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The Mutation in Chd7 Causes Misexpression of Bmp4 and Developmental Defects in Telencephalic Midline

  • Xuan Jiang
    Affiliations
    Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing, China
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  • Yue Zhou
    Affiliations
    Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing, China
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  • Li Xian
    Affiliations
    Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing, China
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  • Weiqian Chen
    Affiliations
    Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing, China
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  • Hanwei Wu
    Affiliations
    Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing, China
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  • Xiang Gao
    Correspondence
    Address reprint requests to Xiang Gao, Ph.D., Model Animal Research Center, 12 Xuefu Rd, Nanjing 210061, China
    Affiliations
    Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing, China

    Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, Wenzhou Medical College, Wenzhou, China
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      Mutations in chromosome-helicase-DNA-binding protein 7 (CHD7) are identified as the main cause for CHARGE syndrome (coloboma, heart anomaly, choanal atresia, retardation, genital and ear anomalies). Most patients (55% to 85%) with CHARGE syndrome display developmental defects in the central nervous system (CNS), of which pathology and molecular mechanisms remain unclear. In this study, we report a novel mutant mouse strain carrying a nonsense mutation, COA1, in exon4 of Chd7 gene. Chd7COA1/+ mice phenocopied human CHARGE syndrome and displayed developmental defects in the telencephalic midline, including dilated third and lateral ventricles, reduced cerebral cortex, and corpus callosum crossing failure. Programed cell death in the telencephalic midline zone of Chd7COA1/+ embryos was impaired, consistent with the incomplete telencephalic medial invagination in Chd7COA1/+ embryos. Interestingly, expression of Bmp4, a signal well known to induce forebrain midline cell fate and apoptosis, was down-regulated and also expanded in the forebrain of Chd7COA1/+ embryos. Furthermore, in vitro studies suggested that CHD7 may directly regulate Bmp4 expression by binding with an enhancer element downstream of the Bmp4 locus. These studies provide novel insight into pathogenesis of CNS anomalies in CHARGE syndrome.
      CHARGE syndrome is a recognizable genetic pattern of birth defects, including ocular coloboma, heart defects, choanae atresia, retarded growth and development, genital hypoplasia, and ear abnormalities, which affects ∼1 in 12,000 to 1 in 8500 newborns.
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      However, the pathology and cause of the forebrain defects are poorly understood.
      Most persons (60% to 80%) with CHARGE syndrome have been identified with chromodomain-helicase-DNA-binding protein 7 (CHD7) mutations, including nonsense, missense, and deletion mutations.
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      Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues.
      Genomic chromatin immunoprecipitation (ChIP) on tile microarrays and ChIP followed by massively parallel DNA sequencing analyses have shown that Chd7 protein binds to genomic sites with enhancer features; colocalizes with OCT4, SOX2, NANOG, SMAD1, and STAT3; and correlates with embryonic stem cell-specific gene expression in mouse embryonic stem cells, but Chd7 does not regulate self-renewal, pluripotency, or somatic reprogramming of mouse embryonic stem cells.
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      Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome.
      and it is also required for development and maintenance of gonadotropin-releasing hormone (GnRH) neurons
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      Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome.
      Chd7 has also been shown to promote neural crest gene expression in Xenopus
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      and proneural gene expression in the inner ear of mice.
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      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      Flies with reduced kismet protein (the Drosophila homolog of CHD7) expression exhibit defects in learning, memory, and gross motor coordination.
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      Kismet/CHD7 regulates axon morphology, memory and locomotion in a Drosophila model of CHARGE syndrome.
      In molecular level, Chd7 forms variable complexes and regulates downstream genes in a tissue- and stage-specific manner. In human neural crest-like cells, CHD7 associates with BRG-1 containing the SWI/SNF chromatin remodeling complex to control multipotent neural crest formation.
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      CHD7 cooperates with PBAF to control multipotent neural crest formation.
      In mouse neural stem cells, Chd7 activates disease-associated genes through a SOX2-CHD7 complex.
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      Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.
      In HeLa cells, CHD7 interacts with CHD8.
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      CHD8 interacts with CHD7, a protein which is mutated in CHARGE syndrome.
      The forebrain is developed from the embryonic telencephalon. Shortly after neural tube closure, the telencephalon is divided into left and right hemispheres by medial invagination. The medial invaginated tissues develop into the telencephalon midline. Defects in midline induction lead to a single telencephalic “holosphere” termed holoprosencaphaly. In the development of telencephalon, three patterning centers work synergistically to regulate regional specification and morphogenesis of the midline.
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      Coordinate regulation and synergistic actions of BMP4 SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles.
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      Dosage of Fgf8 determines whether cell survival is positively or negatively regulated in the developing forebrain.
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      Coordinate expression of Fgf8 Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles.
      The ventral patterning center expresses Shh, which induces the ventral cell fate.
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      Loss of Shh leads to up-regulation of bone morphogenetic protein (BMP) signaling and ablation of Fgf8 in the telencephalon.
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      Coordinate regulation and synergistic actions of BMP4 SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles.
      The dorsal patterning center expresses Bmp and Wnt proteins to promote midline induction, choroidal versus cortical specification, and the hippocampus development.
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      Coordinate regulation and synergistic actions of BMP4 SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles.
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      BMP signaling is required locally to pattern the dorsal telencephalic midline.
      Ectopic expression of Bmp4 on explants of lateral telencephalon induces midline features (apoptosis and Msx1 expression) and inhibits lateral features (proliferation and Foxg1 expression).
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      In this study, we used a Chd7 haploinsufficiency mouse model to study the pathogenesis of the CNS abnormalities in CHARGE syndrome. We observed Chd7 expression in dorsal midline neural epithelial cells. The Chd7COA1/+ embryos have expanded midline cell fate and incomplete medial invagination, leading to midline development defects that recapitulate conditions for patients with CHARGE syndrome. Moreover, Chd7 haploinsufficiency leads to down-regulated and expanded Bmp4 expression in the telencephalic midline of Chd7COA1/+ embryos. In cells of neuroblastoma origin, CHD7 regulates Bmp4 expression via binding with an element downstream of the Bmp4 locus. Our study characterizes forebrain structural and patterning defects in a mouse model of CHARGE syndrome, and it indicate that the forebrain anomalies in CHARGE syndrome result from dysregulated Bmp4 expression.

      Materials and Methods

      Mice

      The Chd7COA1/+ mice were originally identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen among the progenies of ENU-injected C57BL/6J male and C57BL/6J female mice.
      • He F.
      • Wang Z.
      • Zhao J.
      • Bao J.
      • Ding J.
      • Ruan H.
      • Xie Q.
      • Zhang Z.
      • Gao X.
      Large-scale screening of disease model through ENU mutagenesis in mice.
      Mice were maintained in a specific pathogen-free animal facility credited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal welfare and experimental procedures were approved by the Animal Care and Use Committee of the Model Animal Research Center, Nanjing University. For each assay, littermate wild-type mice or embryos were used as controls. Embryonic age was established by considering noon of the day that the vaginal plug was observed as E0.5.

