Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7Whi/+ and Chd7Whi/Whi) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7Whi/Whi embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the pathogenesis of this multiple malformation syndrome.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Aramaki M, Udaka T, Kosaki R, Makita Y, Okamoto N, Yoshihashi H, Oki H, Nanao K, Moriyama N, Oku S, Hasegawa T, Takahashi T, Fukushima Y, Kawame H, Kosaki K (2006) Phenotypic spectrum of CHARGE syndrome with CHD7 mutations. J Pediatr 148:410–414. doi:10.1016/j.jpeds.2005.10.044
Bajpai R, Chen DA, Rada-Iglesias A, Zhang J, Xiong Y, Helms J, Chang CP, Zhao Y, Swigut T, Wysocka J (2010) CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463:958–962. doi:10.1038/nature08733
Bergman JE, Janssen N, Hoefsloot LH, Jongmans MC, Hofstra RM, van Ravenswaaij-Arts CM (2011) CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype. J Med Genet 48:334–342. doi:10.1136/jmg.2010.087106
Borchers A, David R, Wedlich D (2001) Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification. Development 128:3049–3060
Bosman EA, Penn AC, Ambrose JC, Kettleborough R, Stemple DL, Steel KP (2005) Multiple mutations in mouse Chd7 provide models for CHARGE syndrome. Hum Mol Genet 14:3463–3476. doi:10.1093/hmg/ddi375
Chalouhi C, Faulcon P, Le Bihan C, Hertz-Pannier L, Bonfils P, Abadie V (2005) Olfactory evaluation in children: application to the CHARGE syndrome. Pediatrics 116:e81–e88. doi:10.1542/peds.2004-1970
da Huang W, Sherman BT, Lempicki RA (2009a) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13. doi:10.1093/nar/gkn923
da Huang W, Sherman BT, Lempicki RA (2009b) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. doi:10.1038/nprot.2008.211
Dode C, Hardelin JP (2009) Kallmann syndrome. Eur J Hum Genet 17:139–146. doi:10.1038/ejhg.2008.206
Gammill LS, Bronner-Fraser M (2003) Neural crest specification: migrating into genomics. Nat Rev Neurosci 4:795–805. doi:10.1038/nrn1219
Hall BK (2000) The neural crest as a fourth germ layer and vertebrates as quadroblastic not triploblastic. Evol Dev 2:3–5
Hanchate NK, Giacobini P, Lhuillier P, Parkash J, Espy C, Fouveaut C, Leroy C, Baron S, Campagne C, Vanacker C, Collier F, Cruaud C, Meyer V, Garcia-Pinero A, Dewailly D, Cortet-Rudelli C, Gersak K, Metz C, Chabrier G, Pugeat M, Young J, Hardelin JP, Prevot V, Dode C (2012) SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome. PLoS Genet 8:e1002896. doi:10.1371/journal.pgen.1002896
Harland RM (1991) In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol 36:685–695
Hawker K, Fuchs H, Angelis MH, Steel KP (2005) Two new mouse mutants with vestibular defects that map to the highly mutable locus on chromosome 4. Int J Audiol 44:171–177
Hrabe de Angelis MH, Flaswinkel H, Fuchs H, Rathkolb B, Soewarto D, Marschall S, Heffner S, Pargent W, Wuensch K, Jung M, Reis A, Richter T, Alessandrini F, Jakob T, Fuchs E, Kolb H, Kremmer E, Schaeble K, Rollinski B, Roscher A, Peters C, Meitinger T, Strom T, Steckler T, Holsboer F, Klopstock T, Gekeler F, Schindewolf C, Jung T, Avraham K, Behrendt H, Ring J, Zimmer A, Schughart K, Pfeffer K, Wolf E, Balling R (2000) Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat Genet 25:444–447. doi:10.1038/78146
Janssen N, Bergman JE, Swertz MA, Tranebjaerg L, Lodahl M, Schoots J, Hofstra RM, van Ravenswaaij-Arts CM, Hoefsloot LH (2012) Mutation update on the CHD7 gene involved in CHARGE syndrome. Hum Mutat 33:1149–1160. doi:10.1002/humu.22086
Jongmans MC, Admiraal RJ, van der Donk KP, Vissers LE, Baas AF, Kapusta L, van Hagen JM, Donnai D, de Ravel TJ, Veltman JA, Geurts van Kessel A, De Vries BB, Brunner HG, Hoefsloot LH, van Ravenswaaij CM (2006) CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene. J Med Genet 43:306–314. doi:10.1136/jmg.2005.036061
Jongmans MC, van Ravenswaaij-Arts CM, Pitteloud N, Ogata T, Sato N, Claahsen-van der Grinten HL, van der Donk K, Seminara S, Bergman JE, Brunner HG, Crowley WF Jr, Hoefsloot LH (2009) CHD7 mutations in patients initially diagnosed with Kallmann syndrome: the clinical overlap with CHARGE syndrome. Clin Genet 75:65–71. doi:10.1111/j.1399-0004.2008.01107.x
Kim HG, Kurth I, Lan F, Meliciani I, Wenzel W, Eom SH, Kang GB, Rosenberger G, Tekin M, Ozata M, Bick DP, Sherins RJ, Walker SL, Shi Y, Gusella JF, Layman LC (2008) Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome. Am J Hum Genet 83:511–519. doi:10.1016/j.ajhg.2008.09.005
Kirby ML, Hutson MR (2010) Factors controlling cardiac neural crest cell migration. Cell Adh Migr 4:609–621
