Systematic disruption of zebrafish fibrillin genes identifies a translational zebrafish model for Marfan syndrome

Systematic disruption of zebrafish fibrillin genes identifies a translational zebrafish model for Marfan syndrome | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Systematic disruption of zebrafish fibrillin genes identifies a translational zebrafish model for Marfan syndrome View ORCID Profile Karo De Rycke , View ORCID Profile Marina Horvat , Lisa Caboor , Petra Vermassen , Griet De Smet , View ORCID Profile Marta Santana Silva , View ORCID Profile Wouter Steyaert , View ORCID Profile Matthias Van Impe , View ORCID Profile Patrick Segers , View ORCID Profile Julie De Backer , View ORCID Profile Patrick Sips doi: https://doi.org/10.1101/2025.06.21.659830 Karo De Rycke 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Karo De Rycke Marina Horvat 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marina Horvat Lisa Caboor 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium BSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Petra Vermassen 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium BSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Griet De Smet 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium BSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marta Santana Silva 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marta Santana Silva Wouter Steyaert 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wouter Steyaert Matthias Van Impe 2 Biophysical Models for Medical Applications (BioMMedA), Institute of Biomedical Engineering and Technology (IBiTech), Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthias Van Impe Patrick Segers 2 Biophysical Models for Medical Applications (BioMMedA), Institute of Biomedical Engineering and Technology (IBiTech), Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Segers Julie De Backer 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium 3 Department of Cardiology, Ghent University Hospital , Corneel Heymanslaan 10, Ghent B-9000, Belgium MD, PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Julie De Backer Patrick Sips 1 Center of Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University , Corneel Heymanslaan 10, Ghent B-9000, Belgium PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick Sips For correspondence: patrick.sips{at}ugent.be Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Background Fibrillins are essential components of the extracellular matrix. Marfan syndrome (MFS), the most common fibrillinopathy, is characterized by severe cardiovascular complications, including cardiac valve abnormalities, myocardial dysfunction, arrhythmias, and, most commonly, thoracic aortic disease. Unfortunately, no definitive medical cure is available. Objectives To establish a zebrafish model of MFS, to enhance understanding of the cardiovascular consequences of fibrillin impairment and identify novel therapeutic targets. Methods CRISPR/Cas9 technology was used to systematically target all zebrafish fibrillin genes. The cardiovascular phenotype was investigated using fluorescent microscopy at embryonic stages and cardiac ultrasound, histology, and synchrotron X-ray imaging in adults. RNA sequencing and drug testing were performed during early development. Results Fibrillin-2b mutant ( fbn2b -/- ) zebrafish had a reproducible phenotype, with a subset of embryos showing endocardial detachment leading to early mortality. Interestingly, the remaining fbn2b -/- zebrafish developed dilation of the bulbus arteriosus, a structure analogous to the aortic root in humans, and survived normally to adulthood. Adult fbn2b -/- zebrafish displayed cardiac valve abnormalities. Transcriptomic analysis of fbn2b -/- embryos suggested the involvement of extracellular matrix remodeling and immune-related pathways. Administration of nebivolol and losartan did not improve the phenotype of fbn2b -/- larvae. Zebrafish lacking fibrillin-1 and/or fibrillin-2a did not show any phenotype. Conclusion Our fbn2b -/- zebrafish model recapitulates key aspects of human cardiovascular manifestations of MFS and can therefore be considered a novel relevant animal model for MFS. Studying this model allows us to broaden the knowledge of the underlying mechanisms of the disease and discover much-needed disease-specific treatment options. CONDENSED ABSTRACT Fibrillin defects lead to severe cardiovascular complications in Marfan syndrome (MFS), including aortic dilation, dissection, and rupture. To model MFS, we generated zebrafish mutants lacking various fibrillin genes. Among these mutant lines, only fibrillin-2b-deficient zebrafish exhibited cardiovascular phenotypes mimicking human disease. Multimodal imaging revealed early cardiac defects, bulbus arteriosus dilation, and valve abnormalities. Transcriptomic analysis identified altered regulation of pathways related to extracellular matrix homeostasis and immune system activation. Compound testing demonstrated the model’s potential for drug discovery. This zebrafish model, recapitulating key cardiovascular features of MFS, provides a valuable platform to investigate disease mechanisms and identify novel treatment strategies. INTRODUCTION Fibrillin microfibrils are fundamental components of the extracellular matrix that contribute to the integrity of connective tissue in various organs, including blood vessels, lungs, skin, skeleton, and eyes. 1 These microfibrils can either directly provide stress-bearing structural support to the tissue or play an essential role as a scaffold for tropoelastin deposition, leading to the formation of elastic fibers that provide tensile strength to the extracellular matrix. 2 , 3 In addition to their structural role, fibrillin microfibrils are also crucial for tissue mechanobiology and homeostasis through the regulation of the bioavailability of growth factors of the transforming growth factor-β (TGF-β) and bone morphogenetic protein family, and by interactions with cell surface receptors such as integrins. 4 The human genome contains three fibrillin isoforms: fibrillin-1 ( FBN1 ), −2 ( FBN2 ) and −3 ( FBN3 ). Pathogenic variants in FBN1 and FBN2 have been associated with connective tissue disorders, including Marfan syndrome (MFS, OMIM #154700) and Beals-Hecht syndrome, also known as congenital contractural arachnodactyly (OMIM #121050), respectively. 5 – 8 MFS is an autosomal dominant inherited disorder with pleiotropic manifestations, including skeletal abnormalities (e.g. skeletal overgrowth, joint laxity), ocular manifestations (e.g. ectopia lentis), and skin abnormalities (e.g. striae). 1 In addition to the cardinal cardiovascular manifestations – namely thoracic aortic aneurysm and dissection (TAAD) and mitral valve disease – impaired myocardial function and arrhythmias occur more frequently in patients with MFS. These cardiovascular manifestations contribute to significant morbidity and an increased risk of early mortality in patients with MFS. 9 , 10 Patients with congenital contractural arachnodactyly have a MFS-like skeletal phenotype, but the eyes and aorta are typically not affected. 11 Nevertheless, some cases have been described where pathogenic variants in human FBN2 were linked to aortic dilation, indicating a level of functional overlap between different fibrillins. 12 – 14 Noteworthy, syndromes with opposite phenotypes (e.g. short instead of tall stature) have been observed as well in patients harbouring pathogenic variants in specific domains of FBN1 and FBN2 . 15 To date, the importance of FBN3 remains less well characterized. However, some associations of FBN3 variants were reported with polycystic ovary syndrome, Bardet-Biedl syndrome, and Weill-Marchesani syndrome. 16 , 17 Previous research on MFS and other fibrillinopathies has significantly advanced our understanding of their genetic basis and the importance of fibrillin proteins for maintaining extracellular matrix integrity and biomechanical signalling. Nevertheless, the precise molecular mechanisms linking these fibrillin defects to the complex cardiovascular manifestations remain incompletely understood. There is thus a particular need for more flexible in vivo models to address this knowledge gap. During the last decades, zebrafish ( Danio rerio ) has emerged as a versatile animal model to complement established mammalian models. Although the cardiovascular system of teleosts has a less complex architecture than its mammalian counterpart, mainly due to the lack of pulmonary circulation, it has nonetheless proven to be a valuable model for studying cardiovascular diseases. 