Fig 2: CHD7 regulates Class 3 Semaphorin expression in the developing heart. (A) Real-time PCR comparing expression of Class 3 Semaphorin genes in Chd7+/+;Mesp1-Cre and Chd7fl/fl;Mesp1-Cre dissected hearts at E11.5 and E13.5. Supplemental Table S7 shows the microarray results for these genes for comparison. (B) At E13.5, trabecular expression of Sema3A was seen by ISH on control Chd7fl/fl sections, with significant reduction in Chd7fl/fl;Mesp1-Cre hearts (red stars). (C) ISH at E11.5 showed Sema3C expression in the myocardial cuff (blue arrows), which was strongly downregulated at the distal end of the OFT in Chd7fl/fl;Mesp1-Cre hearts (red arrows). Sema3C expression in the cardiac neural crest was also slightly reduced in mutants (white arrows). (D) ENCODE data around the Sema3c locus was used to design primer sets (denoted in red) to overlap with areas of DNase I hypersensitivity and histone modification marks associated with suspected promoter and enhancer regions. (E) ChIP-PCR demonstrated CHD7 binding around the promoter of Sema3C at E11.5 using Primer pair 1. (F) DNase I hypersensitivity assay showed greater chromatin compaction in Chd7fl/fl;Mesp1-Cre hearts (dotted line) than littermate controls (solid line) at the Sema3c promoter. Little change was seen at the Gapdh or Nanog promoters, although Gapdh displayed greater hypersensitivity, correlating with its constitutive expression. Individual replicates are shown, as it was not possible to normalise across separate experiments. ? p<0.05, ?? p<0.01 (unpaired studentttest). Scale bars represent0.2 mm. oft indicates outflow tract; rv, right ventricle; lv, left ventricle.

Fig 3: Pharmacological responses of chd7 mutants and amelioration of neuronal network development by ephedrine PCR proof of a 700 bp deletion in the chd-7 gene in chd-7(gk290) mutant worms.Lifespan analyses of chd-7(gk290) Caenorhabditis elegans mutants treated with ephedrine (green) compared with control DMSO (black). Log-rank test was performed for statistical analyses. (N = 3, n = 50; *P < 0.05).Survival rate of chd7 -/- zebrafish mutants treated with ephedrine (blue) compared with untreated mutants (red). N = 3, n = 60.Acetylated tubulin staining in non-treated and ephedrine-treated chd7 -/- zebrafish mutants showing rescue of the severely affected outbranching structure of Vth cranial nerves. Graphs showing quantitative analyses of percentage and mean total length of peripheral projections per zebrafish in controls and mutants without and with ephedrine treatment (n = 5; ***P < 0.001; **P < 0.005; one-way ANOVA). Data information: Data are presented as mean ± SEM. Scale bar = 50 µm. n is the number of fish or worms used. N is the number of experimental repeats.

Fig 4: Generation and characterization of chd7 mutants Translation following genome editing resulted in a premature stop codon (*) in chd7 -/- fish.qPCR analysis of RNAs from 3dpf larvae shows a significant reduction of chd7 mRNA expression in both chd7 +/- and chd7 -/- compared with wild-type (N = 5). ****P < 0.0001, one-way ANOVA.Examination of brain tissues in chd7 +/+ (left images) and chd7 -/- (right images) by H&E staining at 5 dpf. Zebrafish brains were sectioned at telencephalic (1,1’), diencephalic (2, 2’), mesencephalic (3,3’) and rhombencephalic levels (4,4’). Levels of sections are indicated in the sketch of a sagittal view of a 5 dpf zebrafish brain (top image). The scale bar is 0.12 mm. P: pallium, S: subpallium, Po:preoptic region, Tel: Telencephalon; TeO: tectum opticum (or OT: optic tectum), m: medial tectal proliferating zone, DT: dorsal thalamus, PTd: dorsal part of posterior tuberculum, PTv: ventral part of posterior tuberculum, MO: medulla oblongata, Hyp: hypothalamus, CeP: cerebellar plate. chd7 -/- mutant fish displayed features of CS such as heart defects (red arrow) at a low penetrance (N = 3). ****P < 0.0001; Student’s t-test.Alcian blue staining of 6dpf larvae showing craniofacial defects at Meckel’s cartilage (red arrow). Data information: Data are presented as mean ± SEM. n is the number of fish used. N is the number of experimental repeats.

Fig 5: CHD7 regulates PAQR3 expression in human cells qPCR analysis of PAQR3 expression in a CHD7 mutation-positive patient compared with parental controls set (N = 4; ****P < 0.0001; Student’s t-test). Fold change was calculated according to the 2-??Ct method, using HPRT1 and RPS1 as housekeeping genes for normalization. All data were expressed as mean fold change ± SD across replicates, relative to control parents set to 1 (dotted line). N is the number of experimental repeats.Schematic view of PAQR3 exon 1 and proximal promoter on chromosome 4 (hg19 assembly), obtained with the UCSC genome browser (https://genome.ucsc.edu/) and showing the sequence amplified in ChIP-qPCR assays in LCLs (thick black line) along with a previously described CHD7 ChIP-seq peak (thick grey line) and signal (green) in H1-hESC (ENCODE3).ChIP-qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7 mutation-positive patient (N = 3) compared with parental controls (N = 6); *P < 0.05; Student’s t-test. All data were expressed as mean fold change ± SD. N is the number of experimental repeats.