TRAF7 mutations cause congenital heart defects: reduction of TRAF7 in zebrafish (D–H) and Xenopus tropicalis (I–P) as well as overexpression of mutant TRAF7 in X. tropicalis causes developmental defects (Q–U). (A) Clinical manifestations of patients harboring inherited TRAF7 heterozygous developmental mutations. (B and C). Sanger sequencing traces of patients 1-02030 and 1-03358 and their clinically unaffected parents. The mutations are indicated in bold, and asterisk marks the mutated residue. (D–F) Injection of control (D–D’’) or splice-site (E–E’’’) TRAF7 MO in tg(kdrl:GFP;gata-1:dsRed) embryos results in pronounced heart looping defects at 36 hpf. (F). Quantification of embryos displaying cardiac looping defect ****: P < 0.0001 (Fisher’s exact test; n= # of embryos). (G and H). Traf7 morphants exhibit reduced sox10 expression, disorganized pharyngeal arches. (H) Uninjected zebrafish (Tg:kdr:GFP, sox10:mRFP) embryo at 30 hpf showing pharyngeal arches 1 to 6 (p1 to p6). The otic placode (op) and common cardinal vein (CCV) are also labeled. (I) TRAF7 morphant at the same stage. Note the reduced size of the pharyngeal arches, disorganization of p3 to p6, and reduced Sox10 expression. Green channel: endothelial cells; magenta: Sox10-expressing cells. (I) Distribution of heart position in control and TRAF7 1-d postfertilization morphants. Control: Left = 97.29% ± 3.29; middle= 0.83% ± 1.67; right= 1.88 ± 2.19, from 112 embryos total. TRAF7: Left = 76.31% ± 9.32; Middle = 3.07% ± 5.10; Right = 20.62 ± 9.99, from 112 embryos total. Two-way ANOVA with Bonferroni’s multiple-comparison test, P = 0.0004 for left heart position. J−K. Injection of control (J) or splice-site (J’) TRAF7 MO in one-cell stage Xenopus embryos results in extensive pericardial edema (asterisk). Optical coherence tomography (OCT) highlights the edema (asterisk) and malformed heart (K), reflected in a significant reduction of the shortening fraction (L) at 3 dpf (stage 46) (Movie S3). Mann–Whitney test (scatter plot mean ± SD; n = 17, TRAF7 splice-site MO injected; n = 41, control MO injected.) M–N’ TRAF7 CRISPR (CR#1) injection in one-cell stage Xenopus embryos results in pronounced heart looping defects at 48 hpf (N, N’) as compared to controls (M, M’) (Movie S4). OFT: outflow tract, V: ventricle. O. In situ hybridization analysis of Xenopus embryos (stage 16 to 18, 15 hpf): Unilateral injection (Inj.) with splice-site TRAF7, MO at two-cell stage shows disrupted expression of neural crest markers Sox10 and Twist on the injected side when compared to the internal uninjected control. (P) Quantification of embryos displaying cardiac looping ****: P < 0.0001 (Fisher’s exact test; n= # of embryos). (Q) Quantification of control or TRAF7 splice-site MO-injected embryos analyzed for Sox10 and Twist expression. n = number of embryos. ****: P < 0.0001 (Fisher’s exact test; n = # of embryos). (R–T’) Severe pericardial edema in Xenopus embryos following injection of TRAF7 mRNA encoding mutant form V442M (T) but not WT (S) as compared to uninjected embryos (R). Quantification of embryos displaying pericardial edema (T’), ***: P < 0.001 (Fisher's exact test, n = # of embryos). (U) In situ hybridization analysis of Xenopus embryos (stage 17, 18 hpf): Decreased expression of the neural crest markers Twist and Sox10 following unilateral injection of TRAF7 V442M mRNA at the 2-cell-stage. (V). Quantification of control or TRAF7 splice-site MO-injected embryos analyzed for Sox10 and Twist expression. A = Control, B = WT TRAF7, C = V442M TRAF7. ***:P < 0.001: Pairwise Fisher’s exact test with FDR correction, n = # of embryos.
