Human Genetics

, Volume 133, Issue 8, pp 997–1009

CHD7, the gene mutated in CHARGE syndrome, regulates genes involved in neural crest cell guidance

  • Yvonne Schulz
  • Peter Wehner
  • Lennart Opitz
  • Gabriela Salinas-Riester
  • Ernie M. H. F. Bongers
  • Conny M. A. van Ravenswaaij-Arts
  • Josephine Wincent
  • Jacqueline Schoumans
  • Jürgen Kohlhase
  • Annette Borchers
  • Silke Pauli
Original Investigation

DOI: 10.1007/s00439-014-1444-2

Cite this article as:
Schulz, Y., Wehner, P., Opitz, L. et al. Hum Genet (2014) 133: 997. doi:10.1007/s00439-014-1444-2

Abstract

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.

Supplementary material

439_2014_1444_MOESM1_ESM.pptx (791 kb)
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
439_2014_1444_MOESM2_ESM.pptx (3.2 mb)
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
439_2014_1444_MOESM3_ESM.pptx (513 kb)
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. (A,C,E,G) 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
439_2014_1444_MOESM4_ESM.pptx (261 kb)
Supplementary material 4 (PPTX 261 kb) Figure S4. Repetition of the experiment shown in Figure S3 using 1.5 ng hCHD7 RNA

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yvonne Schulz
    • 1
  • Peter Wehner
    • 2
  • Lennart Opitz
    • 4
  • Gabriela Salinas-Riester
    • 4
  • Ernie M. H. F. Bongers
    • 5
  • Conny M. A. van Ravenswaaij-Arts
    • 6
  • Josephine Wincent
    • 7
  • Jacqueline Schoumans
    • 7
    • 8
  • Jürgen Kohlhase
    • 9
  • Annette Borchers
    • 2
    • 3
  • Silke Pauli
    • 1
  1. 1.Institute of Human GeneticsUniversity Medical Center GöttingenGöttingenGermany
  2. 2.Department of Developmental Biochemistry, Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), GZMBUniversity of GöttingenGöttingenGermany
  3. 3.Department of Biology, Molecular EmbryologyPhilipps-University MarburgMarburgGermany
  4. 4.Department of Developmental BiochemistryUniversity Medical Center GöttingenGöttingenGermany
  5. 5.Department of Human GeneticsRadboud University Nijmegen Medical CenterNijmegenThe Netherlands
  6. 6.Department of GeneticsUniversity Medical Centre Groningen, University of GroningenGroningenThe Netherlands
  7. 7.Department of Molecular Medicine and Surgery and Center for Molecular MedicineKarolinska University HospitalStockholmSweden
  8. 8.Cancer Cytogenetic Unit, Department of Medical GeneticsUniversity Hospital of LausanneLausanneSwitzerland
  9. 9.Center for Human GeneticsFreiburgGermany