We previously reported that morpholino silencing of adrb1 rescued CVP cavernomas in zebrafish embryos⁸. Because of morpholinos potential for off target effects^{12, 13}, we sought to confirm the β1AR’s potential involvement in CCM pathogenesis by inactivating adrb1. We used CRISPR-Cas9 to generate an 8-bp deletion resulting in a premature stop codon at 57 bp (Figure 1A). To exclude potential off-target effects, the top 20 potential off-target sites predicted by Cas-OFFinder (www.rgenome.net/cas-offinder) (Supplementary Table 2) were sequenced and were not mutated (data not shown). We intercrossed adrb1^+/- F1 offspring resulting in adrb1^-/- zebrafish embryos that displayed reduced cardiac contractility (Supplementary Video 1) and decreased heart rate (Supplementary Figure 1A). Similar to Adrb1^-/- mice¹⁴, adrb1^-/- zebrafish exhibited a blunted chronotropic response to a β1AR agonist, isoprenaline hydrochloride (Figure 1B). Adrb1^-/- embryos had no obvious defects in vascular development and they survived to adulthood and were fertile.

We intercrossed adrb^+/- individuals and injected one-cell stage embryos with ccm2 CRISPR and blindly scored the presence of CVP cavernomas at 48hpf. As expected we observed CVP cavernomas in 28% of adrb1^+/+ embryos. In sharp contrast only 3% of adrb1^-/- embryos exhibited cavernomas (Fig. 1C and D) indicating that loss of β1AR prevents CVP cavernomas. these observations demonstrate that the β1AR is required for embryonic CVP cavernoma formation.

Adult ccm2 CRISPR zebrafish display highly penetrant CCMs throughout the central nervous system³. Brains from adult ccm2 CRISPR fish on adrb1^-/- (12 brains) or wild type (13 brains) background were treated with CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis)¹⁵, and these transparent brains were then scanned with light-sheet microscopy and lesions were enumerated and volumes were estimated with NIH ImageJ. While the typical multi-cavern CCMs were observed in brains on wild type background appearing as blood filled dilated vessels (Figure 2A through C), ccm2 CRISPR adrb1^-/- fish exhibited an 87% reduction in lesion volume (Figure 2D through G). Thus, genetic inactivation of β1AR prevented CCMs.

The non-selective β blocker propranolol reduces lesion volume in murine CCM models^{8, 16}. To ascertain whether propranolol had a similar effect in the zebrafish model, the chemical treatment was started from larval stage at concentration which allows the fish to develop to two months for CCM inspection. We added 12.5µM propranolol to or vehicle control to fish water of ccm2 CRISPR zebrafish starting at 3 weeks of age. The water was refreshed daily with drug or vehicle until fish were sacrificed and brains were examined as described (Figure 3A). Quantification based on light-sheet scanning of zebrafish brains (Figure 3B) showed that compared to vehicle-treated controls (Figure 3C, D, and E), propranolol-treated groups displayed a 94% reduction in CCM lesion volume (Figure 3F, G, and H). Similarly, administration of 50µM racemic metoprolol a β1-selctive antagonist produced a similar (98%) reduction in lesion volume (Figure 3B, I, J, and K). Importantly, neither drug at the doses administered reduced the growth of the fish or the volume of their brains. In sum, both genetic and pharmacological loss of β1 adrenergic receptor signaling markedly reduces the lesion burden in the zebrafish ccm2 CRISPR model of CCM.

Inactivation of CCM genes leads to increased endothelial KLF2 expression^{17, 18}, a transcription factor important in cavernoma formation^{3, 19}. Nevertheless, silencing of adrb1 did not prevent the expected increased endothelial klf2a¹⁷ expression in ccm2 morphant Tg(klf2a:H2b-EGFP) fish in which the nuclear EGFP expression is driven by the klf2a promoter (Figure 4A and B). We previously reported that mosaic expression of KLF2a occurs in ccm2 CRISPR fish and is sufficient to drive cavernoma formation when blood is flowing³. Nevertheless, the adrb1 morphant (Figure 4C) and control morphant (Figure 4D) ccm2 CRISPR Tg(klf2a:H2b-EGFP) embryos both displayed a mosaic increase of klf2a reporter (Figure 4E). Similarly, merely slowing the heart and reducing contractility with 2,3-butanedione monoxime (BDM)²⁰ also prevented CVP cavernomas (Fig 4F, and Supplementary Figure 2). Thus, silencing β1AR does not prevent the generalized increase in endothelial KLF2a in ccm2 morphants nor does it prevent the mosaic KLF2a increase in ccm2 CRISPR zebrafish.