18 , 19 Zebrafish have several unique characteristics which make them an attractive disease model: (1) optical transparency during early development, enabling easy intravital microscopic observation, (2) suitability for high-throughput applications due to low cost and high fecundity, and (3) high genetic similarity to humans with over 70% of human coding genes having at least one zebrafish orthologue. 20 For cardiac pathologies specifically, 96% of the genes known to cause cardiomyopathies are conserved and highly expressed in the zebrafish heart. 21 These distinctive traits make zebrafish an invaluable model for cardiovascular research, particularly for small-molecule high-throughput drug screens to discover novel therapeutic targets. 19 Due to their genetic tractability, zebrafish can also be used to assess the physiological effects of variants of uncertain significance, which would be a significant contribution to improved patient management. 22 , 23 Like humans, the zebrafish genome contains three fibrillin genes, termed fibrillin-1 ( fbn1 ), -2a ( fbn2a ), and -2b ( fbn2b ), though conflicting annotations have been used in different publications and databases. A phylogenetic study conducted by Piha-Gossack et al. 24 has revealed only small evolutionary changes in fibrillin protein structure among different species. The ancestral fibrillin gene already contained most key elements except for the unique characteristic domain located at the N-terminus and specific RGD (Arg-Gly-Asp) motifs. In this study, we systematically disrupted the three different fibrillin genes in zebrafish using CRISPR-Cas9 to examine their impact on the development and function of the cardiovascular system. Our findings revealed multiple phenotypes that are pertinent to MFS. MATERIAL AND METHODS All materials and methods are described in detail in the Supplemental Methods. RESULTS Protein homology between human and zebrafish fibrillin isoforms The protein structure of the human fibrillin family has been well-documented and shows that the different fibrillin genes share an almost identical organization of functional domains. To analyze the zebrafish fibrillin gene family, we used the genomic fbn2a and fbn2b sequences, which were mapped and annotated in the GRCz11 assembly of the zebrafish genome sequence. A genome assembly gap, however, prevented correct annotation of the first 32 exons of the zebrafish fbn1 gene. Recently, a full-length cDNA sequence prediction was assigned to fbn1 (XM_073930076.1), and this sequence, which aligned with our own cDNA sequencing data, was used for in-depth analysis (Supplemental Figure 1). The zebrafish fibrillins present a high level of homology to human fibrillin proteins with an average overlap in amino acid sequence of 70% ( Figure 1B ). Further supporting this, InterProScan analysis revealed a highly conserved domain organization between species, with the characteristic 4-cysteine domain, two hybrid domains, and seven 8-cystein or TGF-β binding-like domains present in all identified zebrafish fibrillin proteins ( Figure 1A ). Download figure Open in new tab Figure 1 Overview of the three fibrillin isoforms in zebrafish. (A) Schematic representation of the protein domains identified in all three zebrafish fibrillin isoforms (fibrillin-1, 2a, and 2b). Unique Pro/Gly/Glu-rich regions and the various RGD-motifs are indicated, of which some are exclusively present in zebrafish (*). CRISPR/Cas9-induced recombination sites are indicated with a red arrow; mutation sites in previously reported zebrafish models are also annotated with a black arrow. (B) Heatmap showing the percentage of amino acid sequence similarity between human fibrillin proteins (Y-axis) and their zebrafish orthologs (X-axis). Sequence similarity was calculated using Clustal Omega. (C) Comparison of the relative contribution of proline (Pro) and glycine (Gly) in the Pro/Gly-rich region between human and zebrafish fibrillins. (D) mRNA expression pattern of the fibrillin isoforms in WT zebrafish embryos at 1, 2, 3, 5 and 7 dpf (n = 3-5 for each developmental stage). Data are expressed as a mean ± SEM. Statistical test analysis: one-way ANOVA followed by Dunnett’s multiple comparisons test on log-transformed data. ****p < 0.0001, ***p < 0.001 and **p < 0.01. AA = amino acid, ZF = zebrafish. A characteristic domain within all fibrillins is the proline and/or glycine-rich region immediately following the first TGF-β binding-like domain. Comparing these domains between species reveals similarities in amino acid composition between FBN1 and fbn1 , FBN2 and fbn2b , and, to some extent, FBN3 and fbn2a ( Figure 1C ). Furthermore, the RGD-integrin binding sites present in all human fibrillin isoforms are highly conserved in the zebrafish fibrillins, except for zebrafish fibrillin-2a, which lacks all RGD motifs. Interestingly, fibrillin-1 in zebrafish contains an extra RGD motif in the third TGF-β binding-like domain. The protein structures of all three zebrafish fibrillins are summarized in Figure 1A . Expression pattern of the different fibrillin isoforms in wild-type (WT) zebrafish We used real-time qPCR to measure expression levels of fbn1 , fbn2a and fbn2b in WT whole embryo tissue at different developmental stages (1, 2, 3, 5 and 7 dpf). Our results showed that both fbn1 and fbn2a are expressed more abundantly later in development, whereas fbn2b expression levels are highest at the early stages and gradually decrease with age ( Figure 1D ). Normal cardiovascular development in zebrafish lacking fbn1 and/or fbn2a Four independent fbn1 knock-out (KO) zebrafish models were generated, carrying either a frameshift deletion or insertion in exon 2, 34, or 38, as summarized in Supplemental Table 1. Surprisingly, all fbn1 homozygous mutant ( fbn1 -/- ) zebrafish survived normally to adulthood, without any cardiovascular phenotype during development ( Figure 2A, 2D ). Loss of fbn2a , with or without fbn1 deficiency, also did not influence cardiovascular development or survival ( Figure 2A ). Real-time qPCR expression analysis demonstrated a significant reduction in fbn1 mRNA expression in fbn1 -/- zebrafish compared to WT siblings, observed in both 5 dpf whole larvae ( Figure 2B ) and in adult (9 mpf) ocular, skin, and muscle tissues ( Figure 2C ). fbn2a and fbn2b mRNA expression was not affected in fbn1 mutants (Supplemental Figure 2). Transthoracic echocardiography performed on adult fbn1 and/or fbn2a mutant zebrafish of various ages (6 – 19 mpf) also demonstrated no significant abnormalities in any measured cardiovascular parameters (Supplemental Tables 6 and 7). Download figure Open in new tab Figure 2 Cardiovascular architecture in fbn1 and/or fbn2a mutants. Fluorescent images of the vasculature of fbn1 -/- (Cmg80) zebrafish with or without the additional loss of fbn2a at 8 dpf, showed no phenotypic differences from WT. ( A – left and right) Ventral view of the ventral aorta (VA) and BA of 6-8 dpf WT, fbn1 -/- and fbn1 -/- ;fbn2a -/- larvae respectively. (A - middle) Lateral view of the distal part of the dorsal aorta (DA) merging into the caudal aorta (CA) as well as the posterior cardinal vein (PCV) merging into caudal vein (CV) in 6 dpf WT and fbn1 -/- larvae. ( B) RT-qPCR analysis of fbn1 expression in 5 dpf WT and fbn1 -/- (Cmg80) larvae (n = 7). Each data point represents the mean of two technical repeats. ( C) RT-qPCR analysis of fbn1 expression in eye, skin, and muscle tissue of 9 mpf WT and fbn1 -/- (Cmg80) zebrafish (n = 5-7). (D) Quantification of BA diameters in 7 dpf fbn1 -/- (Cmg80) and matched WT controls during minimal (min) and maximal (max) distension (n = 12 – 13). Data are expressed as mean ± SEM. Statistical analysis: unpaired t-test (B), two-way ANOVA (C, D). ***p<0.001, ***p<0.01. Variable endocardial phenotype in fbn2b -/- zebrafish Next, we generated a 4 bp frameshift deletion located in exon 4 of the fbn2b gene, resulting in a premature termination codon (p.Cys153*). Real-time qPCR expression analysis of fbn2b -/- larvae (1, 2, 3, 5 and 7 dpf) showed a strong and significant reduction of fbn2b mRNA expression compared to WT siblings, likely due to nonsense-mediated decay ( Figure 3E + Supplemental Figure 3). fbn1 and fbn2a mRNA expression was not affected in fbn2b mutants (Supplemental Figure 3). Starting at 24 hpf, fbn2b homozygous mutant ( fbn2b -/- ) zebrafish embryos can be distinguished from their heterozygous and WT siblings by the presence of fin fold atrophy. In approximately 40% of fbn2b -/- offspring, marked pericardial edema was observed, associated with gaps in the endocardium which progress to complete endocardial detachment in the atrium, similar to what was previously reported for the scotch tape ENU fbn2b mutant ( Figure 3A ). 