Koestner U, Shnitsar I, Linnemannstons K, Hufton AL, Borchers A (2008) Semaphorin and neuropilin expression during early morphogenesis of Xenopus laevis. Dev Dyn 237:3853–3863. doi:10.1002/dvdy.21785
Lalani SR, Safiullah AM, Fernbach SD, Harutyunyan KG, Thaller C, Peterson LE, McPherson JD, Gibbs RA, White LD, Hefner M, Davenport SL, Graham JM, Bacino CA, Glass NL, Towbin JA, Craigen WJ, Neish SR, Lin AE, Belmont JW (2006) Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation. Am J Hum Genet 78:303–314. doi:10.1086/500273
Morgan D, Bailey M, Phelps P, Bellman S, Grace A, Wyse R (1993) Ear-nose-throat abnormalities in the CHARGE association. Arch Otolaryngol Head Neck Surg 119:49–54
Ogata T, Fujiwara I, Ogawa E, Sato N, Udaka T, Kosaki K (2006) Kallmann syndrome phenotype in a female patient with CHARGE syndrome and CHD7 mutation. Endocr J 53:741–743
Opitz L, Salinas-Riester G, Grade M, Jung K, Jo P, Emons G, Ghadimi BM, Beissbarth T, Gaedcke J (2010) Impact of RNA degradation on gene expression profiling. BMC Med Genomics 3:36. doi:10.1186/1755-8794-3-36
Pinto G, Abadie V, Mesnage R, Blustajn J, Cabrol S, Amiel J, Hertz-Pannier L, Bertrand AM, Lyonnet S, Rappaport R, Netchine I (2005) CHARGE syndrome includes hypogonadotropic hypogonadism and abnormal olfactory bulb development. J Clin Endocrinol Metab 90:5621–5626. doi:10.1210/jc.2004-2474
Randall V, McCue K, Roberts C, Kyriakopoulou V, Beddow S, Barrett AN, Vitelli F, Prescott K, Shaw-Smith C, Devriendt K, Bosman E, Steffes G, Steel KP, Simrick S, Basson MA, Illingworth E, Scambler PJ (2009) Great vessel development requires biallelic expression of Chd7 and Tbx1 in pharyngeal ectoderm in mice. J Clin Invest 119:3301–3310. doi:10.1172/JCI37561
Sanlaville D, Verloes A (2007) CHARGE syndrome: an update. Eur J Hum Genet 15:389–399. doi:10.1038/sj.ejhg.5201778
Sanlaville D, Etchevers HC, Gonzales M, Martinovic J, Clement-Ziza M, Delezoide AL, Aubry MC, 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 (2006) Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development. J Med Genet 43:211–217. doi:10.1136/jmg.2005.036160
Schwanzel-Fukuda M, Pfaff DW (1989) Origin of luteinizing hormone-releasing hormone neurons. Nature 338:161–164. doi:10.1038/338161a0
Siebert JR, Graham JM Jr, MacDonald C (1985) Pathologic features of the CHARGE association: support for involvement of the neural crest. Teratology 31:331–336. doi:10.1002/tera.1420310303
Smith WC, Harland RM (1991) Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell 67:753–765
Teixeira L, Guimiot F, Dode C, Fallet-Bianco C, Millar RP, Delezoide AL, Hardelin JP (2010) Defective migration of neuroendocrine GnRH cells in human arrhinencephalic conditions. J Clin Invest 120:3668–3672. doi:10.1172/JCI43699
Van Meter TD, Weaver DD (1996) Oculo-auriculo-vertebral spectrum and the CHARGE association: clinical evidence for a common pathogenetic mechanism. Clin Dysmorphol 5:187–196
Vissers LE, van Ravenswaaij CM, Admiraal R, Hurst JA, de Vries BB, Janssen IM, van der Vliet WA, Huys EH, de Jong PJ, Hamel BC, Schoenmakers EF, Brunner HG, Veltman JA, van Kessel AG (2004) Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat Genet 36:955–957. doi:10.1038/ng1407
Williams MS (2005) Speculations on the pathogenesis of CHARGE syndrome. Am J Med Genet A 133A:318–325. doi:10.1002/ajmg.a.30561
Wincent J, Holmberg E, Stromland K, Soller M, Mirzaei L, Djureinovic T, Robinson K, Anderlid B, Schoumans J (2008) CHD7 mutation spectrum in 28 Swedish patients diagnosed with CHARGE syndrome. Clin Genet 74:31–38. doi:10.1111/j.1399-0004.2008.01014.x
Yazdani U, Terman JR (2006) The semaphorins. Genome Biol 7:211. doi:10.1186/gb-2006-7-3-211
Young J, Metay C, Bouligand J, Tou B, Francou B, Maione L, Tosca L, Sarfati J, Brioude F, Esteva B, Briand-Suleau A, Brisset S, Goossens M, Tachdjian G, Guiochon-Mantel A (2012) SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development. Hum Reprod 27:1460–1465. doi:10.1093/humrep/des022
The authors thank the Helmholtz Zentrum München, Germany and K.P. Steel (Sanger Centre, Cambridge, United Kingdom) for providing the Whirligig mouse line; J. Wysocka (Department of Developmental Biology, Stanford University School of Medicine, Stanford, California) for providing the hCHD7 full-length plasmid; W. Engel for his helpful discussions and his support; J. Mänz and U. Lenz for excellent technical assistance; S. Wolf and L. Piontek for excellent animal care and we thank all patients and their parents for participating in this research project. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) [to S.P]. Part of this work was supported by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain [to A.B.].
