Figure 3

Figure 3 Cbx7a/CBX7 contributes to pathological gene expression in CCM, impacting cardiovascular defects in zebrafish mutants.

(A) Functional clustering of gene ontology terms for deregulated genes identified in RNA-sequencing datasets from ccm2^m201 in comparison with wild-type (wt) and ccm2^m201;cbx7a^pbb62 double mutants in comparison with ccm2^m201 mutants. Differentially-expressed genes were identified by using DEseq2 (Galaxy Version 2.11.40.7) and a Wald statistic for pairwise comparisons. (B) Schematic overview of candidate genes related to CCM pathology gene ontology terms. Shown are fold-changes of expression levels in wild-type, ccm2^m201, and ccm2^m201;cbx7a^pbb62 double mutants. (C) Quantification of wnt9b and wnt9a mRNA levels in ccm2^m201 and krit1^ty219c extracted hearts. wnt9b is upregulated in both mutants while wnt9a is upregulated only in krit1^ty219c mutants (n = 3–4 replicates per each group, and each replicate is a pool of 50–100 hearts). Statistical testing is based on one-way ANOVA with Tukey’s multiple comparisons testing (s.e.m. bars indicated). (D, E) Representative images of whole-mount in situ hybridizations of wnt9b expression in the 56 hpf zebrafish heart. In wild-type, wnt9b is expressed in a defined domain confined to the atrioventricular canal (AVC) (D). In Tg(fli1a:Gal4FF)^ubs3;Tg(UAS:klf2a)^ig1 double transgenic embryos [Tg(fli1a>klf2a)] with an endothelial-specific overexpression of Klf2a, the expression of wnt9b is expanded into the cardiac chambers (E). (F–I) Functional rescue of endocardial phenotypes through genetic depletion of Wnt9b in zebrafish ccm2^m201 mutant embryos. Shown are maximum projections of confocal image z-stacks of zebrafish hearts at 56 hpf, with the endocardium being marked by Tg(kdrl:EGFP)^s843 expression. (F) The AVC region forms correctly in wild-type hearts (red arrowhead). The expression of ALCAM in endocardial AVC cells (F’; white arrows) is lost in ccm2^m201mutants (G’). Upon depletion of wnt9b using an antisense oligo morpholino, the overall morphology of ccm2^m201 mutant hearts is improved, the AVC region restored (H; red arrowhead), and ALCAM expression at the endocardial AVC is restored (H’; white arrows). Knock-down of wnt9b alone does not affect AVC formation (I; red arrowhead) and ALCAM expression (I’; white arrows). (J–M) Representative fluorescence microscopy images of CCM2-deficient (CCM2-KO) and wild-type (WT) iPSC-derived ECs immunostained for F-Actin (J, K), and VE-Cadherin (L, M). (N) Schematic overview of WNT9A and WNT9B mRNA levels in primary human lesion material from familial and sporadic CCM patients as compared to healthy brain material and in human iPCS-derived endothelial cells depleted of CCM2 as compared to non-edited cells. In all cases, WNT9A and WNT9B are upregulated. (O–Q) Quantifications based on qRT-PCR of angpt1 and tek/tie2 mRNA levels in ccm2^m201 and ccm2^m201;cbx7a^pbb62 double mutants (O) and in cbx7a antisense morpholino-injected Tg(fli1a>klf2a) (P, Q) (n = 4–5 replicates and each replicate contains 8–10 pooled embryos). Statistical testing is based on student T-test (s.e.m. bars indicated). (R, X) Shown are maximum projections of confocal image z-stacks of zebrafish hearts at 48 hpf in which the endocardium is marked by Tg(kdrl:EGFP)^s843 expression. Wild-type embryos treated with DMSO form a normal AVC (R; red arrowhead) and express ALCAM in endocardial cells at the AVC (R’; white arrows). Endocardial defects in ccm2^m201 mutants include the loss of the AVC constriction (S) and ALCAM expression (S’). (T) ccm2^m201 mutant embryos treated with the Tie1/2 signaling inhibitor BAY826 have a normalized cardiac morphology, show a restoration of the AVC region (T; red arrowhead) and endocardial ALCAM expression (T’; white arrows). (U) Treatment of wild-type embryos with BAY826 has only a mild effect on cardiac morphology as AVC (U; red arrowhead) and expression of ALCAM in endocardial AVC cells (U’; white arrows) are present. (V–X) The genetic depletion of zebrafish tek and tie1 genes suppresses the cardiac ballooning phenotype in krit1 morphants. At 56 hpf, wild-type embryos injected with an antisense morpholino against krit1 lose that AVC constriction and exhibit strongly ballooned hearts, which is a characteristic feature of the cardiac phenotype in zebrafish ccm mutants (W). The genetic depletion of tek/tie2 and tie1 in krit1 morphants restores the AVC constriction and suppresses cardiac ballooning (X, red arrowhead). (Y) Quantifications of the krit1 morphant cardiac phenotype in different tek^hu1667;tie1^bns208 mutant combinations. The penetrance of krit1 morphant cardiac phenotypes decreases with lowered copy numbers of wild-type tek/tie2 and tie1 alleles and is most strongly suppressed by the combined loss of both Tie1/2 receptors. Numbers in boxes are embryos with a rescued heart / total number of embryos with the respective genotype. AVC atrioventricular canal, endo endocardium, myo myocardium, Ven ventricle, Atr atrium. Red arrowheads indicate AVC, while white arrows indicate the presence of ALCAM expression in AVC endocardial cells. Experiments in zebrafish were done in three biological replicates. Scale bars are (D, E, J–M) 100 µm, (F–I; R–U; V–X) 50 µm, (F’–I’; R’–U’) 20 µm. Source data are available online for this figure.