      Mutation Mapping and Genotyping

      Mutation mapping of COA1 mice was performed as previously described.
      • Birren B.
      • Green E.D.
      Genome Analysis: A Laboratory Manual.
      COA1 mice of a C57BL/6J background were crossed to C3H/Hej mice to get the F1 generation. F1 mice with the circling phenotype were backcrossed to C57BL/6J mice to generate N2. The tail DNA samples of N2 mice were extracted, and the polymorphic microsatellite markers chosen from the Mouse Genome Databases (http://www.broad.mit.edu, last accessed June 30, 2009; the Mouse Genome Informatics database is currently housed at http://www.informatics.jax.org) were amplified by PCR. The length polymorphism differences between C57BL/6J and C3H/Hej can be distinguished by electrophoresis with 4% agarose gel. The chromosome that the mutation is located on could be determined by the linkage of the abnormal phenotype in N2 mice and microsatellite markers that represent a C57BL/6J background.
      Each exon of Chd7 was amplified by PCR with Taq polymerase (Biocolor BioScience & Technology, Shanghai, China) and sequenced (primers are listed in Table 1).
      Table 1Primers Used to Sequence the First Four Exons of Chd7
      Primer namePrimer sequence
      Chd7-2-1f5′-AGGCTCCACATTTGCTAAGG-3′
      Chd7-2-1r5′-TGTCTGCTTCTCGGTCCCAC-3′
      Chd7-2-2f5′-CTACAGATTGGTGGGACCGAG-3′
      Chd7-2-2r:5′-AGTGTCCTGGATGCTCGTTC-3′
      Chd7-2-3f5′-CCATCACGGAGGAACGAGCATC-3′
      Chd7-2-3r5′-GAGCCACCGAAGCATCTCC-3′
      Chd7-2-4f5′-TAGGGATGAAACCCGAGATGG-3′
      Chd7-2-4r5′-GTCCCATCTTCAAGGTTTATCAC-3′
      Chd7-2-5f5′-CCCACCAATAAGAAACATTCC-3′
      Chd7-2-5r5′-TCCGCTTTGGAAGTGCCATC-3′
      Chd7-2-6f5′-ACACCACTACTGCTTCAAGTCAAG-3′
      Chd7-2-6r5′-ACAGTAGGACTACCACCTGTCAAC-3′
      Chd7-3-f5′-ACCATCAGCCATTGACTCCT-3′
      Chd7-3r5′-TAGCTTTGCACTGTATGAGCC-3′
      Chd7-4f5′-CGAAGGTGTACGCTAAGTGATG-3′
      Chd7-4r5′-GTTTCTGTGGGCTCTGGCTC-3′
      Chd7-5f5′-AGGAAAGGAGGAAGGAAAGC-3′
      Chd7-5r5′-GACACGTCAGCCAACATCAG-3′
      Chd7-6f5′-GGATGCGTTGTGGCTTGAGG-3′
      Chd7-6r5′-GCAGCAAACCACCTGGACGA-3′
      Chd7-7f5′-GGGGTCAGGTTGTTGTGAATG-3′
      Chd7-7r5′-AGGCAGCAAAGAAACCATACCA-3′
      Chd7-8f5′-AAAATGGGTAATGGATAAAGCC-3′
      Chd7-8r5′-AAGCCAACAGGAAGAATGAGAAAC-3′
      Chd7-9f5′-TGTGGCTCTGACTCTTCACTG-3′
      Chd7-9r5′-GAAACACTTTGGGTGCTCTG-3′
      Chd7-10f5′-AAAATGTGGGCAGTTGAGTCTTC-3′
      Chd7-10r5′-AAGTTCCCAGCAAACAGATATC-3′
      Yolk sacs of the specimens were lysed at 55°C overnight in lysis buffer (0.5 mol/L KCl, 0.1 mol/LM Tris, pH 8.5, 1% NP40, 1%Tween-20, 200 μg/mL Proteinase K). Proteinase K was inactivated at 95°C, and 1 μL was applied for PCR with the primer Chd7-4f and Chd7-4r (Table 1). The PCR products were examined by denaturing high-performance liquid chromatography (DHPLC wave 3500; Transgenomic, New Haven, CT). The presence of a single base mutation causes denaturation of a 273-bp hybrid at a column temperature of 61°C and displayed a two-peak pattern, whereas the wild type remained intact at 61°C and displayed a single peak pattern. For genotyping of embryos from Chd7COA1/+ crossed to Chd7COA1/+, Chd7COA1/+, PCR products displayed two-peak patterns, whereas both Chd7COA1/COA1 and wild-type PCR products displayed single-peak patterns. The Chd7COA1/COA1 and wild-type PCR products were then mixed with the same amount of wild-type PCR products. The mixture was subsequently denatured at 95°C, renatured by cooling to room temperature, and finally examined by DHPLC. Two-peak patterns indicated homozygous genotype.

      Histologic Analysis

      Embryos were harvested at E12.5 to E17.5 in ice-cold PBS, and yolk sacs were taken for genotyping. The specimens of E12.5 to E14.5 were fixed in Bouin's fixture for 6 hours, E15.5 for 12 hours, and E17.5 for 16 hours. Fixed tissue was transferred to 70% ethanol, dehydrated, and embedded in paraffin (327212; Sigma-Aldrich, Indianapolis, IN). H&E staining was subsequently performed on 7-μm dewaxed sections.

      Immunofluorescence

      For immunofluorescence, frozen sections were dried at room temperature for 30 to 40 minutes. Sections were then blocked in block buffer (5% bovine serum albumin, 5% normal goat serum, and 0.1% Tween-20 in PBS) and incubated with primary antibody at 4°C overnight. The next day, sections were washed in PBS and incubated with secondary antibody at room temperature for 1 hour. In the end, after washing with PBS, sections were mounted in 50% glycerol and photographed with the use of a confocal microscopy (Leica, Nussloch, Germany).

      In Situ Hybridization

      For RNA whole-mount in situ hybridization, embryos were harvested, and yolk sacs were taken for genotyping. cDNA fragments corresponding to Chd7 transcripts were PCR-amplified from mouse testis cDNA with the Specific PCR primers that were described previously
      • Bosman E.A.
      • Penn A.C.
      • Ambrose J.C.
      • Kettleborough R.
      • Stemple D.L.
      • Steel K.P.
      Multiple mutations in mouse Chd7 provide models for CHARGE syndrome.
      and subcloned into PBS vector. The cDNA fragment of Shh was a gift from Prof. Janet Rossant (Samuel Lunenfield Research Instituet, Toronto, ON, Canada). The cDNA fragment of Wnt7b was PCR-amplified from mouse kidney cDNA with the following specific PCR primers: wnt7bisf, 5′-CCCAAGCTTAGCATTGTCATCCGTGGTGG-3′ (forward); and wnt7bisr, 5′-GGAATTCCCGCTGCGTTGTACTTCTCCT-3′ (reverse). The cDNA fragments of Msx1 and Msx2 were gifts from Dr. Abate-Shen (Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ). The cDNA fragments of Ttr, Lhx2, and Foxg1 were gifts from Dr. Edwin S. Monuki (Department of Pathology and Laboratory Medicine, University of California Irvine School of Medicine, Irvine, CA). Digoxigenin-labeled RNA probes were transcribed in vitro with SP6 (10810274001; Roche, Indianapolis, IN) or T7 (10881775001; Roche) or T3 (11031163001; Roche) RNA polymerase- and digoxigenin-labeled mix (11277073910; Roche).
      For comparative studies, a minimum of four wild-type and four Chd7COA1/+ embryos were used for each age and probe. Embryos from timed mating were dissected in ice-cold PBS. Embryos of E10.5 to E12.5 were fixed at 4°C in 4% paraformaldehyde in PBS for 12 hours, embryos of E9.5 for 5 hours, and embryos of E8.5 for 1 hour.
      After whole-mount in situ hybridization, embryos were soaked in 30% sucrose in PBS at 4°C, embedded in OCT compound (Leica), and then sectioned (40 μm thick).

      Cell Culture, Transfection, and Luciferase Assay

      SHSY5Y cells (Cell Bank of Shanghai Institutes For Biological Science, CAS, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium/F12 (1:1) supplemented with 10% fetal calf serum and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) in an atmosphere of 5% CO2 at 37°C. Small-interfering RNAs (siRNAs) against Chd7 and control siRNA were designed by the company BiboBio (Guangzhou, Guang Dong, China). The plasmids (∼0.8 μg/well) were transfected into cells plated on 24-well plates at 1.5 × 105 per well, and siRNA (25 pmol per well) was transfected at 1.0 × 105 per well, using lipofectamine 2000 (11668019; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Luciferase assays were performed 24 hours after plasmid transfection with the use of a commercially available kit (Dual Luciferase Reporter Assay System; E1960; Promega, Madison, WI) according to the manufacturer's instructions. Transfection efficiency was normalized by the ratio of the firefly luciferase activity to the cotransfected Renilla luciferase activity. In siRNA transfection assay, plasmids were transfected 36 hours after siRNA transfection, and luciferase activity was measured 24 hours after plasmids transfection. All of the data were collected from at least three independent repeated assays.

      ChIP

      ChIP was performed according to the EZ-Chip kit manufacture's protocol (Upstate Biotechnology, Charlottesville, VA). In brief, nuclear extract of SHSY5Y was homogenized and cross-linked with 1% formaldehyde. The following antibodies were used: anti-CHD7 (ab31824; Abcam Inc., Cambridge, MA), anti-RNA polymerase II and normal rabbit IgG, the latter two supplied in the kit as a positive and a negative control, respectively. Products were evaluated for specificity by PCR with the primers listed in Table 2. All of the bands were analyzed and normalized by their corresponding inputs.
      Table 2Primers Used in Cloning Fragments 1 to 8 and ChIP-PCR
      Primer namePrimer sequence
      Fragment 1-f5′-CTAATAACCTAGAAAGCCTGG-3′
      Fragment 1-r5′-CTTTGTCCAGTGGGTTCATA-3′
      Fragment 2-f5′-TTATGAACCCACTGGACAAAG-3′
      Fragment 2-r5′-GTACAGCCGAAAGAGTCAGTTC-3′
      Fragment 3-f5′-AAATGTCTGACACTGGCGAATA-3′
      Fragment 3-r5′-GGTCCCAGCCATGTGACCATA-3′
      Fragment 4-f5′-CGTTACTGAAGAAAGAAACCCTT-3′
      Fragment 4-r5′-ATTTAAGCCAGCAGACACCCT-3′
      Fragment 5-f5′-CGTTCCTCTGGTAGATGATTA-3′
      Fragment 5-r5′-GCTGAAATATCAATTAGAAATGC-3′
      Fragment 6-f5′-TAGCCCTAACCACAGAAGAAG-3′
      Fragment 6-r5′-TTTGATTCTTTGGGCTTTACC-3′
      Fragment 7-f5′-GTAAAGCCCAAAGAATCAAAG-3′
      Fragment 7-r5′-CAGATAAAGGGAATCAACCAA-3′
      Fragment 8-f5′-GTTTGAAGACTTGGTTGATTCCCTT-3′
      Fragment 8-r5′-TTCTGAGCTTTAGGTTGGAGGT-3′

      Cloning of the Potential Regulatory Fragments and BMP4 Promoter

      BMP4 promoter reporter constructs were generated by cloning a 1.3-kb fragment upstream of the transcription starting site of human Chd7 gene into the reporter gene plasmid pGL3 basic (Invitrogen). Eight potential downstream regulatory fragments were amplified with the primers listed in Table 2 and cloned into the pGL3 basic vector containing the BMP4 promoter or pGL3-promoter vector which contains a minimal SV40 promoter.