25 fbn2b -/- zebrafish with this severe phenotype do not survive past 6-8 dpf due to vascular embolism. The remaining fbn2b -/- larvae exhibit a milder phenotype where the endocardium remains attached to the myocardium, circulation is maintained, and normal survival to adulthood is observed ( Figure 3A ). Interestingly, we observed that a subset of fbn2b -/- embryos with severe pericardial edema (approximately 30%) seems to recover between 3-5 dpf, reverting to the milder phenotype ( Figure 3B ). Download figure Open in new tab Figure 3 Cardiovascular development in fbn2b mutants. Representative images of the diverse phenotypes observed in 2-8 dpf fbn2b -/- (cmg96) mutants and WT controls. (A - left) Lateral whole-embryo view of 3 dpf larvae using brightfield microscopy. Accolade indicates finfold atrophy, black arrowhead indicates severe pericardial edema. (A - middle) Reconstructed 3D in vivo two-photon fluorescent images of the non-beating heart of 2 dpf Tg(kdrl:GFP) WT and fbn2b -/- zebrafish. Endocardial detachment (asterisk) is observed in the atrium of the fbn2b -/- zebrafish with pericardial edema. (A - right) Ventral view of 8 dpf Tg(kdrl:GFP) WT and fbn2b -/- with preserved endocardial integrity. White arrowhead indicates dilated BA. (B) Phenotypic distribution (%) of fbn2b -deficient zebrafish presenting a mild (M) or severe (S) pericardial phenotype and their dynamics over time (3, 4 and 5 dpf) (n = average of 19 clutches). (C) Quantification of the average heart rate in beats per min (bpm) of 3 dpf fbn2b -/- with preserved endocardial integrity and matching controls (n = 13). (D) Quantification of BA diameters at 7 dpf during minimal (min) and maximal (max) distension (n = 11-15). (E) Caudal vein formation in Tg(kdrl:GFP) WT and mild fbn2b -/- zebrafish at 24 and 48 hpf after exposure to 0.01% DMSO vehicle (top) or 10 mM 2,3-butanedione monoxime (BDM) to inhibit cardiac contraction (bottom). White arrow indicates abnormal development of the caudal vein. Exposure to BDM leads to more severe caudal vein dilatation (white line) in fbn2b -/- embryos than in WT controls at 48 hpf (qualitative analysis). (F) mRNA expression levels of fbn2b in WT, fbn2b +/- , and fbn2b -/- (mild or severe) zebrafish at 5 dpf (n = 4-5). Each datapoint represents the mean of two technical replicates. Statistical analysis: unpaired t-test (C), two-way ANOVA (D), one-way ANOVA followed by Tukey multiple comparison’s test on log-transformed data. Data are expressed as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. BA = bulbus arteriosus, v = ventricle. Bulbus arteriosus and cardiac function in fbn2b -/- zebrafish larvae Interestingly, the surviving fbn2b -/- zebrafish in which the endocardium remains attached develop dilatation of the bulbus arteriosus (BA), a structure that is considered to be evolutionarily related to the aortic root and ascending aorta in humans, starting at 5 dpf ( Figure 3A, 3D ). We also tested several cardiac function parameters using brightfield microscopy and found a mild but significant increase in heart rate in mild fbn2b -/- (without pericardial edema) compared to WT controls at 3 dpf ( Figure 3C , Supplemental Figure 4). Venous phenotype in fbn2b -/- zebrafish larvae Besides the BA phenotype, we also discovered that the caudal vein of fbn2b -/- zebrafish initially develops as a dilated, cavernous venous structure lacking vessel integrity. While this phenotype persists in the fbn2b mutants with the severe endocardial phenotype, we found that, in the mild fbn2b mutants, the caudal vein remodels appropriately ( Figure 3E ). This led us to investigate the role of blood flow dynamics further by pharmacologically inhibiting cardiac contraction using a myosin inhibitor. This prevented the resolution of the pronounced caudal vein dilatation in the mild fbn2b -/- zebrafish ( Figure 3E ). qPCR confirms the absence of compensation mechanisms by fbn1 and/or fbn2a To test whether other fibrillin isoforms compensate for a lack of fbn2b expression in the fbn2b -/- zebrafish, the expression levels of fbn1 , fbn2a and fbn2b were measured in WT, fbn2b +/- , fbn2b -/- mild and fbn2b -/- severe whole embryos during early development (1, 2, 3, 5 and 7 dpf). No significant differences in fbn2b expression levels were observed between the mild and severe fbn2b -/- phenotypes ( Figure 3F , Supplemental Figure 3). Also, no differences were observed in fbn1 and fbn2a expression levels between fbn2b -/- and matching controls, nor between fbn2b -/- larvae with a mild versus severe phenotype (Supplemental Figure 3). This suggests that increased expression of fbn1 or fbn2a does not compensate for the lack of fbn2b expression. Triple fibrillin knockouts do not survive to adulthood Since loss of fbn1 and/or fbn2a does not lead to a phenotype, we investigated whether additional loss of fbn2b could unmask a functional role for these fibrillins. Consistent with the single fbn2b KO phenotype, triple knock-out (TKO) mutants exhibited 100% penetrance of finfold atrophy, and a subset of TKO larvae demonstrated severe pericardial edema due to complete endocardial detachment while the remaining TKO had a milder phenotype. ( Figure 4A, 4B ). Kaplan-Meier survival analysis of offspring from a fbn1 -/- ;fbn2a -/- ;fbn2b +/- incross up to 14 dpf showed slightly lower survival rates in TKO in comparison to siblings, although statistical significance was not reached ( Figure 4C ). To date, we have, however, never detected an adult TKO zebrafish despite genotyping approximately 300-400 adult fish raised from fbn1 -/- ;fbn2a -/- ;fbn2b +/- incrosses, indicating premature mortality of zebrafish with this genotype. We next assessed the standard length (SL) at 3 dpf and found that TKO mutants are significantly shorter than their fbn1 -/- ;fbn2a -/- ;fbn2b +/+ siblings ( Figure 4E ). Finally, measurements of the BA diameter at 7 dpf revealed a significant increase in the minimally distended diameter in TKO larvae compared to fbn1 -/- ;fbn2a -/- ;fbn2b +/+ controls. In the maximally distended state, a larger variability was observed in the TKO group ( Figure 4D ). Download figure Open in new tab Figure 4 Phenotypic features of TKO larvae. Representative images of the phenotypes observed in 3-7 dpf fbn1 -/- ;fbn2a -/- ;fbn2b +/+ , fbn1 -/- ;fbn2a -/- ;fbn2b +/- , and fbn1 -/- ;fbn2a -/- ;fbn2b -/- larvae. (A – left) Lateral whole-embryo view of a 5 dpf triple fibrillin knockout (TKO) without complete endocardial detachment and sibling controls, using brightfield microscopy. Accolade: finfold atrophy. (A – right) Fluorescent ventral images of 7 dpf Tg(kdrl:GFP) TKO and sibling controls with preserved endocardial integrity. White arrowhead indicates dilated BA. (B) Phenotypic distribution (%) of TKO zebrafish presenting a mild (M) or severe (S) pericardial phenotype and their dynamics over time (3 and 5 dpf) (n = 21). (C) 14-day Kaplan-Meier survival curve of the offspring of an incross of fbn1 -/- ;fbn2a -/- ;fbn2b +/- zebrafish (n = 21-40). (C) Quantification of BA diameters at 7 dpf during minimal (min) and maximal (max) distension (n = 17-19). (D) Quantification of standard length at 3 dpf (n = 17 – 36). Statistical analysis: one-way Anova followed by Dunnett’s multiple comparison’s test. All data are expressed as a mean ± SEM. *p<0.05. BA = bulbus arteriosus, dpf = days post fertilization. Transcriptomic analysis in fbn2b -/- embryos suggests involvement of extracellular matrix remodeling and immune system activation We conducted bulk RNA sequencing on whole fbn2b -/- and WT embryos at 1 and 2 dpf, to identify early transcriptional changes during the development of the early cardiovascular phenotypes. Differential expression analysis (adjusted p-value (FDR) 1) identified 42 and 361 differentially expressed genes, at 1 and 2 dpf, respectively ( Figure 5A, 5C , Supplemental Table 8). At 1 dpf, gene ontology enrichment analysis of the differentially expressed genes revealed significant enrichment in pathways related to metabolism, biosynthesis, and endothelial and cardiac tissue development (FDR < 0.05) ( Figure 5B, 5D ). The latter transcriptional changes were notably driven by strong downregulation of the fbn2b transcript (log 2 FC-3.22). By 2 dpf, the transcriptional profile shifted, with gene ontology analysis highlighting enrichment of immune-related processes and defence mechanisms. In particular, several components of the complement system were significantly upregulated in fbn2b -/- mutants, including c4b (log 2 FC +1.37), c6 (log 2 FC +3.69), cfb (log 2 FC +1.11), c7a (log 2 FC +0.94), and c7b (log 2 FC +3.18). Additionally, matrix metalloproteinases were markedly increased, with elevated expression of mmp9 (log 2 FC +2.77), mmp13a (log 2 FC +2.57) and mmp13b (log 2 FC +2.85) ( Figure 5 ). Download figure Open in new tab Figure 5 Transcriptomic analysis of fbn2b +/+ and fbn2b -/- siblings at 1 and 2 dpf. (A, C) Volcano plots illustrating differentially expressed genes (DEG) in fbn2b -/- zebrafish compared to WT siblings at 1 dpf and 2 dpf, respectively. Upregulated genes are red and downregulated genes are blue, with genes of importance annotated. Thresholds: FDR ≥ 0.05 and |log2FC| ≥ 1. (C, D) GO enrichment analysis with top 8 hits presented as a barplot. The x-axis and y-axis represent the gene count and pathway, respectively. Thresholds: FDR ≥ 0.05 and |log2FC| ≥ 1. Echocardiographic and synchrotron imaging in adult fbn2b -/- zebrafish hearts reveal cardiac rhythm and morphological abnormalities Using an optimised in-house cardiac ultrasound setup, we assessed the cardiovascular phenotype of adult fbn2b -/- zebrafish in vivo . We detected significant dilation of BA of 6 and 8-month-old fbn2b mutants compared to WT zebrafish ( Figure 6B, 6D ). Additionally, the ventricles of the fbn2b mutants exhibited mild dilation ( Figure 6B, 6C ). Analysis of color flow Doppler (CFD) recordings revealed increased blood inflow and outflow areas in fbn2b -/- zebrafish, indicating that AV and BV valve openings are larger than in WT zebrafish. A small number of fbn2b -/- zebrafish showed valvular regurgitation ( Figure 6F, 6G ). Download figure Open in new tab Figure 6 Cardiac abnormalities of adult fbn2b -/- zebrafish. (A) Schematic representation of the two-dimensional transthoracic echocardiography of adult zebrafish. The zebrafish is anesthetized, positioned with its ventral side upwards, and submerged in water. The echocardiography measurements are made with an ultrasound probe, in abdominocranial axis view for CFD analysis (red), and in longitudinal view for ventricle and BA measurements (dark red). (B) Tracings of the posterior walls of the ventricle (blue) and BA (purple) of WT (left) and fbn2b -/- zebrafish (right) (6 and 8 mpf). (C) Dimensions of the ventricle during diastole (area;d, volume;d) and systole (area;s, volume;s). (D) Measurements of BA volume, area, and diameter while in maximal relaxation (at the time of BV valve contraction). (E) Representative 20-second cardiac rhythm tracings with the associated qualitative score (top). Distribution of cardiac rhythm scores for different genotypes (n = 12) (bottom). (F) CFD recordings of inflow (orange) and outflow (blue) of blood into/from the ventricle in WT (top) and fbn2b -/- zebrafish (bottom), with an example of regurgitant blood flow seen in some mutants (2/14). (G) Quantification of inflow (orange) and outflow (blue) areas and regurgitation fractions from the CFD data. Data are expressed as mean ± SEM. Statistical test analysis: unpaired t-test (C, D, G – inflow area), Mann-Whitney test (G – outflow area, regurgitation fraction IN, regurgitation fraction OUT), Fischer’s exact test (E). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns = non-significant. ACX = abdominocranial view, BA = bulbus arteriosus, LAX = longitudinal axis view, V = ventricle, WT = wild type. We scored fbn2b -/- and WT zebrafish cardiac rhythm based on 20-second-long echocardiography recordings. fbn2b -/- zebrafish showed cardiac rhythm disorders, observed as more irregular or skipped heartbeats ( Figure 6E ). By automated processing of PWD measurements, we observed no significant variations in cardiovascular parameters of systolic, diastolic, and valve function among fbn2b mutants compared to WT controls (Supplemental Table 6). When comparing the volumes of the atrium, ventricle, or BA between fbn2b -/- and WT zebrafish based on synchrotron imaging, we found no statistically significant differences. This ex vivo method couldn’t recapitulate the BA dilation, seen on echocardiography performed in vivo , likely due to a lack of intraluminal pressure postmortem. However, while comparing the 3D heart models of mutants and WT, we noticed increased variability in atrial volumes in fbn2b -/- hearts compared to WT hearts (F test: P = 0.011) (hearts - Supplemental Figure 6; BA – Supplemental Figure 9). Abnormal cardiac valve architecture in adult fbn2b -/- zebrafish Through histological staining for elastin of heart sections from 6 and 8-month-old fbn2b mutant zebrafish, we found a cardiac valve phenotype that is common in all fbn2b mutants. Specifically, all fbn2b -/- zebrafish display abnormalities in the bulboventricular (BV) valve, with one or both leaflets either more thickened or asymmetrically shaped, compared to the valves of WT zebrafish ( Figure 7A ). An altered morphology was also found in the atrioventricular (AV) valves of a small subset of fbn2b -/- zebrafish (2/12 samples), which showed excessive hypertrophy of the valve interstitial cells, leading to increased leaflet length and thickness. Download figure Open in new tab Figure 7 Abnormal cardiac valve architecture of adult fbn2b -/- zebrafish. (A) Histological staining for elastin (purple) of AV (left, purple arrow) and BV valves (right, blue arrow) of WT and fbn2b -/- zebrafish (6 and 8 mpf). Extensive hypertrophy of the AV valve as shown in the representative fbn2b -/- image is found in a subset of mutants (2/12), while the BV valve leaflets are asymmetrical and abnormal in shape in all mutants. (B) Representative images of zebrafish hearts obtained by synchrotron X-ray scanning. Cardiac valves are shown in colour (AV valve – purple, BV valve – blue). (C) Schematic representation of synchrotron X-ray imaging method of zebrafish samples. (D) Representative 3D models of WT (left) and fbn2b -/- zebrafish (right) (16 and 18 mpf). Enlarged models of cardiac valves are shown to display the differences between WT and mutants. (E) Volume, length, and thickness measurements of AV (purple) and BV (blue) valves, obtained from the 3D models. Data are expressed as a mean ± SEM. **p<0.01, *p<0.05, ns = non-significant. Statistical test analysis: Mann-Whitney (AV valve volume), unpaired t-test (BV valve volume, BV leaflet length, BV leaflet thickness). A = atrium, AV = atrioventricular valve, BA = bulbus arteriosus, BV = bulboventricular valve, V = ventricle, WT = wild type. Next, we studied the valves’ 3D structure through synchrotron X-ray imaging of hearts from fbn2b -/- and WT zebrafish ( Figure 7D , AV valves – Supplemental Figure 7; BV valves – Supplemental Figure 8). We found that fbn2b -/- zebrafish have larger BV valve volumes, with the same tendency seen for AV valves. Indeed, the morphology of the AV valves in a subset of fbn2b -/- was extremely aberrant, with abnormal folding and fusing of various leaflet segments. Measurement of the length and thickness of individual BV leaflets confirmed abnormalities seen on histology, with leaflets of fbn2b -/- being longer and thicker than WT ( Figure 7E ). Similar response to β - adrenergic receptor modulation in early development of WT and fbn2b -/- larvae We tested β-adrenergic receptor modulation in WT and fbn2b -/- zebrafish (with a mild phenotype) in the first days of development. Epinephrine similarly elevated heart rate in WT and fbn2b -/- zebrafish, although the increase did not reach statistical significance in fbn2b -/- , likely due to the increased variability in this group (Supplemental Figure 10, top). Administration of the β-adrenergic