February 18, 2010;
CHD7 cooperates with PBAF to control multipotent neural crest formation.
Heterozygous mutations in the gene encoding the CHD
(chromodomain helicase DNA-binding domain) member CHD7
, an ATP-dependent chromatin remodeller homologous to the Drosophila trithorax
-group protein Kismet, result in a complex constellation of congenital anomalies called CHARGE syndrome, which is a sporadic, autosomal dominant disorder characterized by malformations of the craniofacial structures, peripheral nervous system
, ears, eyes
. Although it was postulated 25 years ago that CHARGE syndrome results from the abnormal development of the neural crest
, this hypothesis remained untested. Here we show that, in both humans and Xenopus, CHD7
is essential for the formation of multipotent migratory neural crest
), a transient cell population that is ectodermal in origin but undergoes a major transcriptional reprogramming event to acquire a remarkably broad differentiation potential and ability to migrate throughout the body, giving rise to craniofacial bones and cartilages, the peripheral nervous system
, pigmentation and cardiac structures. We demonstrate that CHD7
is essential for activation of the NC
transcriptional circuitry, including Sox9
. In Xenopus embryos, knockdown of Chd7
or overexpression of its catalytically inactive form recapitulates all major features of CHARGE syndrome. In human NC
associates with PBAF (polybromo- and BRG1
-associated factor-containing complex) and both remodellers occupy a NC
-specific distal SOX9
enhancer and a conserved genomic element located upstream of the TWIST1
gene. Consistently, during embryogenesis CHD7
and PBAF cooperate to promote NC
gene expression and cell migration. Our work identifies an evolutionarily conserved role for CHD7
in orchestrating NC
gene expression programs, provides insights into the synergistic control of distal
elements by chromatin remodellers, illuminates the patho-embryology of CHARGE syndrome, and suggests a broader function for CHD7
in the regulation of cell motility.
Disease Ontology terms:
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Figure 2. CHD7 and its ATP-ase function are required for neural crest migration in vivo. (A) CHD7 mRNA expression during Xenopus embryogenesis. CHD7 expression was visualized by RNA in situ hybridization at indicated stages of development, showing diffuse pattern at the gastrula stage, but expression localized to the neural (arrow 1), neural crest (arrow 2) and preplacodal ectodermal (arrow 3) tissues at the late neurula stage. At the tailbud stages, CHD7 is expressed in the pharyngeal arches (arrow 4) and optic placode (arrow 5), as well as alongside the neural tube (arrow 6).
(B) Morpholino mediated knockdown of CHD7 protein levels in Xenopus laevis embryos. Embryos were injected on both sides at the two-cell stage with morpholino oligonucleotide (MO) targeting either CHD7 or BRD7 at 3.3 uM concentration. Nuclei were extracted from these as well as uninjected control embryos at neurula stage. Whole nuclear lysates were normalized for protein concentrations and analyzed by immunoblotting with α-CHD7xl and α-RNA pol II antibodies.
(C) Neural crest migration defect in CHD7 MO and hCHD7 ATPK998R injected embryos. Two-cell stage embryos were injected with mRNA encoding a photo-activatable protein Kaede alone (a and b), Kaede and CHD7 MO (3.3 uM) (c), Kaede, CHD7 MO, and hCHD7 wt mRNA (1 ng) (d), or Kaede and hCHD7 ATPK998R mRNA (9 ng) (e). At the neurula stage embryonic structures corresponding to a subset of the anterior neural and neural crest tissues were UV-irradiated to photo-convert Kaede protein from green to red fluorescence (schematics in Supplementary Figure 8A). Cell migration to the pharyngeal arches (PA) was assayed at the tailbud stage (orange arrows).
(D) Effect of CHD7 knockdown on expression of transcription factors involved in neural crest formation. Two cell-stage embryos were injected with CHD7 MO at 3.3 uM asymmetrically into a single blastomere and analyzed by whole mount RNA in situ hybridization at neurula stage to visualize expression patterns transcription factors controlling: neural plate border territory specification (Msx1, Zic1, Pax3), maintenance of the competent border (MycII) and early neural crest formation (Sox9, Twist, Slug). Sox9 is detected in two major expression domains: the neural crest (blue arrow) and prospective otic placode (red arrow).
(E) hCHD7 mRNA rescues defects in Sox9 and Twist expression. Two cell-stage embryos were co-injected with CHD7 MO (3.3 uM) and wild type full-length hCHD7 mRNA (1 ng) asymmetrically into a single blastomere. At late neurula stages the embryos were analyzed by whole mount RNA in situ hybridization. Representative examples of full and partial rescue are shown.
