Fibrillin defects lead to severe cardiovascular complications in Marfan syndrome (MFS), including aortic dilation, dissection, and rupture. To model MFS, zebrafish mutants lacking various fibrillin genes were generated. Among these mutant lines, only fibrillin-3-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. This zebrafish model, recapitulating key cardiovascular features of MFS, provides a valuable platform to investigate disease mechanisms and identify novel treatment strategies.

Thoracic aortic dissection (TAD) associates with a high mortality rate. Treatment options are limited and mainly consist of surgical repair at critical aortic diameters as current pharmacological interventions are unable to stop disease progression. Despite the existence of different mouse models for thoracic aortic aneurysm (TAA) and TAD, the underlying disease mechanisms remain elusive. In humans, loss-of-function of SMAD3 or SMAD6 increases the risk for TAA. We therefore targeted both ohnologs of smad3 and smad6 in zebrafish in order to further investigate their contribution to aortic homeostasis. We found an increased diameter of the ventral aorta in smad3a−/−;smad3b−/− double knockout (smad3a/b DKO) zebrafish larvae, while smad6a−/−;smad6b−/− (smad6a/b DKO) zebrafish larvae have a reduced aortic diameter. Smad3a/b DKO survive normally to adulthood, but smad6a/b DKO die before the age of 8 months due to dissections and ruptures in the ventral aorta. Smad6a/b DKO zebrafish also show hypoplasia of the aortic arches and the distal part of the ventral aorta. Surprisingly, the smad3a−/−;smad3b−/−;smad6a−/−;smad6b−/− quadruple knockout (qKO) zebrafish model has normal survival and a milder vascular phenotype compared to the smad6a/b DKO. RNA sequencing of zebrafish larvae indicates upregulation of pathways related to melanogenesis, ribosome, blood vessel development and carboxylic acid transport, and downregulation of negative regulation of endopeptidase activity and immune system. Transcriptomic data of damaged aorta compared with healthy control aorta identifies significant differences in oxidative phosphorylation, mitochondrial function, extracellular matrix and the citrate cycle. In conclusion, data from our novel zebrafish models of thoracic aortic dissection and rupture indicate that SMAD3 function has an important modifying effect on the severe aortic manifestations induced by loss of SMAD6. Legend “TAA” thoracic aortic aneurysm, “TAD” thoracic aortic dissection, “BAV” bicuspid aortic valve, “LOF” loss-of-function. Figure created with BioRender.

Marfan syndrome (MFS) is a multisystemic connective tissue disorder caused by pathogenic variants of the gene encoding fibrillin-1, an important glycoprotein of the extracellular matrix. Among its diverse symptoms, the development of an ascending thoracic aortic aneurysm (ATAA) is the most concerning. An ATAA can fail due to dissection or rupture, both associated with substantial morbidity and mortality. Therefore, MFS patients typically receive medical treatment to slow aneurysm progression and reduce the risk of failure. However, the cellular mechanisms underlying ATAA development in MFS remain incompletely understood, reflected in suboptimal medical treatment options. To address this, we introduce a multiscale computational model of ATAA development in MFS mice as a reproducible, time- and cost-efficient complement to traditional animal experiments. The model implements a bidirectional coupling between a tissue-scale framework for aneurysm growth and remodeling and a cell-scale mechanobiological model for the ascending thoracic aorta. We calibrate and validate against experimental data from mouse studies capturing ATAA progression over time at both the tissue and cellular scales, either with or without pharmacological treatments. Following strong qualitative agreement with experimental observations, we employ the model for an in silico pharmacological treatment trial by simulating the inhibition or activation of various cell-scale model nodes. The simulations identify four novel medical treatments predicted to reduce the long-term failure risk of MFS-induced ATAAs, with inhibition of p38 mitogen-activated protein kinase emerging as the most promising option. Although simplified, the proposed model provides a robust, modular framework that can be readily extended or adapted in future research.