receptor antagonist nebivolol consistently lowered heart rate in both WT and fbn2b -/- zebrafish during early development. In contrast, atenolol showed no significant impact on heart rate in either genotype (Supplemental Figure 10, bottom). Drug treatment with losartan and nebivolol does not improve the phenotype in fbn2b -/- larvae To verify the effects of β-adrenergic receptor and angiotensin II receptor blockade on the phenotype of our fbn2b -/- zebrafish model, we administered nebivolol at 1 µM and tested varying concentrations of losartan (100 – 1000 µM). There was no statistically significant improvement in the phenotypic distribution of any compound-treated fbn2b -/- zebrafish group at 3, 4, and 5 dpf compared to solvent-treated controls ( Figure 8A ). Similarly, no improvement of the dilated BA phenotype was observed in any group after 7 days of drug treatment ( Figure 8B ). Download figure Open in new tab Figure 8 Effects of angiotensin II receptor blocker and β-adrenergic receptor blocker in the fbn2b -/- zebrafish model. (A) 1 dpf fbn2b -/- zebrafish embryos were treated with 100-1000 µM losartan, an angiotensin II receptor blocker (purple-blue shading, n = 12 – 15), and 1 µM nebivolol (red), a β-adrenergic blocker, with solvent as a control (DMSO – grey, n = 70). Distribution of the pericardial phenotype is shown at 3, 4, and 5 dpf. (B) At 8 dpf, the diameter of BA was measured, both in maximal (left) and minimal distension (right). Data are expressed as mean ± SEM. ns = non-significant. Statistical test analysis: Kruskal-Wallis test with Dunn’s multiple comparisons test (A), Fisher’s exact test (B). A = atrium, BA = bulbus arteriosus, M = mild pericardial phenotype, S = severe pericardial phenotype. DISCUSSION Due to sustained efforts in translational research, the life expectancy of individuals with MFS has improved dramatically over the past three decades—an achievement that stands out in the field of cardiovascular medicine. 26 This progress has been driven by advances in diagnostics, longitudinal care, and medical as well as surgical interventions, some of which are underpinned by mechanistic insights gained from mouse models. 27 These models have been instrumental in identifying key pathways involved in aortic disease progression and in testing therapeutic strategies. Yet, despite these successes, fundamental questions remain unresolved, particularly concerning early developmental processes in MFS, tissue-specific dynamics, functional effects of genetic variants, and targetable disease mechanisms. There is a growing need for complementary model systems that can capture aspects inaccessible in murine models and that allow higher-throughput, in vivo functional studies in a vertebrate context. We believe that these issues can be addressed by the new zebrafish model presented in this study, which demonstrated that disruption of fbn2b in zebrafish results in a consistent, early-onset Marfan syndrome-like phenotype, including aortic dilatation, valvular defects, and arrhythmia. To the best of our knowledge, this study is the first to investigate the functional orthology between human and zebrafish fibrillins in vivo , establishing a foundation for utilizing zebrafish to model MFS and to broaden our understanding of other human fibrillinopathies. Zebrafish fbn1 deficiency: a critical look at cardiovascular phenotype reproducibility The high level of conservation between zebrafish and human fibrillin genes/proteins implies a conserved biological function. However, our findings suggest that the zebrafish fbn1 isoform may not be the direct functional ortholog of human FBN1 , as loss of fbn1 did not result in a cardiovascular phenotype. These results stand in contrast to earlier studies that described mild cardiovascular abnormalities in fbn1 -deficient zebrafish. 28 – 30 Specifically, the study by Chen et al. made a morpholino knockdown of fbn1 and observed abnormal vessel dilatation in the head, and a lack of venous plexus remodeling. 28 However, these effects were dose-dependent, with significant abnormalities only seen at relatively high morpholino concentrations. This raises concerns about potential off-target effects and dose-dependent toxicity. 31 Another study by Yin et al. using heterozygous CRISPR-generated fbn1 +/- mutants with frameshift indels in exon 40 (which is identified as exon 16 in their manuscript, as an incomplete fbn1 gene sequence was used for reference) showed limited data suggesting phenotypic alterations including body elongation, reduced pigmentation, and altered cardiac blood flow. 29 More recently, an independent group modeled a likely pathogenic nonsense variant identified in a consanguineous family, by CRISPR/Cas9-based indel generation in exon 60 of fbn1 (labeled as exon 19 in the study, as, again, an incomplete zebrafish fbn1 sequence was used). The authors reported a range of phenotypes in their fbn1 +/- mutants, including pericardial edema, tail curvature, and aortic arch bleeding, but no quantitative data was presented showing the frequency of these phenotypes. Other cardiovascular phenotypes described in this study included decreased cardiac function, angiogenesis abnormalities, and increased dorsal aortic diameter, although these phenotypes were not clearly reported. 30 We were unable to replicate these results in repeated rigorous experiments with any of our four independently generated fbn1 mutant zebrafish lines. Considering the known influence of genetic background on the cardiovascular severity phenotype in mouse models of MFS, 32 it is conceivable that differences in zebrafish strains used in these studies (TU versus AB in our study) may partly explain the divergent outcomes of fbn1 deficiency. However, it is unlikely that strain differences alone account for all observed inconsistencies. We therefore encourage further independent investigations to resolve these discrepancies. Lack of compensatory mechanisms underscores fibrillin gene complexity in zebrafish Another potential reason why complete loss of fbn1 in our models does not lead to a reproducible phenotype could be that compensatory mechanisms are activated. It has previously been shown that CRISPR-induced frameshift mutations leading to premature stop codons can lead to transcriptional adaptation by increasing the expression of genes with a substantial sequence similarity. 33 However, while our qPCR analysis indicated a clear downregulation of fbn1 expression, signifying nonsense-mediated decay, we did not observe compensatory upregulation of other fibrillin genes. Therefore, we hypothesize that the lack of any detectable cardiovascular phenotype in our fbn1 -/- zebrafish rather underscores the complexity of the interspecies functional orthology within the fibrillin gene family. Lower homology of zebrafish fbn2a to human fibrillins We also investigated the cardiovascular impact of fbn2a deficiency, both independently and in combination with the loss of fbn1 , but again did not observe any abnormalities. This result is consistent with the lower amino acid homology of fbn2a with human fibrillin genes. Interestingly, fibrillin-2a lacks all RGD motifs, which are known to mediate binding affinity towards various integrins, 34 while it does contain a novel proline/glutamine-rich domain not found in any other fibrillins. These unique structural differences likely reflect a greater evolutionary distance from the other fibrillins and suggest a unique functional role for zebrafish fibrillin-2a, which may be less relevant to humans. In contrast to fbn1 and fbn2a: fbn2b loss leads to developmental defects Unlike loss of fbn1 and/or fbn2a , disruption of the fbn2b gene consistently resulted in an early and reproducible cardiovascular phenotype reminiscent of MFS, persisting into adulthood. This finding suggests that fbn2b plays a prominent role during early zebrafish development, functionally resembling human FBN2 , although the effects during early development have a lasting impact throughout the zebrafish’s lifespan. This notion aligns well with our qPCR data, which demonstrate that expression of fbn2b precedes fbn1 during zebrafish embryogenesis, similar to FBN2 and Fbn2 in humans and mice, respectively, suggesting a conserved role for fbn2b in early developmental processes. 