Figure 3. Overexpression of CHD7 ATPase mutant in Xenopus recapitulates CHARGE traits
(A) Craniofacial defects in Xenopus embryos expressing ATPaseK998R mRNA. Two-cell stage embryos were symmetrically injected with 9 ng of either ATPaseK998R or control mRNA (Kaede) on both sides and allowed to develop to the late tadpole stage (stage 45) for analysis of craniofacial cartilage by Alcian Blue staining and dissection. M: Meckel's cartilage, C: ceratohyle, B: basohyl, BA: branchial arch.
(B) Coloboma of the eye with microphtalmia. Two-cell stage embryos were injected with ATPaseK998R mRNA asymmetrically into a single blastomere and allowed to develop to the late tadpole stage (stage 45). The eyes from uninjected and injected side were imaged under a stereomicroscope under the same magnification. Black arrows indicate the coloboma, a fissure that has not completely closed.
(C) Defects in the formation of the otolith, a structure analogous to human ear. Four cell embryos were injected in one of the dorsal blastomeres with ATPaseK998R mRNA and raised to early tadpole stages (stage 40-44?). The left and right otolith was imaged at the same magnification. White arrows indicate three parts of the properly developed otolith.
(D) The truncus arteriosus and cardiac outflow tract are abnormally positioned in ATPaseK998R expressing tadpoles. Transverse section through the stage 45 tadpoles expressing control mRNA (left panels) or ATPaseK998R mRNA (right panels). A: atrium, OFT: outflow tract, TA: truncus arteriosus, V: ventricle. Upper left section: The TA is located to the right and superior to the ventricle of the heart. Arrowheads indicate valves within the truncus arteriosus. Upper right section: Arrowhead indicating the TA in the ATPaseK998R mutant heart that is located to the right, but inferior to the ventricle of the heart. Lower left section: A relatively posterior transverse section of the control tadpole showing the normal connection of cardiac OFT to the ventricle of the heart. OFT is directly to the right and superiorly. Arrowheads indicate valves at the atrioventricular junction. Lower right section: Transverse section through a similar region of the ATPaseK998R expressing tadpole heart showing the aberrant orientation of cardiac OFT which is directed to the right and inferiorly.
Figure 4. CHD7 and PBAF bind distal regulatory elements upstream of NC transcription factors and synergistically regulate NC gene expression and migration in vivo. a, In hNCLCs CHD7 and BRG1 co-occupy the NCspecific distal enhancer controlling SOX9 expression. Top: schematic representation of the SOX9 locus, showing the relative positions of primer sets (P1–P3) used for ChIP–quantitative polymerase chain reaction (qPCR) analyses, NC-specific (NCE) and notochord, gut and pancreas-specific (NGPE) SOX9 distal enhancers, as identified in ref. 9. Bottom: ChIP–qPCR analyses of H3K4me1, H3K4me3, CHD7 and BRG1 levels at P1–P3 genomic regions in hNCLCs. b, In hNCLCs CHD7 and BRG1 co-occupy a conserved distal element located upstream from the TWIST1 TSS. Top: schematic representation of the TWIST1 locus, showing the relative positions of primer sets (P1 and P2) used for ChIP–qPCR analyses. The conservation index for 31 eutherian mammals (ENSEMBL) is shown below. Bottom: ChIP–qPCR analyses of H3K4me1, H3K4me3, CHD7 and BRG1 levels at P1 and P2 genomic regions. In a and b the y axes show the percentage of input recovery; error bars represent s.d. from three technical replicates of a representative experiment. Ctrl, control. c, Synergistic effect of Chd7 and Brd7 MOs on Twist expression. Twist in situ hybridization analyses of early-tailbud embryos injected with the indicated doses of Chd7 and/or Brd7 MOs into a single DA blastomere at the eight-cell stage. All injection volumes were kept constant. Representative images of the injected (top) and uninjected (bottom) side of the same embryo are shown. Asymmetry in Twist expression is quantified in the bottom graph. d, Synthetic effect of Chd7 and Brd7 MOs on NC migration. Analysis of cell migration to pharyngeal arches in tadpoles derived from eight-cell stage embryos co-injected with fluorescent lineage tracer and indicated MOs into a single DA blastomere. Quantification of cell migration to pharyngeal arches (PA) is shown in the bottom graph. P values in c and d were calculated by Fisher’s exact test for count data. e, Model of CHD7 function in neural crest formation. We propose that CHD7 and PBAF synergistically, and in a tissue-specificmanner, regulate activity of distal elements controlling expression of critical NC transcription factors. Activation of NC transcriptional circuitry in turn permits gene expression reprogramming, leading to epithelial–mesenchymal transition (EMT), acquisition of multipotency and migratory potential.