Aortic dissection (AD) is characterized by separation within the medial layers of the aortic wall. Pathogenic variants in the fibrillin-1 gene (FBN1), which cause Marfan syndrome, represent a major genetic cause of AD. In a recently established Fbn1G234D/G234D mouse model, intimomedial tears develop at 3 weeks of age, and 50% of mice die by 5 weeks from aortic rupture. Despite this severe phenotype, the magnitude and expansion of AD lesions, as well as the molecular alterations within the medial layers remain incompletely understood. In this study, we used three-dimensional propagation-based X-ray phase-contrast synchrotron imaging for reconstruction of the ascending aortas, together with single-cell RNA sequencing (scRNA-seq) analysis in Fbn1G234D/G234D mice. Synchrotron imaging revealed 1-2 elastic lamellar breaks evolved into widespread disruptions spanning the entire elastic lamellae, accompanied by localized adventitial thickening. scRNA-seq analysis followed by immunofluorescence staining showed upregulation of fibronectin (Fn1) in Fbn1G234D/G234D smooth muscle cells (SMCs). Consistently, increased FN1 expression was observed in human non-heritable AD samples. Furthermore, enhanced expression of fibronectin receptors and activation of focal adhesion kinase signaling suggested augmented extracellular matrix-SMC interactions during disease progression. These findings indicate that AD progression involves coordinated medial structural failure, adventitial remodeling, and fibronectin-associated SMC dysfunction.

Vascular smooth muscle cell (VSMC) plasticity is implicated in extracellular matrix (ECM) turnover and arterial failure. The osteochondrocytic phenotypes of synthetic VSMCs are thought to drive glycosaminoglycan (GAG) accumulation and swelling typically seen in connective tissue disease and hypertension. A central question is whether this phenotype switching under non-homeostatic conditions is a cause or effect of those conditions. We implement a cause-effect association between ECM damage, lost cell mechanosensitivity, and cell phenotype modulation using the Constrained Mixture Model, to simulate the evolution of VSMC population over time. We modelled a cylindrical bi-layer of media and adventitia of a mouse common carotid artery and simulated remodelling in response to initially compromised ECM, concurrent with varying degrees of hypertension. In normo- and moderately hypertensive ECM disruption, physiological remodelling restores mechanical homeostasis to cells with slightly altered mechanical properties. Alternatively, severe hypertension yields complete medial degeneration. Complete loss of stored elastic energy is observed, with stiffened arteries yielding characteristically high pulse wave velocities (PWVs). Early intervention recovering hypertensive to normotensive pressure, as well as enhanced adventitial collagen turnover, are shown to prevent medial degeneration. Our model thus offers a tool to better understand the relationship between ECM damage, arterial failure, and hypertension.

Abstract Background Aortic aneurysm and dissection are the primary causes of morbidity and mortality in Marfan syndrome (MFS), a connective tissue disorder caused by pathogenic variants in the fibrillin-1 gene. While timely surgical repair significantly increases life expectancy, lethal aortic dissection still occurs. This underscores the urgent need for versatile animal models to investigate disease mechanisms and identify new therapeutic targets. Purpose After systematically disrupting all fibrillin genes in zebrafish, we identified fibrillin-2b-deficient (fbn2b-/-) zebrafish as a suitable model for MFS. This zebrafish model provides a powerful platform for evaluating existing therapies and identifying novel drug candidates. Methods fbn2b-/- zebrafish were subjected to detailed cardiovascular phenotyping by fluorescent microscopy in embryonic stages, and cardiac ultrasound, histology, and synchrotron X-ray imaging in adults. We screened over 1500 FDA-approved compounds in fbn2b-/- zebrafish using a genetically encoded reporter for detecting cardiovascular stress. Positive hits were further assessed for phenotypic rescue. Additionally, we tested a β-adrenergic receptor blocker and an angiotensin receptor blocker, both commonly prescribed to patients with MFS. Results Approximately 60% of fbn2b-/- zebrafish embryos develop atrial endocardial detachment and pericardial edema by 2-3 dpf. The majority of these embryos progress to vascular embolism, loss of blood flow, and death at 7-9 dpf. The remaining fbn2b-/- zebrafish survive normally but develop dilation of the bulbus arteriosus during larval stages, which persists into adulthood. Adult fbn2b-/- zebrafish also show cardiac valve abnormalities. The high-throughput drug screen identified a number of promising candidate compounds, although a drug leading to robust phenotypic rescue is yet to be discovered. β-adrenergic and angiotensin receptor inhibition failed to reverse the mutant phenotype. Nevertheless, this outcome is consistent with clinical observations suggesting that these drugs do not directly target the underlying pathophysiological mechanisms driving the core cardiovascular manifestations of MFS. Interestingly, when assessing heart rate modulation in fbn2b-/- larvae, we observed an exaggerated negative chronotropic response to β-adrenergic receptor inhibition, indicating a potentially elevated baseline sympathetic tone. Conclusions The fbn2b-/- zebrafish model effectively recapitulates different aspects of the cardiovascular pathology observed in patients with MFS. This allows us to do in vivo compound screens to investigate underlying disease mechanisms and search for new therapeutic targets. While neither current clinical treatments nor compounds identified through the first round of unbiased high-throughput drug screening produced strong phenotypic rescue, these findings underscore the complexity of the disease and the pressing need for continued efforts to identify effective treatments.