35 CRISPR-generated fbn2b mutant: a phenotypic comparison with existing ENU models The fbn2b mutant zebrafish generated in this study, carrying a premature stop codon in exon 4 (c.458_461del, p.C153*), show a morphological phenotype largely reminiscent of previously reported ENU mutant lines, puff daddy ( pfd gw1 – p.G161*) 25 and scotch tape ( sco te382 – p.C1312F ) 36 . These ENU mutants have either nonsense or missense mutations, resulting in either a loss of protein or an altered protein structure, respectively. Similar to the pfd gw1 and sco te382 mutants, our fbn2b homozygous mutants display a characteristic finfold atrophy, which does not appear to impact their overall fitness. In contrast to the pfd gw1 mutant, our CRISPR-induced fbn2b mutant line did not develop a notochord phenotype, which was also not present in the sco te382 mutant . It was later discovered that, besides mutations in fbn2b , mutations in the pku300 gene also contribute to the cardiovascular defects in the sco te382 mutant. 36 Similarly, it cannot be excluded that confounding genetic factors also influence the observed notochord deformities in pfd gw1 . This might explain why our novel CRISPR/Cas9-generated fbn2b mutant model with a frameshift mutation in almost the same location as pfd gw1 does not present with a notochord phenotype. This underscores that, while forward genetic screens are valuable in identifying novel genes within developmental pathways, thorough validation is necessary to exclude the possibility that multiple genes contribute to the observed phenotype. Consistent with observations in the sco te382 mutant (but unlike pfd gw1 ), the severe pericardial edema phenotype was not completely penetrant in our fbn2b -/- zebrafish. We hypothesize that the progression to irreversible, lethal endocardial detachment is a stochastic process. As described previously for the sco te382 mutant 25 , all fbn2b -/- zebrafish likely experience compromised endocardial integrity, leading to increased intercellular gap formation. Only a subset reaches a critical threshold in the size of these gaps, leading to the complete detachment of the endocardium from the underlying cardiac jelly. 36 Interestingly, a subset of our CRISPR-induced fbn2b -/- mutants initially develops pericardial edema around 2 dpf, which appears to resolve by 5 dpf in approximately 30% of the cases. We hypothesize that in these fbn2b -/- zebrafish the enlarged endocardial gaps, which are formed dynamically during growth of the cardiac chambers in early development and result in fluid leak into the pericardium, are closed sufficiently during further cardiac developmental stages in these embryos, preventing complete endocardial detachment, restoring the integrity of the endocardial barrier function and ensuring survival. 25 BA dilatation in fbn2b mutants reflects aortic pathology seen in MFS The fbn2b -/- zebrafish without a pericardial phenotype at 5 dpf survive to adulthood and are fertile. Interestingly, we found that these zebrafish show a dilated BA phenotype that emerges during the larval stage and persists into adulthood. The specific dilation of the BA observed in our fbn2b -/- model is particularly relevant as the BA is anatomically and functionally related to the aortic root and ascending aorta in humans, 37 , 38 which is typically dilated in patients with MFS. 39 The elastic outflow properties of the BA are evolutionarily conserved, supporting its role as a ‘Windkessel’ organ. Like the aortic root, the BA functions as an elastic reservoir, absorbing the pulsatile output of the ventricle and ensuring continuous blood flow throughout the circulation. 40 , 41 Several cases of human FBN2 defects leading to aortic dilatation have been reported 12 – 14 , further supporting the indication of a functional overlap between different fibrillins and thus underscoring the potential clinical relevance of this zebrafish model. It is important to note that we do not have evidence that BA dilation in our fbn2b -/- zebrafish progresses to dissection and fatal aortic rupture at later stages. The only zebrafish line so far known to develop progressive ventral aortic dissection and rupture is a model with defects in the TGF-β effector proteins Smad3 and Smad6. 42 Dilation of the BA was also observed in previously generated zebrafish mutant lines deficient in various mediators of the TGF-β signaling pathway. Zebrafish lacking both Tgfbr1 paralogues ( alk5a -/- , alk5b -/- ) show a severe dilation of the developing BA and ruptures in its endocardial layer starting at 4 dpf, which results in severe pericardial edema, retrograde blood flow, and ultimately mortality by 7 dpf. 43 Zebrafish double mutant for the latent TGF-β-binding proteins 1 (ltbp1) and 3 (ltbp3) ( ltbp1 -/- , ltbp3 -/- ) exhibit BA and ventricular dilation during larval stages, pericardial edema, aortic regurgitation, and premature death by 8 dpf. 44 These phenotypes are similar to the fbn2b -/- larvae with a severe phenotype, which also die by 8 dpf. These similarities highlight the relevance of fibrillin and TGF-β interactions, and the consequences of their disruption, which are central to the pathogenesis of MFS. 44 fbn2b deficiency induces elevated heart rate and arrhythmias We also report a slightly elevated heart rate in our fbn2b -/- embryos compared to their WT siblings. Although common MFS mouse models (e.g. Fbn1 C1041G+ ) are not known to show significant differences in heart rate 45 , a study in human induced pluripotent stem cell-derived cardiomyocytes showed that cells carrying a pathogenic FBN1 variant had a higher intrinsic beating rate. 46 Of note, zebrafish cardiac electrophysiology differs significantly from that of mice and can even be considered physiologically more similar to humans. 47 , 48 In MFS patients, cardiac arrhythmias, frequently associated with mitral valve prolapse, are well-recognized clinical features 49 , which have been replicated in mouse models. 50 Our adult fbn2b -/- zebrafish model recapitulates this aspect, as they exhibit a higher frequency of irregular or skipped heartbeats compared to WT siblings. Given the clinical relevance of cardiac arrhythmias in MFS, 49 , 51 , 52 our fbn2b -/- zebrafish model can be valuable for further elucidating the underlying physiological mechanisms involved. Interestingly, fbn2b -/- larvae had a stronger negative chronotropic effect upon administration of the β-adrenergic receptor blocker nebivolol. This observation suggests that fbn2b -/- zebrafish may have an intrinsically elevated sympathetic tone, which warrants further investigation. fbn2b’s role in endothelial mechanosensing Similar to observations in the pfd gw1 mutant, 25 we found that the posterior caudal vein in our fbn2b -/- zebrafish initially develops as a dilated structure with compromised vessel integrity. This phenotype resolves during further development in fbn2b -/- without persistent endocardial detachment, unless cardiac contraction is pharmacologically inhibited, resulting in loss of blood flow. This suggests that fibrillin mediates blood flow-induced biomechanical signaling crucial for vascular development, aligning with fibrillin’s established role in mechanosensing. 53 , 54 In MFS mice ( Fbn1 C1041/G+ ), it has been shown that morphological defects in endothelial cells are most pronounced at aneurysm-prone regions exposed to elevated mechanical stress. 55 , 56 Since vascular smooth muscle cells are not recruited to the zebrafish arterial wall until after 3 dpf, the early vascular defects observed here are likely the result of endothelial dysfunction. 57 Total fibrillin deficiency in zebrafish TKO zebrafish, lacking any functional fibrillin gene, showed cardiovascular phenotypes during early development, which are similar to the single fbn2b KO. Furthermore, they showed decreased general fitness as indicated by a shorter standard length and slightly decreased survival during larval stages. Interestingly, TKO do not survive to adulthood, suggesting that the additional loss of fbn2b unmasked essential functions of fbn1 and fbn2a . This also implies that loss of only one or two zebrafish fibrillins can generally be compensated by the other fibrillin(s) to guarantee (at least partial) survival. However, the essential role of fbn2b in cardiovascular homeostasis can not be compensated by the other fibrillins. In mice, Fbn1 functionally compensates for Fbn2 loss during embryogenesis in a dosage-sensitive manner. Nearly all Fbn1 -/- ;Fbn2 -/- embryos and approximately half of Fbn1 +/- ;Fbn2 -/- embryos fail to survive gestation, underscoring the critical role of Fbn1 dosage in development. 