      Antibody

      Antibodies and reagents used are listed as follows: rabbit anti-CHD7 (ab31824; Abcam Inc.), mouse anti-proliferating cell nuclear antigen (sc9857; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-bromodeoxyuridine (BrdU; Roche), mouse anti-β-actin (A5441; Sigma-Aldrich), goat anti-mouse IgG (horseradish peroxidase-conjugated; 31439; Pierce Biotechnology, Rockford, IL), goat anti-rabbit IgG (horseradish peroxidase-conjugated; A9169; Sigma-Aldrich), mouse IgG (12-371B; Millipore, Billerica, MA), rabbit IgG (PP64B; Upstate Biotechnology), rabbit anti-Sox2 (ab97959; Abcam Inc.), rabbit anti-glial fibrillary acidic protein (GFAP; ab7260; Abcam Inc.), and NorthernLights 557-conjugated mouse monoclonal anti-Oligodendrocyte Marker O4, sheep anti-rabbit IgG (Cy 3-conjugated; C2306; Sigma-Aldrich).

      Results

      A Novel Mouse Strain with Circling Behavior and Eye Defects Caused by Chd7 Mutation

      In an ENU mutagenesis-screening experiment,
      • He F.
      • Wang Z.
      • Zhao J.
      • Bao J.
      • Ding J.
      • Ruan H.
      • Xie Q.
      • Zhang Z.
      • Gao X.
      Large-scale screening of disease model through ENU mutagenesis in mice.
      we identified a mutant mouse line with head bobbing and circling phenotypes. This mutation was referred to as COA1 (circling and ocular abnormality). In addition to circling behavior, the COA1-mutant mice displayed hyperactivity and growth retardation and showed slimmer figures compared with the wild-type littermates (Figure 1, A–E and G).
      Figure thumbnail gr1
      Figure 1Identification of mutation COA1 in the mouse Chd7 gene. A and D: The Chd7COA1/+ mouse (D) has a hunched head compared with the wild-type mouse (A). B and E: Coronal section show Chd7COA1/+ mouse (E) has dilated lateral and third ventricles, with severe hippocampus atrophy compared with the wild-type mouse (B). C: Chd7COA1/+ mice display loss of olfactory bulb (middle) or smaller olfactory bulb (left) compared with the brain of wild-type mice (right). F: Chd7COA1/+ mice exhibit shortened life span than wild-type mice. G: Chd7COA1/+ mice have lower body weight than the wild-type control. Left panel shows body weight of male mice, and right panel shows body weight of female mice. H: Partial electropherograms obtained by direct sequencing of PCR products show the nonsense mutation of Chd7 (2155A→T). I: Western blot analysis of Chd7 protein level in E10.5 embryonic lysates with the indicated antibodies. J: Mouse Chd7 genomic structure is shown with all of the predicted functional domains; two chromodomains (blue), SNF2-like ATPase (orange), DNA-binding domain (green), and BRK domain (purple) are indicated. The asterisk indicates the mutation COA1.
      With a panel of markers polymorphic between the C57BL/6J and the C3H/Hej strains (http://www.broad.mit.edu, last accessed June 30, 2009; the Mouse Genome Informatics database is currently housed at http://www.informatics.jax.org), the genomic interval associated with the COA1 phenotype was mapped to the proximal region of chromosome 4. Subsequent sequencing of candidate genes found an A-to-T nonsense transition in exon 4 of the Chd7 gene (Figure 1H). To characterize the COA1 mutation, we performed Western blot analysis with anti-CHD7 antibody. No Chd7 protein was detected from Chd7COA1/COA1 embryos, and reduced Chd7 protein was detected from Chd7COA1/+ embryos, indicating COA1 mutation was a null mutation (Figure 1I). In humans, haploinsufficiency for CHD7 causes CHARGE syndrome,
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Chromodomain proteins in development: lessons from CHARGE syndrome.
      • Vissers L.E.
      • van Ravenswaaij C.M.
      • Admiraal R.
      • Hurst J.A.
      • de Vries B.B.
      • Janssen I.M.
      • van der Vliet W.A.
      • Huys E.H.
      • de Jong P.J.
      • Hamel B.C.
      • Schoenmakers E.F.
      • Brunner H.G.
      • Veltman J.A.
      • van Kessel A.G.
      Mutations in a new member of the chromodomain gene family cause CHARGE syndrome.
      so Chd7COA1/+ mice are a good model for human CHARGE syndrome.
      In the genotyping assay of postnatal day 1 pups from Chd7COA1/+ crossing Chd7+/+, 33 were wild type and only 15 were Chd7COA1/+ (Table 3). To determine the possible embryonic lethality, embryos of E15.5 to E18.5 were dissected and genotyped by DHPLC. At E15.5, the number of Chd7COA1/+ and wild-type embryos was 18 and 15, respectively, in line with the expected ratio of 1:1. In a later stage, however, the number of Chd7COA1/+ and wild-type embryos was 24:33 at E17.5 and 14:31 at E18.5 (Table 3), suggesting partial embryonic lethality at later gestation. The homozygous Chd7COA1/COA1 mutants can only survive to E10.5, consistent with other reports on Chd7-mutant mice.
      • Hurd E.A.
      • Capers P.L.
      • Blauwkamp M.N.
      • Adams M.E.
      • Raphael Y.
      • Poucher H.K.
      • Martin D.M.
      Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues.
      Table 3Embryos of Male Chd7COA1/+ × Female C57BL/6J Mice
      GenotypeE15.5E17.5E18.5P1
      COA1/+18241415
      +/+15333133
      Total33574548
      The number of embryos (E15.5 to E18.5) and P1 pups of different genotypes are shown.
      For live-born Chd7COA1/+ mutants, only 55% of them survived to the sexual mature stage (Figure 1F). Chd7COA1/+ mice had lower body weight than did their wild-type littermates (Figure 1G). Similar to human patients with CHARGE syndrome and other Chd7-mutant mouse models, the Chd7COA1/+ mice also exhibited eye defects, hyperactivity, hearing loss, and lower fertility.
      • Bosman E.A.
      • Penn A.C.
      • Ambrose J.C.
      • Kettleborough R.
      • Stemple D.L.
      • Steel K.P.
      Multiple mutations in mouse Chd7 provide models for CHARGE syndrome.
      • Hall B.D.
      Choanal atresia and associated multiple anomalies.

      Chd7 Is Expressed during Early Developmental Stage of CNS

      CNS abnormalities, especially forebrain defects, are frequently reported in patients with CHARGE syndrome.
      • Sanlaville D.
      • Etchevers H.C.
      • Gonzales M.
      • Martinovic J.
      • Clement-Ziza M.
      • Delezoide A.L.
      • Aubry M.C.
      • Pelet A.
      • Chemouny S.
      • Cruaud C.
      • Audollent S.
      • Esculpavit C.
      • Goudefroye G.
      • Ozilou C.
      • Fredouille C.
      • Joye N.
      • Morichon-Delvallez N.
      • Dumez Y.
      • Weissenbach J.
      • Munnich A.
      • Amiel J.
      • Encha-Razavi F.
      • Lyonnet S.
      • Vekemans M.
      • Attie-Bitach T.
      Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development.
      • Asakura Y.
      • Toyota Y.
      • Muroya K.
      • Kurosawa K.
      • Fujita K.
      • Aida N.
      • Kawame H.
      • Kosaki K.
      • Adachi M.
      Endocrine and radiological studies in patients with molecularly confirmed CHARGE syndrome.
      Consistently, Chd7 expression has been reported in both mouse and human adult brains.
      • Vissers L.E.
      • van Ravenswaaij C.M.
      • Admiraal R.
      • Hurst J.A.
      • de Vries B.B.
      • Janssen I.M.
      • van der Vliet W.A.
      • Huys E.H.
      • de Jong P.J.
      • Hamel B.C.
      • Schoenmakers E.F.
      • Brunner H.G.
      • Veltman J.A.
      • van Kessel A.G.
      Mutations in a new member of the chromodomain gene family cause CHARGE syndrome.
      • Bosman E.A.
      • Penn A.C.
      • Ambrose J.C.
      • Kettleborough R.
      • Stemple D.L.
      • Steel K.P.
      Multiple mutations in mouse Chd7 provide models for CHARGE syndrome.
      • Hurd E.A.
      • Capers P.L.
      • Blauwkamp M.N.
      • Adams M.E.
      • Raphael Y.
      • Poucher H.K.
      • Martin D.M.
      Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues.
      • Hurd E.A.
      • Poucher H.K.
      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      However, the Chd7 expression pattern during forebrain morphogenesis in early stages was unknown. Thus, we examined Chd7 expression in continuous developmental stages from E8.5 to E10.5.
      At E8.5, Chd7 was widely expressed, with the highest level in the forebrain neural fold, midbrain neural fold, neuroepithelial prominence, and neural tube (Figure 2, A, C, and E). At E9.5 Chd7 was expressed with a higher level in telencephalon, first branchial arch, optic vesicle, and otocyst (Figure 2B). At E10.5, as development proceeds, the expression of Chd7 at E10.5 was observed in telencephalic vesicles, first branchial arch, optic cup, and nasal process (Figure 2D). Coronal section of E10.5 mice showed that the telencephalic midline also expressed Chd7 (Figure 2F).
      Figure thumbnail gr2
      Figure 2Chd7 is expressed in the CNS since early developmental stage. A, C, and E: At E8.5, Chd7 is highly expressed in forebrain neural fold, midbrain neural fold, neuroepithelial prominence, and neural tube. B: At E9.5, Chd7 is widely expressed, with the highest levels in telencephalon, first branchial arch, optic vesicle, and otocyst. D: At E10.5, the expression of Chd7 mainly concentrates in telencephalic vesicles, first branchial arch, nasal process, and optic cup. F: Coronal section of E10.5 showed that Chd7 is expressed in the dorsal midline (red arrowhead). The level of the section in F is indicated by the black dotted line in D. Ot, otocyst; Op, optic vesicle; Fnf, forebrain neural fold; Mnf, midbrain neural fold; Nt, neural tube; Npp, neuroepithelial prominence.
      In summary, Chd7 is expressed in the telencephalon during early development of CNS, implicating a role of Chd7 during early brain development.

