of thymidine that can label cells in the S-phase. Immunostaining of BrdU showed an increase in BrdU and GFP double-positive cells (Fig 2A–C). Furthermore, Western blotting of isolated NSCs with TCF20 knockdown also revealed that loss of TCF20 leads to an increase of SOX2 protein level (Fig 2D). Then, immunostaining of PAX6 and TBR2 was performed to investigate whether the cell proportions of radial glia cells (RGs) and intermediate progenitor cells (IPs) were disturbed after the loss of TCF20. We found the proportions of GFP+ PAX6+ cells and GFP+ TBR2+ cells both had different degrees of increase when TCF20 was depleted. Meanwhile, the percentage of GFP and proliferation marker SOX2 double-positive cells was also increased in TCF20 knockdown group (Fig 2E–I). The results suggested that loss of TCF20 enhances NPC proliferation. Since we already found that TCF20 knockdown affects cell proliferation, we next wondered what the effect of loss of TCF20 was on the cell cycle? Therefore, we performed a cell cycle exit experiment by co-immunostaining with Ki67 and BrdU. BrdU was injected into pregnant mice at E15, 24 h before sacrificing mother mice. Costaining of BrdU and Ki67 helped us to detect the percent of cells exiting cell cycles in the following 24 h after BrdU administration. The knockdown of TCF20 reduced the proportion of GFP+BrdU+Ki67− cells among the GFP+ BrdU+ cells population (Fig EV2A–C). This result indicated that the loss of TCF20 results in more cells remaining in the cell cycle rather than undergoing differentiation. Figure 2. TCF20 deficits result in abnormal cell proliferation A diagram of the BrdU labeling time point in electroporation experiments. Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm. Statistical analysis of 2-h BrdU labeling experiments. n = 6 brains. Western blotting analysis indicated an increase in the proliferation marker SOX2 in TCF20 depleted NSCs. Immunostaining with PAX6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n = 3 brains for each group. Immunostaining with SOX2 and PAX6 in E16 electroporated embryo brains. White arrows represent GFP, SOX2 double-positive cells; red arrows represent GFP, TBR2 double-positive cells. Scale bar: 50 μm. Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n = 8 brains for each group. Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n = 5 brains for each group. Data information: Bars and error bars represent the means ± SEM. Two-tailed unpaired t-tests were used to analyze the data, n.s. (no significant difference), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). Source data are available online for this figure. Source Data for Figure 2 [embr201949239-sup-0007-SDataFig2.TIF] Download figure Download PowerPoint Given that the knockdown of TCF20 leads to a reduction in GFP+ cells in the CP, a neuronal birthdate experiment was performed to investigate whether the neural differentiation was changed after TCF20 knockdown. IUE was performed with TCF20 knockdown plasmids in E13 embryonic brains. After 24 h, BrdU was injected into mother mice. All electroporated brains were collected at P0 for analysis of terminal mitosis. Staining with a BrdU antibody, we found that the percentage of GFP and BrdU double-positive cells in the CP was dramatically decreased. The data demonstrated that many NPCs could not proceed terminal mitosis and differentiate into neurons on time, which is consistent with the cell cycle exit. Furthermore, co-immunostaining was performed for SATB2, CTIP2, and TBR1. A significant reduction in GFP-positive cells that colocalizing with SATB2, CTIP2, or TBR1 was observed, which means that neural differentiation was disturbed after TCF20 knockdown (Fig 3D–H). Co-immunostaining with CUX1, a marker of upper layer neurons, also revealed that NPCs did not differentiate into neurons as a result of the loss of TCF20 (Fig EV2F and G). Taken together, these data showed that TCF20 knockdown in the cerebral cortex promotes cell proliferation but inhibits cell differentiation. Here, we thought the proliferation and differentiation dysregulation might be the main factor resulting in GFP+ cell abnormal distribution. The other two possibilities were analyzed in further experiments. Figure 3. TCF20 depletion impairs neuronal differentiation A. SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. B. Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP-positive cells. n = 3 brains. C. Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. D, E. Statistical analysis of CTIP2, GFP, and TBR1, GFP double-positive cell ratio in GFP-positive cells. n = 3 brains. F. A diagram of the BrdU labeling time point in electroporation experiments. G. Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. H. Percentage of BrdU and GFP double-positive cells in GFP-positive cells n = 3 brains for each group. Data information: Bars and error bars represent the means ± SEM. Two-tailed unpaired t-tests were used to analyze the data, n.s. (no significant difference), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). Scale bar: (C, G) 25 μm; (A) 50 μm. Download figure Download PowerPoint TCF20 knockout impairs neurogenesis in the embryo cerebral cortex TCF20 knockout mice were generated to further investigate the biological function of TCF20 during brain development. We designed 2 guide RNAs targeting the 2 ends of exon 2 of TCF20, and this strategy would result in the deletion of the majority of the TCF20 CDS area. Approximately 5kbp of genomic DNA was removed through CRISPR technology (Fig 4A). Western blotting and RT–qPCR results both confirmed that TCF20 was successfully deleted in TCF20 KO mice. In addition, immunostaining of brain slices confirmed this result (Fig 4B–F). Figure 4. Deletion of TCF20 impairs neurogenesis A. A diagram of the construction of TCF20 knockout mice. Two guide RNAs targeted the ends of TCF20 exon 2. With the help of cas9, TCF20 exon 2 was deleted from the genome. B. Western blotting analysis of TCF20 in TCF20 WT, HET, KO mice reveals that TCF20 was deleted. C. Statistical analysis of the normalized band intensity of TCF20. n = 3 samples for each genotype. D. RT–qPCR analysis of TCF20 in TCF20 WT, HET, and KO mice reveals that TCF20 was deleted. n = 4 samples. E. Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out. Scale bar: 50 μm. F. Statistical analysis of the normalized fluorescence intensity of TCF20. n = 6 samples for each genotype. G. Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation. H. Immunostaining of different cortical layer markers TBR1, CTIP2, and SATB2, White dash line represents the positive area. Scale bar: 50 μm. I–K. Statistical analysis of different cells layers in TCF20 WT, HET, and KO mice. n = 7 brains of each group. L. TCF20 deletion impairs neurogenesis and results in abnormal cell distribution. Scale bar: 50 μm. M. Statistical analysis of GFP+ distribution in different zones. n = 3 brains for each genotype. Data information: Bars and error bars represent the means ± SEM. Two-tailed unpaired t-tests were used to analyze the data, n.s. (no significant difference), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). Source data are available online for this figure. Source Data for Figure 4 [embr201949239-sup-0008-SDataFig4.TIF] Download figure Download PowerPoint We first detected several proliferation and differentiation makers in the TCF20 knockout mice. The differentiation markers SATB2 and NeuN were decreased with dosage dependence of the loss of TCF20, while the proliferation markers PAX6, SOX2, and PCNA had opposite results along with the loss of TCF20 (Fig 4G). Second, different layer markers CUX1, SATB2, CTIP2, and TBR1 were used to detect the differentiation of E16 TCF20 KO mice embryos. The layer thickness or cell numbers in different layers were decreased in TCF20 KO and HET mice (Figs 4H–K and EV3A and B). Third, in utero electroporation of E13-E16 TCF20 KO embryos with GFP plasmids, and the mice showed that TCF20 dysfunction resulted in abnormal cell distribution, which was similar to what was observed in the TCF20 knockdown experiments. After TCF20 deletion, the proportion of GFP+ cell located in the CP was significantly decreased, while GFP+ cells in the VZ/SVZ and IZ were increased, which indicated that TCF20 deletion promoted NPCs proliferation and inhibited differentiation (Fig 4L and M). And IUE experiment of GFP plasmids in TCF20 KO mice from E15 to P0 showed that most cells with GFP labeling could normally migrate into upper layer, which indicated that cell migration ability might not be affected in the late period after TCF20 deletion. However, because the differentiation and migration were strongly linked, it was hard to clearly distinguish them in our research system. Thus, we thought migration deficits might also be involved in the whole brain development process (Fig EV3C). TUNEL staining of E16 embryo cerebral cortex slices showed no obvious difference between WT and TCF20 KO mice. This result implied that TCF20 deletion did not cause apoptosis (Fig EV3D and E). These results suggested that TCF20 is essential for the balance between neural differentiation and proliferation. The cell cycle exiting result in TCF20 KO mice was similar to that of TCF20 knockdown experiments. Many NPCs still stayed in the cell cycle after TCF20 deletion (Fig EV3H–J). Then, we detected changes of microglia marker IBA1, and no significant differences were found after TCF20 knock out (Fig EV3K and L). Click here to expand this figure. Figure EV3. TCF20 deletion leads to abnormal cell proliferation and differentiation Immunostaining of CUX1. White dash line represents the positive area. Scale bar = 50 μm. Statistical analysis of Cux1-positive layer thickness in TCF20 KO mice and WT mice. n = 5 brains. IUE of GFP plasmids in WT and KO mice from E15 to P0 revealed cell migration did not affected by TCF20 deletion. Scale bar: 50 μm. TCF20 deletion did not induce cell apoptosis as shown in the TUNEL assay; a1 a2 represent 6.5× enlarged as the dashed boxes showed. Scale bar: 50 μm. Statistical analysis of TUNEL+ cells in 1,000 μm cortex. n = 5 brains. Co-immunostaining of Ki67 and BrdU in the cell cycle exit experiment. White arrows represent BrdU+Ki67−GFP+ cells. Scale bar: 50 μm. Statistical analysis of numbers of Ki67+ cells. n = 4 brains Statistical analysis of ratio of Ki67−, BrdU+ cells to BrdU+ cells. n = 4 brains. Immunostaining of microglia marker IBA1. Scale bar = 50 μm Statistical analysis of numbers of IBA1+ cells. n = 5 brains. Download figure Download PowerPoint The dysfunction of TCF20 leads to autistic-like behaviors in adult and pup mice Since the deletion of TCF20 disturbed neurogenesis in the mouse embryo cortex, we wondered whether TCF20 dysfunction would have phenotypes in mice similar to those in humans. Unfortunately, our homozygous TCF20 knockout (KO) mice could not survive to adulthood. During development, the homozygous TCF20 KO mice showed severe global development delay, decreased body weight and brain size from embryo to P14. Over 80% of homozygous TCF20 KO mice died before P14, so we used heterozygous TCF20 KO mice instead to conduct the adult behavioral test (Fig EV4A–I). Besides, heterozygous TCF20 KO mice are closer to the ASD patients in genotype. First, we performed an ultrasonic vocalization test on wild-type (WT), heterozygous TCF20 KO (HET), and homozygous TCF20 KO (KO) pups. Compared with the WT pups, the call numbers and total call durations were significantly reduced in HET and KO pups. However, for the single call duration, only the KO pup group showed a significant decrease. Although many HET pups also had shorter call durations, there was no significant difference. It seemed that KO pups had a worse ability to communication than HET and WT pups (Fig 5A–D). These results suggested that not only KO pups but also HET pups show autism
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