58 fbn2b deficiency triggers the complement pathway Bulk RNA sequencing analysis of our fbn2b mutant model revealed upregulation of several components of the complement signalling pathway during early developmental stages. The complement system is a key component of innate immunity, mediating host defense through a cascade of proteolytic activations that enhance pathogen clearance and modulate inflammation. It can be activated via three distinct pathways: the classical, lectin, and alternative pathway. Increasing evidence implicates the alternative pathway in the pathogenesis of cardiovascular diseases, including aortic aneurysms. For example, activation of the alternative complement pathway contributes to elastase-induced abdominal aortic aneurysm formation, and studies have shown elevated plasma levels of C3a and C5a in patients with TAAD. 59 , 60 Interestingly, genetic ablation of Cfb in Fbn1 C1041/G+ MFS mice reduced the occurrence of TAAD, further supporting a pathogenic role for this pathway. In a related study, the same group demonstrated the C3a-C3aR axis contributes to TAAD via upregulation of MMP2, a key enzyme involved in ECM remodeling. 60 Additionally, genetic variants in C1R have been associated with TAA formation in patients with bicuspid aortic valve, suggesting a broader involvement of the complement system in aortic disease. 61 Collectively, these findings and our current data imply that complement activation may play a role in aneurysm progression in MFS. It is well established that endothelial cells in MFS mice exhibit structural and functional abnormalities that result in endothelial dysfunction, 56 which is also evidenced in our fbn2b -/- zebrafish model by the endocardial and caudal vein phenotypes. We propose that these structural defects may serve as triggers for complement activation, leading to the recruitment of inflammatory cells such as macrophages and neutrophils. These cells are known to secrete matrix metalloproteinase-9 (MMP9), and to a lesser extent MMP13, which contributes to extracellular matrix degradation and disease progression in TAAD, particularly by digesting different collagen types, including the main fibrillar and basement membrane collagens. Interestingly, both mmp9 , mmp13a and mmp13b were increased in the fbn2b -/- RNA sequencing dataset, and could represent downstream targets of immune cell activation, which contribute to aortic wall damage. Targeting the complement cascade - particularly components of the alternative pathway - may therefore represent a promising therapeutic strategy for preventing or mitigating TAAD in MFS. However, further studies are required to determine the most effective and specific targets within the complement system. Collectively, these findings underscore the complex nature of aortic disease in MFS, demonstrating the contribution of various signaling pathways beyond TGF-ß. A deeper understanding of the intricate crosstalk among these pathways is therefore essential and warrants further investigation. Cardiac valve abnormalities in adult fbn2b mutants: parallels with patients with MFS Histological staining for elastin in sections of adult fbn2b -/- zebrafish revealed cardiac valve abnormalities in both the AV and particularly the BV valves. Propagation-based phase-contrast synchrotron imaging confirmed longer and thicker BV valve leaflets in all fbn2b -/- , which was supported by the observation of widened valve openings in vivo using CFD. Similar cardiac valve defects have been shown in zebrafish lacking the gene coding for elastin isoform a ( elna sa12235 ). 62 The phenotypic resemblance between these two zebrafish models suggests a possible shared etiology, potentially reflecting the essential role of fibrillin as a scaffold for elastin fiber formation. On the other hand, it is tempting to speculate that the important role of fbn2b in maintaining an intact endocardial barrier through intercellular contact during early development 25 could be related to the observed valve defects in the adult fbn2b -/- zebrafish. It has been shown that endocardial cell migration significantly contributes to the initial development of the AV valve in early larval stages. 63 Additional studies will be needed to confirm whether loss of fbn2b affects these processes in the development of both the AV and BV valves, which could explain the malformation of valvular leaflets we observed in adult fbn2b -/- zebrafish. Cardiac valve architecture has been closely examined in Fbn1 C1041G/+ and Fbn1 C1041G/ C1041G MFS mouse models as well, both of which show dramatic alterations in mitral valve structure. Notably, Fbn1 C1041G/ C1041G mice display unique malformations where leaflet tips fold back and fuse to more proximal segments, 64 , 65 resembling the abnormalities we observed in the fbn2b -/- zebrafish with an AV valve phenotype. The valve defects identified in our fbn2b -/- zebrafish model further support its validity as a model of the cardiovascular manifestations of MFS. Mitral valve prolapse and tricuspid valve prolapse respectively affect approximately 65% and 35% of patients with MFS. 66 Nevertheless, while valve defects are associated with regurgitation in approximately 40% of cases, this was only observed in a few cases using PWD in our zebrafish model. It is conceivable that the mild valve defects that we observed using histology and 3D modeling may have caused only subtle or intermittent regurgitation that eluded detection by our echocardiography measurements, particularly given the challenges of using this technique in small organisms like zebrafish. 67 Though a subset of patients with MFS develops intrinsic cardiomyopathy characterized by left ventricular dilation and dysfunction, myocardial dysfunction is at most very mild in the majority of individuals. 68 – 71 Echocardiography measurements revealed mild ventricular hypertrophy in our fbn2b -/- zebrafish, while no statistically significant differences were observed between fibrillin mutants and WT zebrafish in any of the 20 PWD parameters investigated. Synchrotron-based 3D reconstructions of the atrium, ventricle, and BA in fbn2b -/- zebrafish did not show any significant differences in volume and diameter compared to WT. It is important to note, however, that synchrotron imaging is performed on fixed samples collected postmortem, where cardiac and vascular structures collapse due to loss of intraluminal blood pressure. This discrepancy could potentially be overcome by vascular corrosion casting. 72 , 73 Interestingly, fbn2b -/- zebrafish show significantly higher variability in atrial volumes compared to WT. Those mutants with the largest atria also exhibit pronounced thickening of the AV valves, suggesting a pathophysiological consequence of prolonged volume overload due to the retrograde blood flow across the valve. 74 However, there were only a limited number of these cases, and echocardiography data is not available for these samples, limiting confirmation. Exploring the efficacy of beta-blockers and angiotensin receptor blockers in fbn2b zebrafish Given that β-adrenergic antagonists are considered standard medical care for MFS patients, 75 we investigated their effects in our zebrafish model. We tested atenolol, the most prescribed β-blocker in MFS with selective anti-β1-adrenergic receptor activity, and nebivolol, a third-generation, also β1-selective β-blocker but with additional vasodilatory effects mediated by nitric oxide release, which has also been tested in MFS patients. 