      Chd7COA1/+ Embryos Exhibit Telencephalic Midline Developmental Defects

      Chd7COA1/+ mice developed hydrocephalus and arhinencephaly (Figure 1, A–E) with different severity. Chd7COA1/+ mice with the most serious phenotype showed no visible olfactory bulb (Figure 1C), devastatingly thinned frontal and parietal cortexes, remarkably expanded lateral ventricles, and dilated third ventricle, accompanied with shrinking of the hippocampus (Figure 1, B and E). Chd7COA1/+ mice with the mild phenotype displayed smaller olfactory bulb and expanded lateral ventricle (Figure 1E).
      To identify the onset of forebrain defects in Chd7COA1/+ mice, we analyzed the mutant embryos at different gestational stages. Gastrulation and neuralation of Chd7COA1/+ embryos was normal with clear segments of prosencephalon, mesencephalon, and rhombencephalon. Both the anteroposterior patterning and mediolateral patterning of the Chd7COA1/+ embryo appeared normal on observing the whole embryos. From E12.5, histologic analysis of Chd7COA1/+ embryos showed abnormalities in the telencephalon, with most severe anomalies related to the midline. At E12.5, the third ventricle of Chd7COA1/+ embryos was slightly expanded (Figure 3A), and mutant embryos displayed shorter invaginated cortical neuroepithelium (cortex) in the dorsal midline region than that of wild-type embryos (Figure 3, A and B). At E13.5, Chd7COA1/+ embryos showed a wider gap in the dorsal midline and expanded third ventricle (Figure 3B). E15.5 Chd7COA1/+ embryos also displayed expanded third and lateral ventricles (Figure 3C). Hippocampus commissure began to develop in both Chd7COA1/+ and wild-type embryos at E15.5 (Figure 3C). In E17.5 Chd7COA1/+ embryos, in addition to expanded third and lateral ventricles, the corpus callosum and hippocampal axons failed to cross the midline, which suggest the cortex and hippocampus neurons failed to project to the opposite hemisphere (Figure 3D). The hippocampus in Chd7COA1/+ embryos showed agenesis compared with the wild-type embryos (Figure 3D). Moreover, the neocortex was also thinner in the mutant embryos (Figure 3D).
      Figure thumbnail gr3
      Figure 3Chd7COA1/+ embryos exhibit telencephalic midline developmental defects. All of the images show coronal sections through the telencephalon. A: At E12.5, Chd7COA1/+ embryos display expanded third ventricle and shorter dorsal invaginated midline neuroepithelium (double-headed arrow) compared with the wild-type embryos. B: At E13.5, Chd7COA1/+ embryos show enlarged third ventricle, shorter dorsal invaginated midline neuroepithelium (double-headed arrow), and an expanded gap (asterisk) in the dorsal midline. C: At E15.5, Chd7COA1/+ embryos exhibit expanded lateral and third ventricles compared with the wild-type embryos. The bottom panel shows that the hippocampus commissure appears in both Chd7COA1/+ and wild-type embryos at E15.5. D: At E17.5, the Chd7COA1/+ embryos exhibit expanded lateral and third ventricles, thinner neocortex. Corpus callosum and hippocampal axons fail to cross the midline in the Chd7COA1/+ embryos (arrow). hp, hippocampus; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; ml, corpus callosum; Ncx, neocortex; TV, third ventricle.
      In conclusion, COA1 mutation resulted in maldevelopment of the telencephalic midline, especially the dorsal midline, including dilated lateral and third ventricles, shorter invaginated dorsal midline cortex, and corpus callosum crossing failure.

      Glial Cells Develop Normally in the Forebrain of Chd7COA1/+ Mice

      Glial cells are nonneuronal cells that function to maintain homeostasis and to supply nutrients and physical support for neurons.
      • Jessen K.R.
      • Mirsky R.
      Glial cells in the enteric nervous system contain glial fibrillary acidic protein.
      In the CNS, glial cells develop from the ventricular zone of the neural tube, consisting of the most abundant cell types in the CNS. Main types of glial cells in the CNS are astrocytes and oligodendrocytes, which play a role in many neurologic diseases.
      • Lu D.C.
      • Zador Z.
      • Yao J.
      • Fazlollahi F.
      • Manley G.T.
      Aquaporin-4 reduces post-traumatic seizure susceptibility by promoting astrocytic glial scar formation in mice.
      • Evans L.T.
      • Morse R.
      • Roberts D.W.
      Epilepsy surgery in tuberous sclerosis: a review.
      Immunofluorescence of GFAP and oligodendrocyte markers O4 was performed to characterize the development of astrocytes and oligodendrocytes in 3-month-old Chd7COA1/+ and wild-type mice. In the coronal forebrain sections, GFAP-positive astrocytes are abundant in the hippocampus, and quantitation analysis showed that the density of astrocytes in the hippocampus was not significantly changed in Chd7COA1/+ mice (see Supplemental Figure S1, A and C, at http://ajp.amjpathol.org). In the forebrain, O4-positive oligodendrocytes were densely concentrated in the corpus callosum of both Chd7COA1/+ and wild-type mice without obvious differences (see Supplemental Figure S1B at http://ajp.amjpathol.org).
      The result of immunofluorescence of GFAP and O4 showed that astrocytes and oligodendrocytes develop normally in the forebrain midline of Chd7COA1/+ mice.

      Chd7COA1/+ Embryos Display Incomplete Midline Invagination and Disrupted Midline Cell Fate Decision

      The defects in the developing telencephalic midline of Chd7COA1/+ embryos indicated abnormal midline signaling. The dorsal telencephalon is composed of three distinct epithelial cell types: the choroid plexus, which produce cerebrospinal fluid; cortical neuroepithelium, which gives rise to neurons and glia of cerebral cortex; and the cortical hem, which forms a boundary between cortical neuroepithelium and the telencephalic choroid plexus epithelium.
      • Grove E.A.
      • Tole S.
      • Limon J.
      • Yip L.
      • Ragsdale C.W.
      The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice.
      • Monuki E.S.
      • Porter F.D.
      • Walsh C.A.
      Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.
      To further characterize the midline patterning defects of Chd7COA1/+ embryos, we examined the expression of molecular markers of the dorsal telencephalon.
      We first examined transthyretin (Ttr), a marker gene specifically expressed in the choroid plexus. At E11.5, Ttr expression was clearly visible by whole-mount in situ hybridization in Chd7COA1/+ embryos but less intensive in wild-type embryos (Figure 4A). Subsequent dissection indicated that the difference in whole-mount staining was because the domain of Ttr-expressing neuroepithelium in mutant embryos did not invaginate as deeply as that of wild-type embryos in the rostral telencephalon (Figure 4C), and Ttr was ectopically expressed in the medial coronal section of Chd7COA1/+ telencephalon (Figure 4C). At E12.5, the choroid plexus in Chd7COA1/+ embryos marked by expression of Ttr was structurally disorganized and displayed mild hyperplasia (Figure 4D). Consistent with the histologic analysis, the dorsal midline of Chd7COA1/+ embryos showed a wide gap (Figure 4D). The cerebral cortex in Chd7COA1/+ embryos marked by Lhx2 failed to involute as deeply as the wild-type embryos (Figure 4E). Moreover, the midline marker, Msx1, was expanded in Chd7COA1/+ embryos, indicating more cells were committed to midline cell fate (Figure 4F). At E12.5, Msx1 was highly expressed at the pineal gland in the telencephalon of mouse embryos
      • Bach A.
      • Lallemand Y.
      • Nicola M.A.
      • Ramos C.
      • Mathis L.
      • Maufras M.
      • Robert B.
      Msx1 is required for dorsal diencephalon patterning.
      (Figure 4F). However, in the E12.5 Chd7COA1/+ embryos, expression of Msx1 displayed a small cleft pattern in the midline (Figure 4F).
      Figure thumbnail gr4
      Figure 4Chd7COA1/+ embryos display choroidal versus cortical patterning defects in the telencephalic midline. A: Whole-mount in situ hybridization of E11.5 embryos showed ectopic expression of Ttr (red arrowhead), a marker of choroid plexus, in Chd7COA1/+ embryos. B: Whole-mount in situ hybridization of E12.5 Chd7COA1/+ and wild-type embryos. C: In the rostral coronal section of E11.5 embryos (upper panel), the distance from expression of Ttr to the dorsal cortex (double-headed arrow) is shorter in Chd7COA1/+ embryos. In the medial coronal section of E11.5 (lower panel), Chd7COA1/+ embryos show ectopic expression of Ttr (red arrowhead). D: The level of coronal sections in D is indicated by white dotted line in B. Choroid plexus is structurally disorganized in Chd7COA1/+ embryos. The dorsal midline of Chd7COA1/+ embryos showed an abnormal wide cleft (asterisk). E: Expression of Lhx2, the cortical cortex marker, involutes in the dorsal midline region of wild-type embryos (red arrowhead) in both the rostral and medial sections, but not in Chd7COA1/+ embryos (red arrowhead). The level of coronal sections (M, medial; R, rostral) in the lower panels is indicated by white dotted line in the upper panel. F: At E11.5, expression of the midline marker, Msx1, is expanded in Chd7COA1/+ embryos (arrow). At E12.5, Msx1 is highly expressed at the pineal gland in wild-type embryos (arrow) but in a cleft pattern in Chd7COA1/+ embryos (arrow).
      Taken altogether, the dorsal midline of Chd7COA1/+ embryos expanded but did not invaginate properly at E11.5. The improper midline invagination and expanded telencephalic midline lead to midline dysmorphogenesis in Chd7COA1/+ embryos.

