Characterizing the cardiovascular phenotype of a new zebrafish model of Marfan syndrome
Authors: Sips P Y , Le Roux R , Caboor L , Vermassen P , Mennens L , De Backer J 
Published: October 2021

Abstract

Background
Marfan syndrome (MFS) is a rare disease caused by a defect in the fibrillin-1 gene (FBN1), with potentially severe cardiovascular manifestations. MFS patients are particularly susceptible to a progressive aortic dilation leading to potential dissection and wall rupture. No causal treatment for the disease is available and current medical treatment is aimed at slowing aortic disease progression to minimize severe complications. When indicated, surgical repair of the aortic defect is performed. Although these strategies have clearly led to improved survival, some patients still present with fatal complications.

Purpose
To generate a new flexible zebrafish model of MFS to gain a better understanding of the underlying pathophysiological mechanisms and to find new treatment options.

Methods
We used the CRISPR/Cas9 method to disrupt the 3 fibrillin genes in zebrafish (fbn1, fbn2a, and fbn2b). The Tg(kdrl:EGFP) reporter was used to visualize cardiovascular structure by fluorescent microscopy up to 8 days post fertilization (dpf). A subset of embryos was treated with the myosin inhibitor 2,3-butanedione monoxime (BDM).

Results
We found that zebrafish lacking fbn1 and/or fbn2a do not show any detectable phenotype during development. No evidence of induction of genetic compensation was found in these mutant lines.
Zebrafish deficient in fbn2b however do show strong phenotypes, including fully penetrant finfold atrophy (Fig. 1A-B and E, arrowhead). On average 50% of homozygous fbn2b mutant (fbn2b−/−) zebrafish embryos show endocardial detachment (Fig. 1C-D; a:atrium, v:ventricle), leading to vascular embolism, pericardial edema (Fig. 1B, arrow), loss of blood flow, and ultimately death at 7–9 dpf. Interestingly, fbn2b−/− without endocardial detachment survive normally, but develop a dilated bulbus arteriosus phenotype during larval stages (Fig. 1F-G, arrow; 1H: diameter during minimal and maximal distension, *: P&lt;0.05 and ***: P&lt;0.001 by Sidak's post-test after Two-Way ANOVA). This anatomical structure is strongly related to the aortic root in humans, which is the predominant location of aortic dilation in MFS.
All fbn2b−/− embryos show abnormal early development of the caudal vein as a cavernous structure lacking vessel integrity (Fig. 2, arrowheads). This phenotype resolves in embryos retaining normal blood flow. We found that fbn2b−/− embryos raised in BDM to inhibit blood flow show a more severe caudal vein phenotype than wild-type (WT) controls (Fig. 2, yellow line: severe vascular dilation).

Conclusion
Loss of fbn2b, but not the other fibrillin genes, in zebrafish results in cardiovascular manifestations overlapping with MFS. These data indicate that fbn2b−/− zebrafish can be a relevant model to explore the mechanisms leading from fibrillin deficiency to the cardiovascular symptoms observed in MFS. Our preliminary results suggest that there is an interplay between fibrillin deficiency and biomechanical signaling.

Funding Acknowledgement
Type of funding sources: Foundation. Main funding source(s): Fund Baillet Latour Grant for Medical Research Figure 1. Phenotype of fbn2b−/− larvaeFigure 2. Effect of blood flow in fbn2b−/−