76 , 77 We found that nebivolol significantly decreased heart rate in WT and, to an even larger extent, fbn2b -/- larvae. Atenolol, however, did not significantly impact heart rate in either genotype. This disparity in the effects of both β-blockers could potentially be explained by differences in pharmacokinetics – it has been shown previously that atenolol has no significant effect on heart rate in zebrafish larvae over a range of doses, while other β-blockers do. 78 Alternatively, it is conceivable that the zebrafish larvae may be more sensitive to modulation by nitric oxide released after nebivolol administration. Next, we tested the effects of nebivolol at the dose that was shown to be bioactive based on the heart rate reduction, and losartan, an angiotensin II type I receptor blocker, another commonly prescribed medication for patients with MFS. 79 We found that neither losartan nor nebivolol had a measurable impact on the fbn2b -/- pericardial edema phenotype distribution or BA dilation. This result however aligns with clinical observations, showing that β-blockers and angiotensin receptor blockers can slow the rate of increase of aortic dilation in patients with MFS, resulting in a delay in the need for prophylactic aortic surgery, but do not abolish the risk of aortic dissection and rupture, which is unfortunately still encountered in patients under close medical management. 77 The findings in our zebrafish model imply that these drugs do not directly address the basic pathophysiological mechanisms leading to the cardinal cardiovascular manifestations of MFS. This further underscores the need for novel therapeutic strategies to eliminate the risk of potentially fatal aortic complications in MFS, which can now be addressed using previously unavailable unbiased approaches in our novel zebrafish model. STUDY LIMITATIONS An inevitable limitation of this study concerns the interspecies relationship between the fibrillin genes in human and zebrafish. There is no definitive explanation why loss of zebrafish fbn2b , rather than fbn1 , gives rise to the observed MFS-like phenotypes, while FBN1 is clearly the primary disease-causing gene in humans. Our data indicate that fbn2b is more important during early development, resulting in the observed phenotypes in larvae, which persist until adulthood. The lack of any (progressive) phenotype in zebrafish lacking fbn1 suggests a compensation by the other fibrillin(s), which is supported by the lack of viability of zebrafish lacking all fibrillin genes. Further studies will, however, be necessary to completely unravel the functional distinctions between the different zebrafish fibrillins. Nevertheless, our data are concordant in showing that loss of fbn2b leads to phenotypes that faithfully mimic the cardiovascular manifestations of MFS. The limitation of our fbn2b -/- zebrafish model is the observed phenotypic heterogeneity during larval stages. A conclusive explanation of the variable outcome of the endocardial phenotype in fbn2b -/- larvae, with only a subset developing a lethal phenotype, remains lacking. Our hypothesis that a stochastic process is at the basis of the fate of the potential progression to complete endocardial detachment can only be proven by exclusion of other possible explanations. Finally, in this study, we focused predominantly on cardiovascular manifestations in our zebrafish model, as those are the chief cause of morbidity and mortality in patients with MFS. Additional studies will be necessary to investigate skeletal and ocular manifestations, which are common features in patients with MFS. CONCLUSIONS Our comprehensive study of zebrafish fibrillins demonstrates that disruption of zebrafish fbn2b leads to a spectrum of cardiovascular abnormalities that resemble those observed in human MFS, including BA dilation, cardiac valve defects, and heart rhythm irregularities. These findings highlight the complex interspecies orthology of fibrillins. Our data further suggest that the early phenotype observed in our model is potentially related to the activation of the innate immune system. The availability of this novel zebrafish model represents a significant new tool for research into the mechanisms of MFS-related cardiovascular manifestations and the identification of new therapeutic targets. CLINICAL PERSPECTIVES COMPETENCY IN MEDICAL KNOWLEDGE Marfan syndrome (MFS) is a life-threatening connective tissue disorder with no effective cure. Understanding of its pathogenesis and available treatment options remains limited, highlighting the urgent need for more versatile animal models. We show that zebrafish with fbn2b deficiency exhibit cardiovascular abnormalities that closely resemble those observed in patients with MFS. Our data suggest that fibrillin defects disturb essential cardiovascular development processes, which is not addressed by currently recommended medical treatments for patients with MFS. TRANSLATIONAL OUTLOOK The availability of a zebrafish model that faithfully recapitulates multiple cardiovascular manifestations of MFS enables novel strategies to pursue better diagnosis and treatment options for patients. The cardiovascular phenotype of fbn2b ⁻/⁻ zebrafish larvae can serve as a valuable readout for unbiased in vivo high-throughput drug screening to uncover new therapeutic targets. The model may also be of value to test the pathogenicity of rare FBN1 variants identified with diagnostic testing. HIGHLIGHTS We disrupted zebrafish fibrillins to create a novel animal model of MFS. We applied detailed cardiovascular phenotyping across both early developmental and adult stages of fibrillin-impaired zebrafish. fbn2b -/- zebrafish exhibit aortic dilation and valvular abnormalities resembling the cardiovascular manifestations observed in patients with MFS. This fbn2b -/- zebrafish model represents a relevant tool for advancing our understanding of MFS pathogenesis and for identifying urgently needed therapeutic strategies. ACKNOWLEDGEMENTS We wish to thank the Zebrafish Facility Ghent Core, particularly Karen Vermeulen for the diligent care of our zebrafish. We also thank our NGS core and Ghent Light Microscopy Core. We acknowledge Diamond Light Source (Harwell Science and Innovation Campus, UK) for time on Beamline I13-2 under Proposal MG32919-1, and Swiss Light Source (Paul Scherrer Institut, Switzerland) for time on beamline TOMCAT X02DA under Proposal 20221979. We would like to thank Violette Deleeuw, Michiel Vanhooydonck, Yousof Mohammad Asaad Abdel-Raouf, Simon D’hulst and Isaac Rodriguez-Rovira for their assistance in acquiring synchrotron data. We acknowledge the use of AI tools (ChatGPT, Copilot and Gemini) for text refinement. Funder Information Declared Research Foundation - Flanders , G0A8322N Baillet Latour Fund Concerted Research Action grant from the Ghent University Special Research Fund , BOF GOA019-21 Interdisciplinary Research grant of the Ghent University Special Research Fund , IOP-038-18 Footnotes Funding support and author disclosures This work was supported by a grant from the Research Foundation Flanders (G0A8322N, to PS, JDB and PS), the 2019 Grant for Medical Research from the Baillet Latour Fund (to JDB), a Concerted Research Action grant from the Ghent University Special Research Fund (BOF GOA019-21 to PS, JDB and PS) and an Interdisciplinary Research grant of the Ghent University Special Research Fund (IOP-038-18 to PS, JDB and PS). The authors have reported that they have no relationships relevant to the contents of this paper to disclose. 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Share Systematic disruption of zebrafish fibrillin genes identifies a translational zebrafish model for Marfan syndrome Karo De Rycke , Marina Horvat , Lisa Caboor , Petra Vermassen , Griet De Smet , Marta Santana Silva , Wouter Steyaert , Matthias Van Impe , Patrick Segers , Julie De Backer , Patrick Sips bioRxiv 2025.06.21.659830; doi: https://doi.org/10.1101/2025.06.21.659830 Share This Article: Copy Citation Tools Systematic disruption of zebrafish fibrillin genes identifies a translational zebrafish model for Marfan syndrome Karo De Rycke , Marina Horvat , Lisa Caboor , Petra Vermassen , Griet De Smet , Marta Santana Silva , Wouter Steyaert , Matthias Van Impe , Patrick Segers , Julie De Backer , Patrick Sips bioRxiv 2025.06.21.659830; doi: https://doi.org/10.1101/2025.06.21.659830 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7616) Biochemistry (17625) Bioengineering (13852) Bioinformatics (41825) Biophysics (21397) Cancer Biology (18524) Cell Biology (25417) Clinical Trials (138) Developmental Biology (13350) Ecology (19858) Epidemiology (2067) Evolutionary Biology (24277) Genetics (15581) Genomics (22459) Immunology (17698) Microbiology (40278) Molecular Biology (17134) Neuroscience (88400) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4812) Physiology (7632) Plant Biology (15106) Scientific Communication and Education (2042) Synthetic Biology (4281) Systems Biology (9807) Zoology (2266)