ABSTRACT Background Marfan syndrome (MFS) is a life-threatening heritable connective tissue disorder caused by pathogenic variants in fibrillin-1, characterized by progressive cardiovascular disease. Current medical therapies slow disease progression but do not prevent major complications, underscoring the need for new treatment strategies and unbiased discovery approaches. Methods We used a zebrafish model of MFS lacking fibrillin-3 ( fbn3 -/- ), which recapitulates key cardiovascular phenotypes including cardiac stress, valvular defects, arrhythmia, and aortic dilation. To enable sensitive, quantitative assessment of cardiac stress, we generated a novel transgenic zebrafish reporter expressing secreted nanoluciferase under control of the stress-responsive nppb promoter. This reporter was combined with morphological phenotyping and bulbus arteriosus (BA) imaging. We evaluated standard MFS therapies, targeted modulators of TGF-β signaling, and performed an unbiased high-throughput drug screen of over 1 500 clinically approved compounds across multiple developmental treatment windows. Results fbn3 -/- larvae exhibited markedly elevated nppb activity that correlated with phenotypic severity and peaked during stages of highest mortality. The nanoluciferase reporter provided a ∼1 000-fold dynamic range, substantially outperforming Firefly luciferase-based assays. Pharmacological inhibition of TGF-β signaling produced transient or deleterious effects, while β-blockers, losartan, and allopurinol failed to consistently improve cardiac stress, pericardial edema, or BA dilation. The unbiased high-throughput drug screen identified a small number of primary and secondary hits; however, none demonstrated reproducible phenotypic rescue upon rigorous multi-dose, multi-time window validation. Conclusions This study establishes a sensitive zebrafish-based platform for early, quantitative assessment of cardiovascular stress in MFS. Our findings highlight the limited efficacy of current therapies, the context-dependent nature of TGF-β modulation, and the biological complexity underlying MFS pathogenesis. Although no definitive therapeutic candidates were identified, this work lays a robust foundation for expanded unbiased discovery efforts aimed at identifying disease-modifying interventions for MFS.

Genomic variants of uncertain significance (VUS) impede clinical decision-making. In this study, we use a knock-in strategy in zebrafish to evaluate the COL1A2 c.2123G>A VUS, identified in a 59-year-old female with recurrent fractures. Using prime editing, we obtained different zebrafish lines respectively harboring the VUS, a known pathogenic variant, or a known benign variant. Comprehensive skeletal phenotyping revealed no significant abnormalities in the zebrafish modeling the benign variant and the VUS, while zebrafish modeling the pathogenic variant showed scoliosis of the vertebral column, vertebral fusions, vertebral compressions, fractures, and increased mineralization of the notochord and intervertebral ligament. Our findings demonstrate for the first time, that COL1A2 variant modeling in zebrafish models informs functional validation and shows potential for elucidating associated pathogenic mechanisms. This approach can be extended to study VUS in other genes. ### Competing Interest Statement The authors have declared no competing interest.

Mycotoxins such as Aflatoxin B1 (AFB1) and Cyclopiazonic Acid (CPA) pose serious health risks, yet their combined toxicity remains poorly understood. This study examined, for the first time, the individual and joint effects of AFB1 and CPA on zebrafish (Danio rerio) embryos, focusing on developmental and cardiac outcomes. Embryos were exposed from 6 h to 4 days post-fertilization to varying concentrations of each toxin and their mixtures. AFB1 caused fin fold wrinkling, underdeveloped pectoral fins, body curvature, growth retardation, and high lethality at elevated doses. CPA induced dose-dependent abnormalities, including cerebral hemorrhage, pericardial edema, fin and body deformities, and increased mortality. Combined exposure produced severe malformations encompassing most defects from either toxin alone, with markedly higher mortality, indicating synergistic toxicity. Cardiac assessments showed that CPA alone mainly reduced heart rate, stroke volume, and cardiac output, while AFB1 alone showed no significant effects on cardiac function. Co-exposure, however, further suppressed cardiac function, revealing enhanced cardiotoxicity. These results highlight the amplified risks of co-exposure to CPA and AFB1 and emphasize the need to account for multi-mycotoxin interactions in food safety assessments. The study also reinforces the value of zebrafish embryos as an in vivo model for mycotoxin toxicity.