      Midline Apoptosis in Chd7COA1/+ Embryos Is Reduced

      Shortly after neural tube closure, the telencephalon undergoes rapid expansion when the progeny of every cell division re-enters the cell cycle,
      • Caviness Jr, V.S.
      • Takahashi T.
      • Nowakowski R.S.
      Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model.
      whereas the telencephalic midline undergoes cell apoptosis. The rapid proliferation in the lateral neuroepithelium and apoptosis in the midline result in medial invagination and lateral vesicle expansion, which eventually shapes the telencephalon.
      • Hentges K.
      • Thompson K.
      • Peterson A.
      The flat-top gene is required for the expansion and regionalization of the telencephalic primordium.
      The insufficient midline invagination in Chd7COA1/+ embryos prompted us to examine the apoptosis and proliferation rate in the embryonic telencephalon. The whole-mount Nile blue (NB) staining was used to measure cell apoptosis and to mark the nonnecrotic cell death. NB staining showed decreased dorsomedial NB-positive cells in E10.5 Chd7COA1/+ embryos (Figure 5, A and B), suggesting less apoptosis in mutant embryos. At E10.5, DNA fragmentation labeled in TUNEL assay in both the dorsal and ventral midline regions was significantly reduced in the mutant embryos (Figure 5, C–D), indicating down-regulated cell apoptosis in the forebrain midline of Chd7COA1/+ embryos. In contrast, the proliferation rate in the mutant was not altered in the midline and lateral neuroepithelia by either BrdU-labeling assay (Figure 5, E–I) or the proliferating cell nuclear antigen staining (see Supplemental Figure S2 at http://ajp.amjpathol.org).
      Figure thumbnail gr5
      Figure 5Midline apoptosis is down-regulated and proliferation rate is reserved in Chd7COA1/+ embryos. A and B: The whole-mount Nile Blue staining showed reduced apoptosis in the dorsal midline region (arrow) of E10.5 Chd7COA1/+ embryos. C and D: At E10.5, TUNEL assay showed less apoptotic cells in the midline of Chd7COA1/+ embryos (D) compared with wild-type control (C). Red: PI staining of the nucleus; green: TUNEL signal. E and F: BrdU assay showed that the proliferation rate of neural epithelium in Chd7COA1/+ embryos (F) was comparable with wild-type embryos (E) at E12.5. G and H: BrdU assay showed that the proliferation rate of neural epithelium was reserved in Chd7COA1/+ embryos (H) compared with wild-type embryos (G) at E10.5. I: Results of quantitation analysis of G and H. NS, no significant difference.
      In summary, the reduced midline apoptosis of Chd7COA1/+ embryos is in accord with inadequate midline invagination, whereas the roughly reserved proliferation rate engenders the normal size of telencephalon in Chd7COA1/+ embryos.

      Bmp4 Signaling Is Expanded and Down-Regulated in the Forebrain Midline of Chd7COA1/+ Embryos

      To decipher the molecular mechanism of midline defects in Chd7COA1/+ embryos, we examined the formation and patterning of the three centers that direct forebrain midline development. BMP4 directly induces the forebrain midline apoptosis and dorsal midline
      • Furuta Y.
      • Piston D.W.
      • Hogan B.L.
      Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development.
      • Currle D.S.
      • Cheng X.
      • Hsu C.M.
      • Monuki E.S.
      Direct and indirect roles of CNS dorsal midline cells in choroid plexus epithelia formation.
      • Fernandes M.
      • Gutin G.
      • Alcorn H.
      • McConnell S.K.
      • Hebert J.M.
      Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly.
      • Hebert J.M.
      • Mishina Y.
      • McConnell S.K.
      BMP signaling is required locally to pattern the dorsal telencephalic midline.
      and promotes the choroidal versus cortical specification and formation of the choroid plexus.
      • Fernandes M.
      • Gutin G.
      • Alcorn H.
      • McConnell S.K.
      • Hebert J.M.
      Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly.
      • Hebert J.M.
      • Mishina Y.
      • McConnell S.K.
      BMP signaling is required locally to pattern the dorsal telencephalic midline.
      • Monuki E.S.
      • Porter F.D.
      • Walsh C.A.
      Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.
      The reduced midline apoptosis, abnormal dorsal chroidal versus cortical patterning, and expanded midline in Chd7COA1/+ embryos prompted us to examine the dorsal Bmp4 patterning center first. At E9.5 and E10.0, Bmp4 expression in the telencephalon was comparable between Chd7COA1/+ and wild-type embryos (Figure 6A). At E10.5, however, as shown by both whole-mount in situ hybridization and subsequent coronal section, expression of Bmp4 in the dorsal telencephalon of Chd7COA1/+ embryos extended laterally and displayed an expanded pattern compared with wild-type embryos (Figure 6B), which is in accord with the expanded midline in E11.5 Chd7COA1/+ telencephalon. In addition, expression level of Bmp4 in the dorsal and anterior telencephalon was down-regulated in Chd7COA1/+ embryos (Figure 6B), consistent with the decreased programed cell death in the telencephalic midline of Chd7COA1/+ embryos.
      Figure thumbnail gr6
      Figure 6Bmp4 expression is down-regulated and expanded in the telencephalon of Chd7COA1/+ embryos. A: At E9.5 and E10.0, expression of Bmp4 in the forebrain of Chd7COA1/+ embryos is comparable with that of wild-type embryos (red arrowhead). B: At E10.5, expression of Bmp4 in the dorsal midline expands in Chd7COA1/+ embryos (red arrowhead in upper panel). The expression level of Bmp4 in both anterior (white arrow) and dorsal (red arrowhead) telencephalon is down-regulated in Chd7COA1/+ embryos compared with wild-type embryos. Coronal section (lower panel) indicates that expression of Bmp4 (between the two black arrows) in Chd7COA1/+ embryos is expanded and down-regulated. (C) At E9.5, expression of Shh in Chd7COA1/+ embryos is the same with that of wild-embryos. Panel E is to dissect the embryo following the black dotted line in C and view from the direction of the arrow in C. E: Expression of Shh in rostral diencephalon ventral midline (red arrowhead) is reserved in Chd7COA1/+ embryos. D: At E10.5, expression of Shh in Chd7COA1/+ embryos is the same with that of wild-type embryos. Panel F is to dissect the embryo following the black dotted line in D and view from the direction of the arrow in D. F: Expression of Shh in rostral diencephalon ventral midline (red arrowhead) and ventral telencephalon (blue arrowhead) are reserved in Chd7COA1/+ embryos. G: At E9.5, Fgf8 is expressed rostrally in Chd7COA1/+ embryos, the same with wild-type embryos. H: At E10.5, Fgf8 is expressed at the ventral midline (black arrowhead) in Chd7COA1/+ embryos, the same with wild-type embryos.
      The patterns of the other two centers were basically unaltered in mutant embryos. At E9.5, expression of Shh in the rostral diencephalon ventral midline, which is crucial for forebrain midline development,
      • Geng X.
      • Speirs C.
      • Lagutin O.
      • Inbal A.
      • Liu W.
      • Solnica-Krezel L.
      • Jeong Y.
      • Epstein D.J.
      • Oliver G.
      Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly.
      was well reserved in Chd7COA1/+ embryos (Figure 6, C and E). At E10.5, expression of Shh in the rostral diencephalon ventral midline and ventral telencephalon was also both reserved in Chd7COA1/+ embryos (Figure 6, D and F). Similarly, expression of Fgf8, which demarcates the commissural plate and is required for patterning telencephalon,
      • Shanmugalingam S.
      • Houart C.
      • Picker A.
      • Reifers F.
      • Macdonald R.
      • Barth A.
      • Griffin K.
      • Brand M.
      • Wilson S.W.
      Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon.
      remained the same between Chd7COA1/+ and wild-type embryos (Figure 6, G and H).
      Because the dorsal Bmp4 signaling center functions to induce the dorsal midline,
      • Currle D.S.
      • Cheng X.
      • Hsu C.M.
      • Monuki E.S.
      Direct and indirect roles of CNS dorsal midline cells in choroid plexus epithelia formation.
      • Hebert J.M.
      • Mishina Y.
      • McConnell S.K.
      BMP signaling is required locally to pattern the dorsal telencephalic midline.
      we predicted that the expanded expression of Bmp4 should define a broader midline feature and thus a reduced lateral feature. Thus, we next examined the lateral ventricle ectoderm maker, Foxg1 and Wnt7b. Foxg1 regulates neuroepithelial cell proliferation rate and the timing of neuronal differentiation in the telencephalon.
      • Xuan S.
      • Baptista C.A.
      • Balas G.
      • Tao W.
      • Soares V.C.
      • Lai E.
      Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.
      The expression of Foxg1 is complementary with Bmp4, and ectopic Bmp4 expression inhibits Foxg1.
      • Furuta Y.
      • Piston D.W.
      • Hogan B.L.
      Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development.
      In line with the expanded expression of Bmp4, Foxg1 expression shrinks in Chd7COA1/+ embryos, making an obtuse angle (Figure 7B), whereas in the wild-type embryos the Foxg1 expression makes an acute angle (Figure 7A). Consistently, another ventricle ectoderm marker, Wnt7b, was expressed with a bigger gap in between the two hemispheres and displayed a flattened mediolateral gradient in Chd7COA1/+ embryos (Figure 7, C and D). We also examined Bmp4 targets, Msx genes. The expression of Msx1 in the midline was expanded and down-regulated in Chd7COA1/+ embryos, and Msx2 was expressed with a wider gap in the midline of Chd7COA1/+ embryos (Figure 7, E–H). Both these observations are in concert with the expanded and down-regulated expression of Bmp4.
      Figure thumbnail gr7
      Figure 7Chd7COA1/+ embryos display expanded midline features and shrunk lateral ventricle features. A and B: Foxg1 is expressed with a wider medial opening (white dotted line in B) in the Chd7COA1/+ embryos, compared with the acute angle opening in wild type (white dotted line in A). C and D: Lateral ventricle marker, Wnt7b, is expressed with an expanded medial cleft and flattened mediolateral gradient in Chd7COA1/+ embryos (D), compared with the wild type (C). E and F: The expression of Msx1 expands in Chd7COA1/+ embryos (arrowhead in F) compared with the wild type (arrowhead in E). G and H: Msx2 is expressed with an expanded gap in the midline of Chd7COA1/+ embryos (arrowhead in H) compared with the wild-type embryos (arrowhead in G).
      Taken all these data together, in Chd7COA1/+ embryos, the expanded expression of Bmp4 specifies more cells to midline fate and thus the lateral fate contracts; meanwhile, the midline apoptosis in Chd7COA1/+ embryos is impaired, which results in incomplete invagination. It may be the combined effect of these two factors that leads to the midline defects in Chd7COA1/+ embryos.