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary material 1 (PPTX 790 kb) Figure S1. Chd7 loss of function results in a downregulation of Sema3a expression in Xenopus laevis. Embryos were injected in one blastomere at the two-cell stage with different Morpholino Oligonucleotides (MO) in combination with lacZ RNA as a lineage tracer. Sema3a expression was detected by whole mount in situ hybridization at neurula stages (stage 20-22). (A, B) Embryos injected with 20 ng of a Control Morpholino (Co MO) show Sema3a expression in the midbrain–hindbrain boundary (mh) (A) as well as in the somites (s) (B). (C–F) Different concentrations of a Chd7 MO were injected. (C, D) Embryos injected with 10 ng Chd7 MO show a reduction in Sema3a expression at the midbrain–hindbrain boundary and in the somites. (E, F) Injection of 20 ng Chd7 MO strongly reduced Sema3a expression. (G) The graph summarizes three independent experiments. Numbers indicate the number of injected embryos. Reduction of Sema3a expression or patterning defects are indicated as mild defects, whereas loss of Sema3a expression is scored as severe defect. Standard error of the means is shown
Supplementary material 2 (PPTX 3229 kb) Figure S2. Chd7 loss of function inhibits Sema3a expression at tailbud stages. Embryos were injected with 10 ng or 20 ng MO in combination with lacZ RNA as a lineage tracer in one blastomere at the 2-cell stage. At tailbud stage 27, the Sema3a expression was analyzed by whole mount in situ hybridization. (A-C) Injected side is shown on the right. (A) Embryo injected with 20 ng control MO (Co MO) shows normal Sema3a expression in the splanchnic mesoderm (sm), the somites (s) and the midbrain–hindbrain boundary (mh). (B) Embryo injected with 10 ng Chd7 MO showing a reduction in Sema3a expression on the injected site. (C) Embryos injected with 20 ng Chd7 MO exhibiting a severe reduction in Sema3a expression. (D) Graph summarizing the percentage of mild and severe defects in Sema3a expression of one representative experiment. Numbers indicate the number of injected embryos
Supplementary material 3 (PPTX 513 kb) Figure S3. The Chd7 MO phenotype can be rescued by injection of human CHD7 RNA, which is not recognized by the Chd7 MO. Embryos were co-injected with 10 ng MO and 1 ng human CHD7 RNA (hCHD7 RNA) in one blastomere at the 2-cell stage and Sema3a expression was analyzed at neurula stages (20-22). One representative rescue experiment is shown. () Anterior view of the embryos. (B,D,F,H) Dorsal view of the embryos, posterior is up. (A-H) shows embryos injected as indicated; controls are uninjected embryos. (I) Graph summarizing the percentage of mild and severe defects in Sema3a expression of one representative rescue experiment. Numbers indicate the number of injected embryos
Supplementary material 4 (PPTX 261 kb) Figure S4. Repetition of the experiment shown in Figure S3 using 1.5 ng hCHD7 RNA
About this article
Cite this article
Schulz, Y., Wehner, P., Opitz, L. et al. CHD7, the gene mutated in CHARGE syndrome, regulates genes involved in neural crest cell guidance. Hum Genet 133, 997–1009 (2014). https://doi.org/10.1007/s00439-014-1444-2
- Neural Crest
- Neural Crest Cell
- Hypogonadotropic Hypogonadism
- Morpholino Oligonucleotide
- Charge Syndrome