Genomic variants of uncertain significance (VUS) impede clinical decision-making. In this study, we employ a knock-in strategy in zebrafish to evaluate the COL1A2 c.2123G>A VUS, identified in a 78-year-old female with atypical femoral fractures. Using prime editing, we generated different col1a2 zebrafish lines respectively harboring the VUS, a known pathogenic variant, and a known benign variant. Comprehensive skeletal phenotyping revealed no significant abnormalities in zebrafish harboring the VUS. In contrast, zebrafish with the pathogenic variant showed an increased eye diameter, scoliosis, vertebral fusions, vertebral compressions, fractures, and increased mineralization of the notochord and intervertebral ligament compared to wild type controls. Our findings represent the first demonstration that COL1A2 variant modeling via prime editing in zebrafish not only aids in functional validation, but also holds promise for uncovering the underlying pathogenic mechanisms. This approach can be applied to investigate VUS in other genes as well.

Thoracic aortic aneurysm and dissection (TAAD) significantly impact cardiovascular morbidity and mortality. A large subset of TAAD cases, particularly those with an earlier onset, is linked to heritable genetic defects. Despite progress in characterizing genes associated with both syndromic and non-syndromic heritable TAAD, the causative gene remains unknown in most cases. Another important bottleneck in the correct and timely diagnosis of TAAD is the large proportion of variants of unknown significance (VUS) that are routinely encountered upon medical genetic testing. Reliable functional modeling data is required to accurately identify new causal genes and to determine the pathogenicity of VUS. To address this gap, our collaborative effort—comprising teams from Yale University, University of Kentucky, and Ghent University—explores a novel approach: modeling TAAD in zebrafish. Leveraging the unique advantages of this animal model promises to allow for accelerated variant pathogenicity assessment, ultimately enhancing patient care. In this review, we critically explore the currently available zebrafish-based approaches that can be used for testing pathogenicity of genes and variants related to TAAD, and we offer an outlook on the implementation of these strategies for clinical applications.

BACKGROUND:Aortic dissection (AD) is the separation of medial layers of the aorta and is a major cause of death in patients with connective tissue disorders such as Marfan syndrome. However, molecular triggers instigating AD, its temporospatial progression, and how vascular cells in each vessel layer interact and participate in the pathological process remain incompletely understood. To unravel the underlying molecular mechanisms of AD, we generated a spontaneous AD mouse model. METHODS:We incorporated a novel missense variant (p.G234D) in FBN1, the gene for fibrillin-1, identified in a patient with nonsyndromic familial AD into mice using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. We performed molecular pathological analyses of the aortic lesions by histology, immunofluorescence staining, electron microscopy, synchrotron-based imaging, and single-cell RNA sequencing. Biochemical analysis was performed to examine the binding capacity of mutant human FBN1G234D (fibrillin 1 Gly234Asp) protein to LTBPs (latent TGFβ [transforming growth factor-beta] binding proteins), and signaling pathways in the mutant aortic wall were examined by the Western blot analysis. RESULTS:Fifty percent of the Fbn1G234D/G234D mutant mice died within 5 weeks of age from multiple intimomedial tears that expanded longitudinally and progressed to aortic rupture accompanied by massive immune cell infiltration. Fbn1G234D/G234D endothelial cells exhibited altered mechanosensing with loss of parallel alignment to blood flow and upregulation of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) as early as 1 week of age. Single-cell RNA sequencing, validated by immunostaining, revealed a cluster of monocyte/macrophage predominantly in the intima at 3 weeks of age before the dissection, and the second cluster of macrophages increased during the progression of intimomedial tears, exhibiting strong CCR2+ (C-C motif chemokine receptor 2 positive) and both M1- and M2-like features. Consistently, upregulation of MMP2/9 (matrix metalloproteinase 2 and 9) was observed. Biochemically, FBN1G234D lost the ability to bind to LTBP-1, -2, and -4, resulting in the downregulation of TGFβ signaling in the aortic wall. CONCLUSIONS:We show that interactions involving endothelial cells and macrophages/monocytes in the intima, where the extracellular matrix (ECM) microenvironment contains reduced TGFβ signaling, contribute to the initiation of AD. Our novel AD mouse model provides a unique opportunity to identify target molecules involved in the intimomedial tears that can be utilized for the development of therapeutic strategies.