      Chd7 Regulates Bmp4 Expression Directly by Binding with an Enhancer Downstream of Bmp4

      We next investigated the potential mechanisms by which CHD7 affects the expression pattern of Bmp4. It was reported that CHD7 might bind sites with enhancer-like features to fine-tune the downstream gene transcription.
      • Schnetz M.P.
      • Bartels C.F.
      • Shastri K.
      • Balasubramanian D.
      • Zentner G.E.
      • Balaji R.
      • Zhang X.
      • Song L.
      • Wang Z.
      • Laframboise T.
      • Crawford G.E.
      • Scacheri P.C.
      Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns.
      • Schnetz M.P.
      • Handoko L.
      • Akhtar-Zaidi B.
      • Bartels C.F.
      • Pereira C.F.
      • Fisher A.G.
      • Adams D.J.
      • Flicek P.
      • Crawford G.E.
      • Laframboise T.
      • Tesar P.
      • Wei C.L.
      • Scacheri P.C.
      CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression.
      In the human genome, a 1.8-kb sequence that locates 72 kb downstream of Bmp4 was identified in a whole-genome ChIP followed by massively parallel DNA sequencing assay as human CHD7 binding element.
      • Schnetz M.P.
      • Bartels C.F.
      • Shastri K.
      • Balasubramanian D.
      • Zentner G.E.
      • Balaji R.
      • Zhang X.
      • Song L.
      • Wang Z.
      • Laframboise T.
      • Crawford G.E.
      • Scacheri P.C.
      Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns.
      Bmp4 was found to be the closest gene to this element. We hypothesized that Chd7 may directly regulate Bmp4 expression via this putatively regulatory element. Comparison analysis between human and mouse genomes showed that the mouse homologue of this element is located 74 kb downstream of mouse Bmp4 and the nearest gene was again Bmp4. Conservation levels analysis of this element among human, rhesus, mouse, dog, elephant, and chicken showed two highly conserved fragments, which we divided into three fragments and named F1, F6, and F7 (Figure 8A; Table 4). Open chromatin analysis by Formaldehyde Assisted Isolation of Regulatory Elements assay in three human-derived cell lines, GM12878, H1-hESC, and K562, showed several protein-depleted regions (open chromatin), F2, F3, F4, F6, and F7, which might be active regulatory elements (Figure 8A; Table 4). The other regions, which may bind with protein, were defined as F1, F5, and F8 (Figure 8A; Table 4). However, chromatin isolated probe-PCR showed that, among the eight fragments, only F5 bound with CHD7 protein specifically (Figure 8B; see Supplemental Figure S3 at http://ajp.amjpathol.org).
      Figure thumbnail gr8
      Figure 8Analysis of the element located at 72 kb downstream of Bmp4. A: Sequence conservation, open chromatin analysis, and definition of eight fragments. Sequence conservation levels for human-rhesus, human-mouse, human-dog, human-elephant, human-opossum, human-platypus, human-chicken, human-lizard, human-Xenopus tropicalis, human-stickleback, and open chromatin analysis based on Formaldehyde Assisted Isolation of Regulatory Elements assay from ENCODE analyzed by the University of California Santa Cruz Blat tool (http://genome.ucsc.edu, last accessed December 15, 2010). F1, fragment 1; F2, fragment 2; F3, fragment 3; F4, fragment 4; F5, fragment 5; F6, fragment 6; F7, fragment 7; F8, fragment 8. B: ChIP assay showed that CHD7 protein bound with fragment 5. IgG was applied as negative control. C: Western blot analysis with nuclear extract of SHSY5Y cells showed that CHD7 was expressed in SHSY5Y cells. D: Luciferase assay for the ability of the eight fragments in the regulatory locus to enhance or repress reporter gene expression driven by Bmp4 promoter. E: Luciferase assay for the ability of the eight fragments in the regulatory locus to enhance or repress reporter gene expression driven by minimal SV40 promoter. F: In SHSY5Y cells, blocking Chd7 reduced the ability of fragment 5 to enhance reporter gene expression driven by Bmp4 promoter. G: Chd7 mRNA expression levels were analyzed by real-time PCR of RNA samples prepared from Chd7 or mock siRNA-transfected SHSY5Y cells (n ≥6). D–G: Values are average ± SEM. *P < 0.05, **P < 0.01.
      Table 4Length and Mouse Genome Coordinates of the Eight Fragments
      Mouse genome coordinatesLength (bp)
      Fragment 1Chr14:53417940–53418137197
      Fragment 2Chr14:53418156–53418299143
      Fragment 3Chr14:53418338–53418499161
      Fragment 4Chr14:53418601–53418745144
      Fragment 5Chr14:53418747–53418946199
      Fragment 6Chr14:53419289–53419463174
      Fragment 7Chr14:53419444–53419613169
      Fragment 8Chr14:53419583–53419777194
      We further tested the ability of the eight fragments to regulate Bmp4 promoter with a luciferase reporter system in a neuroblastoma-derived cell line, SHSY5Y, which expresses Chd7 endogenously (Figure 8C). As shown in Figure 8D, only F5 enhanced the Bmp4 promoter activity, whereas other fragments either repressed or had no effects on the Bmp4 promoter activity. We also examined regulatory function of the eight fragments with the use of the SV40 promoter. As shown in Figure 8E, F2, F3, and F6 enhanced the luciferase gene expression driven by SV40 promoter; F7 repressed the luciferase gene expression driven by SV40 promoter; and F1, F4, F5, and F8 showed no regulatory function to SV40 promoter. We suspected that the regulatory function of these fragments were specific to the Bmp4 promoter.
      To further test whether the enhancer activity of F5 to Bmp4 promoter was via CHD7, luciferase activity was measured when Chd7 was knocked down by siRNA. As shown in Figure 8F and G, partial blockage of Chd7 brought down the enhancer activity of F5 compared with that in cells transfected with mock siRNA.

