Adrb1 signaling is essential for CVP dilation. (A) The targeted adrb1 allele shows an 8-nucleotide deletion producing a pre-stop codon. Adrb1 null cDNA is predicted to encode truncated adrb1 protein. The wild type adrb1 protein contains 390 amino acids, while the predicted adrb1 null protein would contain 2 missense amino acids (gray bar) and would terminate after amino acid 18. (B) Isoprenaline hydrochloride (50 µM) treatment at 72hpf lead to a heart rate increase in zebrafish, while the delta heart rate in adrb1-/- is significantly smaller than that of wild type. Paired two-tailed t test, P<0.0001. (C) After ccm2 CRISPR injection, representative bright field and confocal images of 2dpf embryos show that wild type embryos display CVP dilation, while adrb1-/- embryos were resistant to this defect. Arrowhead and arrows indicate the dilation in CVP. Scale bar: 500 µm (bright field), 100µm (confocal). (D) Paired two-tailed t test shows that CVP dilation is significantly reduced in adrb1-/- embryos compared to control. P=0.0012.

Genetic inhibition of adrb1 signaling could rescue CCM in ccm2 CRISPR zebrafish. (A through F) Representative light sheet microscopy scanning pictures of brains from ccm2 CRISPR adult zebrafish of wild type (A through C) and of adrb1-/- (D through F). Brains from ccm2 CRISPR on wild type background show lesions indicated by arrows (A through C), while brains from ccm2 CRISPR on adrb1-/- do not show lesions (D through F). Scale bar: 1mm. (G) Statistical analysis of lesion volume by unpaired two-tailed t test. P=0.0005.

Both propranolol and metoprolol could rescue CCM in ccm2 CRISPR zebrafish. (A) A diagram outlines the drug treatment experiment, CUBIC treatment and following recording of CCMs in adult zebrafish brain. The chemical treatment was started from week 3 with 12.5 µM propranolol or 50 µM metoprolol, and increased to 25 µM propranolol and 100 µM metoprolol, respectively from week 5. The fish water with chemicals or vehicle control are refreshed on a daily basis. (B) Statistical analysis of lesion volume by one-way ANOVA followed by Tukey’s multiple comparison test. P<0.01. (C through K) Representative light sheet microscopy scanning pictures of brains from ccm2 CRISPR adult zebrafish. In controls without chemical treatment (C, D, and E) there were vascular anomalies indicated by arrows. Neither propranolol (F, G, and H) nor metoprolol (I, J, and K) treated fish showed vascular lesions in the brain. Scale bar: 1mm.

Adrb1 signaling does not alter klf2a expression in ccm2 CRISPR embryos. Tg(klf2a:H2b-EGFP; kdrl:mcherry) embryos were injected and nuclear EGFP signal in mcherry labeled vascular endothelial cells is recorded by confocal. Representative images from each group are shown. (A and B) Neither embryos injected with ccm2 MO together with adrb1 MO (A) nor control MO (B) displayed mosaic pattern of endothelial nuclear EGFP intensity. (C through E) All the embryos are absent of blood flow due to tnnt morpholino injection. Compared to that of control (C), ccm2 CRISPR embryos co-injected with adrb1 MO (D) or control MO (E) both displayed a mosaic increase of nuclear EGFP intensity of vascular endothelial cells. Scale bar:100µm. (F) EGFP intensity of endothelial nuclei from 2 embryos in each group are quantified with Image J. Statistical analysis is performed by one-way ANOVA followed by Tukey’s multiple comparison test. There is no significant difference between ccm2 morphants coinjected with adrb1 MO (A) and control MO (B). There is also no significant difference between ccm2 CRISPR embryos coinjected with adrb1 MO (D) and control MO (E). P>0.05. (F) At 2dpf, 2,3-BDM prevented the CVP cavernoma dramatically. 164 embryos in 2,3-BDM treated group and 177 in control group were used for Two-tailed paired t-test. P=0.0013.