Background Aortic dissection (AD) is the separation of medial layers of the aorta and is a major cause of death in patients with connective tissue disorders such as Marfan syndrome. However, molecular triggers instigating AD, its temporospatial progression, and how vascular cells in each vessel layer interact and participate in the pathological process remain incompletely understood. To unravel the underlying molecular mechanism of AD, we generated a spontaneous AD mouse model.Methods We incorporated a novel missense variant (p.G234D) in FBN1 , the gene for fibrillin-1, identified in a non-syndromic familial AD patient into mice using CRISPR/Cas9 system. We performed histopathological analyses of the aortic lesions by histology, immunofluorescence staining, electron microscopy, synchrotron-based imaging and single-cell (sc)RNA-sequencing. Biochemical analysis was performed to examine the binding capacity of mutant human FBN1G234D protein to latent Tgfβ binding proteins (LTBPs), and signaling pathways in the mutant aortic wall were examined by western blot analysis.Results 50% of the Fbn1 G234D/G234D mutant mice died within 5 weeks of age from multiple intimomedial tears that expanded longitudinally and progressed to aortic rupture accompanied by massive immune cell infiltration. scRNA-sequencing, validated by immunostaining, revealed a significant increase in MHC class II-positive pro-inflammatory macrophages and monocytes at the site of intima tears with upregulation of MMP2/9 and marked disruption of elastic lamina. Subendothelial matrices, such as type IV collagen and laminin, expanded into the medial layer, where fibronectin expression was highly upregulated. Fbn1 G234D/G234D endothelial cells exhibited altered mechanosensing with loss of parallel alignment to blood flow and upregulation of VCAM-1 and ICAM-1, all of which likely contributed to the infiltration of immune cells. Biochemically, FBN1G234D lost the ability to bind to latent TGFβ binding protein (LTBP)-1, -2, and -4, resulting in the downregulation of TGFβ signaling in the aortic wall.Conclusions We show that dynamic interactions involving endothelial cells (ECs) and macrophages/monocytes in the intima, where the ECM microenvironment is altered with the reduced TGFβ signaling, contributes to the initiation of AD. Our novel AD mouse model provides a unique opportunity to identify target molecules involved in the intimomedial tears that can be utilized for development of therapeutic strategies.### Competing Interest StatementThe authors have declared no competing interest.* AD : aortic dissection TAA : thoracic aortic aneurysm TAAD : thoracic aortic aneurysm and dissection AA : aortic aneurysm FBN1 : fibrillin1 TGFβ : transforming growth factor-beta LTBP : latent TGFβ binding protein ECM : extracellular matrix EC : endothelial cell SMC : smooth muscle cell