      Discussion

      In this study we used ENU mutagenesis to identify a novel Chd7 mutant mouse, which phenocopied the human CHARGE syndrome. We characterized the telencephalic midline developmental defects in Chd7 mutants and showed that CHD7 protein directly regulates Bmp4 expression by binding with an enhancer 72 kb downstream of Bmp4, misexpression of which likely underlies the forebrain defects in Chd7COA1/+ mice. Our results showed a previously unappreciated role of CHD7 in forebrain midline development and provided an insight into the pathogenesis underlying the CNS defects in CHARGE syndrome.
      In human patients with CHARGE syndrome, CNS anomalies are one of the leading defects. However, the pathogenesis and molecular mechanism of the CNS defects in CHARGE syndrome remain unknown. Conditional deletion of Chd7 in the developing otocyst leads to reduced vestibulo-cochlear ganglion size and neuron number.
      • Hurd E.A.
      • Poucher H.K.
      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      Chd7 haploinsufficiency results in down-regulation of proliferation in olfactory epithelial neural stem cells and reduced regeneration of olfactory sensory neurons.
      • Layman W.S.
      • McEwen D.P.
      • Beyer L.A.
      • Lalani S.R.
      • Fernbach S.D.
      • Oh E.
      • Swaroop A.
      • Hegg C.C.
      • Raphael Y.
      • Martens J.R.
      • Martin D.M.
      Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome.
      In addition, Chd7Gt/+ mice that are heterozygous for a gene-trapped lacZ allele show impaired cellular proliferation in olfactory placode and decreased gonadotropin-releasing hormone neurons in the nasal region.
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome.
      Here, our data suggest a crucial role of Chd7 in forebrain midline development. To summarize, our study suggests a broad role of CHD7 in promoting neurogenesis, which should account for the CNS defects of patients with CHARGE syndrome. In addition, our study provides further insight into the mechanism of Chd7 function. CHD7 belongs to the CHD family of proteins that contain tandem chromodomains located in the N-terminal region.
      • Woodage T.
      • Basrai M.A.
      • Baxevanis A.D.
      • Hieter P.
      • Collins F.S.
      Characterization of the CHD family of proteins.
      The chromodomain is an evolutionarily conserved sequence that is often involved in regulating gene activity.
      • Jones D.O.
      • Cowell I.G.
      • Singh P.B.
      Mammalian chromodomain proteins: their role in genome organisation and expression.
      Recently, Scacheri and colleagues
      • Schnetz M.P.
      • Handoko L.
      • Akhtar-Zaidi B.
      • Bartels C.F.
      • Pereira C.F.
      • Fisher A.G.
      • Adams D.J.
      • Flicek P.
      • Crawford G.E.
      • Laframboise T.
      • Tesar P.
      • Wei C.L.
      • Scacheri P.C.
      CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression.
      found that CHD7 probably participated in the transcriptional regulation via binding with cis-elements, mostly enhancers. Consistently, in this study, we showed that Chd7 directly regulates Bmp4 expression by binding with an enhancer downstream of Bmp4. In SHSY5Y cells, blockage of CHD7 leads to down-regulated activity of the Bmp4 promoter. Consistently, Chd7 haploinsufficiency leads to reduced expression of Bmp4 in embryonic olfactory placode.
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome.
      We found that Chd7 haploinsufficiency in vivo leads to not only down-regulation but also expansion of Bmp4 expression in the forebrain midline. Among the eight fragments downstream of Bmp4, only F5 showed enhancer activity to Bmp4 promoter; F1, F2, F3, F4, F6, and F7 showed repressive activity to only Bmp4 promoter. It is likely CHD7 affects not only the level but also the spatial pattern of Bmp4 expression, which offers one of the possible explanations for the suppressive activity of F1, F2, F3, F4, F6, and F7 in vitro. It would be interesting to perform more in-depth analysis on the synergistic interaction and function of these fragments to the Bmp4 promoter.
      The role of early Fgf signaling, especially Fgf3, Fgf19, and Fgf8, in the otic development have been firmly established.
      • Alvarez Y.
      • Alonso M.T.
      • Vendrell V.
      • Zelarayan L.C.
      • Chamero P.
      • Theil T.
      • Bosl M.R.
      • Kato S.
      • Maconochie M.
      • Riethmacher D.
      • Schimmang T.
      Requirements for FGF3 and FGF10 during inner ear formation.
      • Rinkwitz S.
      • Bober E.
      • Baker R.
      Development of the vertebrate inner ear.
      • Phillips B.T.
      • Bolding K.
      • Riley B.B.
      Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction.
      Otic induction fails in embryos null for Fgf3 and hypomorphic for Fgf8.
      • Ladher R.K.
      • Wright T.J.
      • Moon A.M.
      • Mansour S.L.
      • Schoenwolf G.C.
      FGF8 initiates inner ear induction in chick and mouse.
      However, conditional deletion of Chd7 in the developing otocyst did not prevent the otic vesicle formation, although semicircular canals and cristae were completely lost.
      • Hurd E.A.
      • Poucher H.K.
      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      In the CHARGE syndrome mouse model Chd7Gt/+ mice also display normal otic induction but develop truncated or hypoplastic lateral semicircular canals.
      • Hurd E.A.
      • Capers P.L.
      • Blauwkamp M.N.
      • Adams M.E.
      • Raphael Y.
      • Poucher H.K.
      • Martin D.M.
      Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues.
      Bmp4 is a key regulator of otic capsule chondrogenesis in high vertebrates.
      • Liu W.
      • Oh S.H.
      • Kang Yk Y.
      • Li G.
      • Doan T.M.
      • Little M.
      • Li L.
      • Ahn K.
      • Crenshaw 3rd, E.B.
      • Frenz D.A.
      Bone morphogenetic protein 4 (BMP4): a regulator of capsule chondrogenesis in the developing mouse inner ear.
      • Liu W.
      • Butts S.
      • Kim H.
      • Frenz D.A.
      Negative regulation of otic capsule chondrogenesis: it can make you Smad.
      • Gerlach L.M.
      • Hutson M.R.
      • Germiller J.A.
      • Nguyen-Luu D.
      • Victor J.C.
      • Barald K.F.
      Addition of the BMP4 antagonist, noggin, disrupts avian inner ear development.
      Therefore, it is plausible to postulate that Bmp4 may mediate the disruption of later otocyst differentiation in CHARGE syndrome if indeed Chd7 regulates BMP4 expression. Martin and colleagues
      • Hurd E.A.
      • Poucher H.K.
      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      did not examine the expression level of Bmp4 and Fgf8 in the otocyst in the Chd7Gt/+ embryos. Consistent with our data, it was reported that the expression of Fgf8 was not altered, whereas Bmp4 is down-regulated in olfactory placode in Chd7Gt/+ or Chd7Gt/Gt embryos compared with wild-type littermates.
      • Layman W.S.
      • McEwen D.P.
      • Beyer L.A.
      • Lalani S.R.
      • Fernbach S.D.
      • Oh E.
      • Swaroop A.
      • Hegg C.C.
      • Raphael Y.
      • Martens J.R.
      • Martin D.M.
      Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome.
      Because Chd7 may regulate downstream genes in a tissue/stage-dependent manner, we could not exclude the possibilities that Fgf8 results in other defects in Chd7 mutant mice.
      A roof-plate (Bmps) Lhx2 signaling pathway exists in cortical patterning.
      • Monuki E.S.
      • Porter F.D.
      • Walsh C.A.
      Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.
      • Cheng X.
      • Hsu C.M.
      • Currle D.S.
      • Hu J.S.
      • Barkovich A.J.
      • Monuki E.S.
      Central roles of the roof plate in telencephalic development and holoprosencephaly.
      Ablation of Bmps in the dorsal midline leads to a reduction of Lhx2-negative domain at the dorsal midline and holoprosencaphaly.
      • Monuki E.S.
      • Porter F.D.
      • Walsh C.A.
      Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.
      • Cheng X.
      • Hsu C.M.
      • Currle D.S.
      • Hu J.S.
      • Barkovich A.J.
      • Monuki E.S.
      Central roles of the roof plate in telencephalic development and holoprosencephaly.
      In addition, high concentration of Bmp4 bath application abolishes Lhx2 expression.
      • Monuki E.S.
      • Porter F.D.
      • Walsh C.A.
      Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.
      Here, in Chd7COA1/+ embryos, expanded expression of Bmp4 suppresses Lhx2 expression in the dorsal midline cortical neuroepithelium, and the altered Bmp4-Lhx2 pathway in Chd7COA1/+ embryos should underlie the midline defects.
      Development of the forebrain, especially the midline development, is an exquisitely organized, complex process and is consequently highly susceptible to disruptions. All of the morphogens crucial in the midline development show dose-dependent features: different combination of wild-type, hypomorphic, and null alleles of Fgf8 result in graded defects in midline development.
      • Storm E.E.
      • Garel S.
      • Borello U.
      • Hebert J.M.
      • Martinez S.
      • McConnell S.K.
      • Martin G.R.
      • Rubenstein J.L.
      Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers.
      Partial blockage of BMP signaling disrupts the specification and differentiation of choroid plexus,
      • Hebert J.M.
      • Mishina Y.
      • McConnell S.K.
      BMP signaling is required locally to pattern the dorsal telencephalic midline.
      whereas both up-regulation and complete blockage of BMP signaling result in midline induction failure.
      • Fernandes M.
      • Gutin G.
      • Alcorn H.
      • McConnell S.K.
      • Hebert J.M.
      Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly.
      • Anderson R.M.
      • Lawrence A.R.
      • Stottmann R.W.
      • Bachiller D.
      • Klingensmith J.
      Chordin and noggin promote organizing centers of forebrain development in the mouse.
      The regulation of morphogens to forebrain midline development is also stage dependent. Temporal perturbation of the Shh signaling pathway created a continuum of holoprosencaphaly-related defects.
      • Cheng X.
      • Hsu C.M.
      • Currle D.S.
      • Hu J.S.
      • Barkovich A.J.
      • Monuki E.S.
      Central roles of the roof plate in telencephalic development and holoprosencephaly.
      • Cordero D.
      • Marcucio R.
      • Hu D.
      • Gaffield W.
      • Tapadia M.
      • Helms J.A.
      Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes.
      Ablation of BMP-producing roof-plate in different stages also leads to phenotypes of different severity.
      • Monuki E.S.
      • Porter F.D.
      • Walsh C.A.
      Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.
      • Cheng X.
      • Hsu C.M.
      • Currle D.S.
      • Hu J.S.
      • Barkovich A.J.
      • Monuki E.S.
      Central roles of the roof plate in telencephalic development and holoprosencephaly.
      Here, in Chd7COA1/+ embryos, the ectopic expression of Bmp4 appears at E10.5 after the rostral-caudal and dorsal-ventral patterning has been established; thus, the midline defects are not severe. In addition, although expression of Bmp4 represses the expression of Shh and Fgf8 in the telencephalon,
      • Ohkubo Y.
      • Chiang C.
      • Rubenstein J.L.
      Coordinate regulation and synergistic actions of BMP4 SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles.
      in Chd7COA1/+ embryos, the ectopic expression of Bmp4 is not broad enough, and the expression of Shh and Fgf8 is reserved, which also makes the midline phenotype mild.
      Targeted disruption of Chordin and Noggin, the BMP antagonists, minimizes the expression of Foxg1,
      • Anderson R.M.
      • Lawrence A.R.
      • Stottmann R.W.
      • Bachiller D.
      • Klingensmith J.
      Chordin and noggin promote organizing centers of forebrain development in the mouse.
      which means ectopic expression of BMPs constricts Foxg1. In Chd7COA1/+ embryos, expression of Bmp4 expands; thus, the expression of Foxg1 shrinks. However, Foxg1 also represses BMP signaling by restricting its expression to the dorsal midline and thus establishes the subpallial fate in the ventral progenitor cells.
      • Dou C.L.
      • Li S.
      • Lai E.
      Dual role of brain factor-1 in regulating growth and patterning of the cerebral hemispheres.
      We therefore cannot rule out the possibility that the shrinkage of Foxg1 contributes to the expansion of Bmp4. Considering the global effects of Chd7 in modifying gene transcription,
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Chromodomain proteins in development: lessons from CHARGE syndrome.
      • Schnetz M.P.
      • Bartels C.F.
      • Shastri K.
      • Balasubramanian D.
      • Zentner G.E.
      • Balaji R.
      • Zhang X.
      • Song L.
      • Wang Z.
      • Laframboise T.
      • Crawford G.E.
      • Scacheri P.C.
      Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns.
      • Schnetz M.P.
      • Handoko L.
      • Akhtar-Zaidi B.
      • Bartels C.F.
      • Pereira C.F.
      • Fisher A.G.
      • Adams D.J.
      • Flicek P.
      • Crawford G.E.
      • Laframboise T.
      • Tesar P.
      • Wei C.L.
      • Scacheri P.C.
      CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression.
      Chd7 may also affect the expression of Foxg1, which then modifies the expression of Bmp4 synergistically with Chd7.
      CHD7 is required for the neural stem cell proliferation in the olfactory epithelium.
      • Layman W.S.
      • McEwen D.P.
      • Beyer L.A.
      • Lalani S.R.
      • Fernbach S.D.
      • Oh E.
      • Swaroop A.
      • Hegg C.C.
      • Raphael Y.
      • Martens J.R.
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      Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome.
      In addition, CHD7 is shown to promote cellular proliferation in early inner ear and embryonic olfactory placode.
      • Hurd E.A.
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      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome.
      However, the forebrain neuroepithelium cellular proliferation of Chd7COA1/+ embryos is not changed compared with the wild type. The roughly reserved proliferation in the Chd7COA1/+ embryos does not conflict with the role of CHD7 as a positive regulator of cell proliferation in previous reports, because CHD7 influences cell proliferation in a dose- and tissue-dependent manner. At E9.5 or E10.5, no change in the number of proliferating cells in the otic epithelium is observed in the Chd7Gt/+ embryos, whereas a significant decrease is observed in the conditional knockout embryos.
      • Hurd E.A.
      • Poucher H.K.
      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      Thus, it is possible that neuroepithelium proliferation rate is down-regulated in Chd7-null embryos but reserved in Chd7COA1/+ embryos. Because of early lethality of Chd7COA1/COA1 embryos, it is worthwhile to examine the neuroepithlium proliferation rate in Chd7 conditional knockout embryos.
      Recent research indicates that CHD7 binds with SOX2 to regulate downstream transcription.
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      • Lenhard B.
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      Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.
      Immunofluorescence of Sox2 shows that the expression of Sox2 is down-regulated in the midline region of Chd7COA1/+ specifically at E10.5 when expression of Bmp4 is dysregulated (see Supplemental Figure S4 at http://ajp.amjpathol.org), consistent with the fact that Bmp4 is less concentrated in the midline structure in mutants. It is possible that Sox2 may participate in the transcriptional regulation of Bmp4, and the regional down-regulation of Sox2 causes the regional down-regulation of Bmp4. Moreover, Sox2 is highly expressed in the neuroepithelium of developing CNS
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      The down-regulation of Sox2 in Chd7COA1/+ indicates the loss of neural progenitors. Further studies to elucidate the regulation between Sox2 and Bmp4 will be extremely worthwhile.
      It has been shown that CHD7 regulates downstream genes in a tissue- and cell-specific manner.
      • Layman W.S.
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      CHD7 is shown to bind with PBAF components to regulate expression of Sox9 and TWIST1 in human neural crest cells.
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      In mouse neural stem cells, Chd7 forms a complex with Sox2 to activate a set of common targets, including Jag1, Gli2, Gli3, and Mycn.
      • Engelen E.
      • Akinci U.
      • Bryne J.C.
      • Hou J.
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      • Demmers J.
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      • Philipsen S.
      • Pevny L.H.
      • Grosveld F.G.
      • Rottier R.J.
      • Lenhard B.
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      Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.
      In mouse mesenchymal stem cells, Chd7 associates with NLK, SETDB1, and peroxisome proliferator-activated receptor-γ to regulate peroxisome proliferator-activated receptor-γ target genes.
      • Takada I.
      • Mihara M.
      • Suzawa M.
      • Ohtake F.
      • Kobayashi S.
      • Igarashi M.
      • Youn M.Y.
      • Takeyama K.
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      • Mezaki Y.
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      Chd7 haploinsufficiency in mice results in reduced expression of Ngn1, Otx2, and Fgf10 in mouse otocyst
      • Hurd E.A.
      • Poucher H.K.
      • Cheng K.
      • Raphael Y.
      • Martin D.M.
      The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.
      ; reduced expression of Fgfr1, Bmp4, and Otx2 in embryonic olfactory placode; and reduced expression of GnRH1 and Otx2 in the adult hypothalamus.
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome.
      Here, we observed that Chd7 haploinsufficiency leads to down-regulation and expansion in expression of Bmp4 in the telencephalic midline. It would be important to explore why the targets of CHD7 vary for different tissues. One of the explanations is that different complexes that CHD7 binds with in different tissues account for the highly variable targets.
      • Layman W.S.
      • Hurd E.A.
      • Martin D.M.
      Chromodomain proteins in development: lessons from CHARGE syndrome.
      Finding the CHD7 complex in the forebrain midline will be helpful to elucidate the regulatory role of CHD7 on transcription and the cause of forebrain defects in CHARGE syndrome.