Zebrafish were maintained and with approval of Institutional Animal Care and Use Committee of the University of California, San Diego. The following mutant and transgenic lines were maintained under standard conditions: Tg(fli1:EGFP)^{y1 27}, Tg(klf2a:H2b-EGFP) ²⁸, and Tg(kdrl:mcherry)^{is5 29}. adrb1^-/- zebrafish was obtained by coninjection of Cas9 protein (EnGen Spy Cas9 NLS, M0646, NEB) with gRNA targeting adrb1. Genotyping of adrb1^-/- was performed with forward primer (5’-AGAGCAGAGCGCGGATGGAA-3’) and reverse primer (5’-GATCCATACATCCAGGCT-3’).

pCS2-nls-zCas9-nls (47929) and pT7-gRNA (46759) were bought from Addgene. The CRISPR RNA (crRNA) sequences used in this study are as follow: ccm2-1 5’- GGTGTTTCTGAAAGGGGAGA-3’, ccm2-2 5’- GGAGAAGGGTAGGGATAAGA-3’, ccm2- 3 5’-GGGTAGGGATAAGAAGGCTC-3’, ccm2-4 5’-GGACAGCTGACCTCAGTTCC-3’, adrb1 5’-GACTCTAAACGCGCCACGG-3’. Target gRNA constructs were generated as described before³⁰. Morpholino sequence used in this study are as follow: adrb1 (5’- ACGGTAGCCCGTCTCCCATGATTTG-3’), ccm2 (5’-GAAGCTGAGTAATACCTTAACTT CC-3’), tnnt2a (5’-CATGTTTGCTCTGATCTGACACGCA-3’), control (5’- CCTCTTACCTC AGTTACAATTTATA-3’).

The pCS2-nls-zCas9-nls plasmid containing Cas9 mRNA was digested with NotI enzyme, followed by purification using a Macherey-Nagel column, serving as the template. The capped nls-zCas9-nls RNA was synthesized using the mMESSAGE mMACHINE SP6 Transcription Kit from ThermoFisher Scientific. The resulting RNA was purified through lithium chloride precipitation, as per the instructions provided in the kit. For gRNA synthesis, the gRNA constructs were linearized using BamHI enzyme and purified using a Macherey-Nagel column. The gRNA was synthesized via in vitro transcription using the MEGAshortscript T7 Transcription Kit from ThermoFisher Scientific. After synthesis, the gRNA was purified by alcohol precipitation, as instructed in the same kit. The concentration of the nls-zCas9-nls RNA and gRNA was measured using a NanoDrop 1000 Spectrophotometer from ThermoFisher Scientific, and their quality was confirmed through electrophoresis on a 1% (wt/vol) agarose gel.

All injections were performed at 1-cell stage with a volume of 0.5nl. The final injection concentrations are as follow: Cas9 protein (10µM), Cas9 mRNA (750ng/μl), gRNA 120ng/μl, adrb1 MO (4ng/µl), ccm2 MO (4ng/µl), tnnt2a MO (5.3 ng/µl), control MO (4ng/µl).

Propranolol (P0995, TCI) (12.5µM) and metoprolol (M1174, Spectrum) (50µM) were used to treat the zebrafish larva from Day 21. Beginning from Day 35, adjusted concentration of propranolol (25µM) or metoprolol (100µM) were used to treat the juvenile fish. The chemicals were dissolved in fish water, and fish water containing chemicals were refreshed daily. Egg water without above chemicals was refreshed daily for fish used as negative control. 2,3-butanedione monoxime (BDM) (6mM) was added to egg water of the ccm2 CRISPR embryos at 25hpf, and CVP cavernoma was observed at 2dpf.

To prepare the embryos for imaging, they were first anesthetized using egg water containing 0.016% tricaine (3-amino benzoic acid ethyl ester) from Sigma-Aldrich. Subsequently, the anesthetized embryos were embedded in 1% low melting point agarose obtained from Invitrogen (product number 16520050). The imaging process was carried out using a Zeiss 880 Airyscan confocal microscope, utilizing the standard Airyscan mode, with a 20x/NA 0.8 objective. The intensity of nuclear EGFP (enhanced green fluorescent protein) was quantified using ImageJ software.

Heartbeat and blood flow were recorded using Olympus MVX10. The embryos were treated with 0.004% tricaine which does not have effect on heartbeat^{31, 32}.

The dissection of zebrafish brains followed the methodology described in a previous study by Gupta et al.³³. The CUBIC method was optimized based on the findings from a previous report by Susaki et al.¹⁵. The brains were fixed in 4% paraformaldehyde (PFA) with a pH of 7.5 for 24 hours and subsequently washed with PBS (phosphate-buffered saline) for an additional 24 hours. Following the PBS wash, the brains underwent CUBICR1 treatment at 37°C in a water bath for 42 hours. For imaging, the samples were placed in CUBICR2 as the imaging medium and imaged using a ZEISS Lightsheet Z.1 microscope. Scanning was carried out utilizing 5X dual illumination optics in combination with a 5X objective.

The statistical analysis was conducted using GraphPad Prism software. P-values were calculated using an unpaired two-tailed Student’s t-test, unless otherwise specified. The bar graphs display the mean values along with their corresponding standard deviations (SD).