Abstract Background Despite improved life expectancy for patients with Marfan syndrome (MFS), significant morbidity and mortality persist. Although primarily attributed to aortic dissection and rupture, recent studies highlight the emerging role of arrhythmias and heart failure as additional contributors to morbidity and mortality in MFS. While severe valvular heart disease is an established cause of heart failure, there is a growing recognition of primary cardiomyopathy in patients with MFS. The mechanisms underlying cardiomyopathy in MFS remain poorly understood. Purpose This study aimed to reveal insights into the ultrastructural myocardial architecture in patients with MFS, thereby elucidating the role of cardiomyopathy in MFS. Methods This study enrolled 44 patients who underwent cardiac surgery at 2 centers (Marfan syndrome patients n=27 (MFS), ascending thoracic aortic aneurysm repair patients n=5 (non-MFS), patients post-orthotopic heart transplant n=12 (controls)). Myocardial biopsy samples were subjected to histopathological analysis using transmission electron microscopy and conventional histology. Results Conventional histological analysis revealed subtle abnormalities in the cardiomyocyte morphology of MFS patients, compared to non-MFS and controls. Overall, a slight increase in interstitial fibrosis and mild myocyte hypertrophy were observed in MFS patients. Ultrastructural examination of both MFS and non-MFS patients revealed significant structural remodeling and degenerative changes in the myocardium. These changes included: extensive myofibril lysis, compromised cell membrane integrity and nuclear chromatin condensation. Degenerative lesions were often accompanied by cytoplasmic debris, lysosomal residual bodies, autophagic vacuoles, and severe mitochondrial pathology. Dense accumulation of glycogen granules and lipofuscin was evident in areas of focal myofibril lysis. Notably, only MFS patients exhibited recruitment of granulocytes, including mast cells, eosinophils and neutrophils. A subset of MFS patients displayed advanced stages of myocardial degeneration. However, these changes did not correlate with clinical myocardial failure. Conclusions To the best of our knowledge, this study represents the first evidence of ultrastructural changes in the myocardium of patients with MFS. Our data indicate various degenerative changes in the myocardial architecture, including structural and metabolic myocardial remodeling, along with inflammatory cell infiltration. We believe that our findings of ultrastructural lesions reflect the complexity of myocardial disease in MFS and emphasize the importance of careful surveillance for cardiomyopathy in MFS patients. Further studies are warranted to confirm whether the observed granulocyte influx is specific to MFS and to clarify its role in MFS myocardial disease pathophysiology.Graphical abstract

ABSTRACT Thoracic aortic aneurysm and dissection (TAAD) associates with a high mortality rate. Despite the existence of different mouse models for TAAD, the underlying disease mechanisms remain elusive. Treatment options are limited and mainly consist of surgical repair at critical aortic diameters as current pharmacological interventions are unable to stop disease progression. In humans, loss of function (LOF) of SMAD3 and SMAD6 impairs vascular homeostasis, increasing the risk for TAAD. We developed a zebrafish model for thoracic aortic dissection/rupture by targeting both ohnologs of smad3 and smad6 . At 10 days post fertilization, we found an increased diameter of the ventral aorta in smad3a −/− ; smad3b −/− double knockout zebrafish, while smad6a −/− ; smad6b −/− double knockout zebrafish have a reduced aortic diameter associated with early mortality. We discovered that a smad3a −/− ; smad3b −/− ; smad6a −/− ; smad6b −/− quadruple knockout (qKO) zebrafish model is viable and survives to adulthood, although exposure to stress leads to sudden death. Histological analysis of the adult ventral aorta shows medial elastolysis, aortic dissections and ruptures at sites exposed to high biomechanical stress. RNA-sequencing of 5 days post fertilization qKO zebrafish indicates a profile of reduced negative regulation of proteolysis and upregulation of melanogenesis, a previously unaddressed pathway in this pathology. We confirm that pharmacological modulation of tyrosinase, the enzyme responsible for the production of melanin, influences aortic morphology. Overall, the qKO mutant, thus far the only known zebrafish model of thoracic aortic dissection and rupture, reveals novel SMAD3/6-dependent pathways that impact thoracic aortic homeostasis, in this way opening avenues for the development of novel treatments in TAAD.

Vascular corrosion casting is a method used to visualize the three dimensional anatomy and branching pattern of blood vessels, guiding insight into health and cardiovascular disease pathogenesis and progression. A polymer resin is injected in the vascular system and, after curing, the surrounding tissue is removed. This corrosion process often deforms or even fractures the fragile cast, resulting in an overall loss of information. Here, we propose a method that does not require corrosion of the tissue, based on in-situ high-resolution computed tomography (micro-CT) scans. Since there is a lack of CT contrast between the polymer cast and the animals’ surrounding soft tissue, we introduce hafnium oxide nanocrystals (HfO2 NCs) as CT contrast agents into the resin. The NCs dramatically improve the overall CT contrast of the cast and allow for straightforward segmentation in the CT scans. We designed the NC surface chemistry to ensure colloidal stability of the NCs in the casting resin, resulting in a homogeneous dispersion that remains stable during casting and curing. Using only 5 m% of HfO2 NCs, high-quality casts of both zebrafish and mouse models could be segmented using CT imaging software, allowing us to differentiate even μm scale details, without having to alter the resin injection method or affecting the resin’s mechanical properties. Our new method of virtual dissection by visualizing casts in-situ using contrast enhanced CT imaging greatly expands the application potential of the technique.