      Acknowledgments

      We thank Drs. Chaojun Li, Chunwen Lin, and Jianxin Hu for their useful suggestions and technical guidance.

      Supplementary data

      • Supplemental Figure S1

        Astrocytes and oligodendrocytes develop normally in Chd7COA1/+ mice. Frozen coronal sections of forebrain of Chd7COA1/+ and wild-type mice were stained with GFAP antibody (red) (A) and oligodendrocyte marker O4 antibody (red) (B) by immunofluorescence at 3 months. A: The density of astrocytes in the hippocampus region is the same between Chd7COA1/+ and wild-type mice. B: The oligodendrocyte marker O4 stained the corpus callosum (white arrow) specifically in the forebrain midline region, and the results are the same between Chd7COA1/+ and wild-type mice. GFAP and O4 are the markers of astrocytes and oligodendrocytes, respectively. C: Result of quantitation analysis for A. N.S., no significant difference.

      • Supplemental Figure S2

        Chd7COA1/+ embryos showed reserved proliferation rate at E12.5. At E12.5, immunohistochemistry of proliferating cell nuclear antigen in the neuroepithelium of Chd7COA1/+ embryos (D) showed no difference from that of wild-type embryos (A). A: The right blue frame is magnified as B. The left blue frame is magnified C. D: The right blue frame is magnified as E. The left blue frame in is magnified as F.

      • Supplemental Figure S3

        ChIP of eight fragments. Anti-RNA polymerase II and primers specific for human GAPDH promoter were used as positive control. Normal rabbit IgG was used as negative control. Among the eight fragments, only fragment 5 can be specifically amplified from the immunoprecipitate of antibody against CHD7.

      • Supplemental Figure S4

        Sox2 is down-regulated in the midline region of Chd7COA1/+ embryos specifically. Frozen coronal sections of telencephalon of Chd7COA1/+ and wild-type (WT) embryos were stained with Sox2 antibody (red) (A) by immunofluorescence at 3 months. The immunofluorescence results indicate that Sox2 is down-regulated in the midline region of Chd7COA1/+ embryos specifically. B is the magnification of the white box region in A.

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