Biology subjects

Willaert, A.

Publications and source records attributed to Willaert, A..

12 recordsLinked to original sources

bioRxiv2025

Evaluating Variants of Uncertain Significance in Adult Knock-in Zebrafish: A Proof of Concept with a COL1A2 Variant

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.

genetics↗

bioRxiv2025

Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish

Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiencies (EE) and potential off-target effects. In this study, we optimized conventional CRISPR/Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing (PE), a recently developed technique that is not yet commonly used. Using next-generation sequencing (NGS), we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of 1) varying Cas9 amounts, 2) HDR templates with chemical modifications to improve integration efficiency, 3) different micro-injection procedures, and 4) synonymous guide-blocking variants in the protospacer sequence. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 and 800 pg. The use of Alt-R HDR templates (IDT) further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. PE, however, increased EE up to fourfold and expanded the F0 founder pool for four targets when compared to conventional HDR editing, with fewer off-target effects. Therefore, PE is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.

genetics↗

bioRxiv2025

Decoding Phenotypic Variability in Osteogenesis Imperfecta: Zebrafish as a Model for Molecular and Ultrastructural Insights.

Phenotypic variability is common in human diseases, even when the same genes are affected. In this study, three zebrafish models of Osteogenesis Imperfecta (OI) with dominant glycine substitutions in type I collagen genes (col1a1amh13/+, col1a1adc124/+, and col1a2mh15/+) were characterized for phenotypic severity and variability, using a newly developed standardized scoring system. Comprehensive analyses of the vertebral columns in these models revealed histological and ultrastructural differences that corresponded with phenotypic severity. Increasing skeletal severity correlated with a higher incidence of skeletal deformities and abnormalities. This, in turn, was associated with thinner bones and increased disorganization of collagen fibrils, fiber accumulation and mineralization, elastin deposits, and increased cell proliferation in the notochord and intervertebral ligament (IVL). Additionally, osteoblast function and bone regenerative capacity were increasingly compromised. These characteristics, combined with genetic information, have the potential to predict the severity of phenotypic outcomes in dominant forms of OI, caused by mutations in type I collagen. A remarkable intra-familial phenotypic variability in the col1a2mh15/+mutant holds potential for future approaches that could help in understanding the underlying mechanisms of this variability and the identification of modifier genes. Finally, through proteomics analysis three potential protein biomarkers (HSP47, Col8a1, and Bcan) were identified, that could serve as indicators of disease severity. These biomarkers not only have diagnostic value, but will allow stratification by OI type, have predictive value towards progression of the clinical presentation and will play a role in treatment guidance. Validation in human tissue samples will further reveal their clinical relevance. Significance StatementPhenotypic variability in human diseases, such as Osteogenesis Imperfecta (OI), remains poorly understood. Using zebrafish models with dominant glycine substitutions in type I collagen, this study links genetic mutations to phenotypic severity through standardized scoring and detailed ultrastructural and molecular analyses. Key findings include skeletal abnormalities, compromised osteoblast function, and intra-familial phenotypic variability, suggesting the role of modifier genes. Proteomics identified three potential biomarkers (HSP47, Col8a1, and Bcan) with diagnostic and prognostic value. These results provide critical insights into genotype-phenotype correlations, offering a foundation for personalized approaches to diagnosis, stratification, and treatment of OI and related disorders.

molecular biology↗

bioRxiv2024

Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility

Heritable Fragile Bone Disorders (FBDs) encompass a spectrum of conditions, from widespread multifactorial to rare monogenic diseases, all characterized by an elevated risk of fractures. The process of validating causative genes and elucidating their pathogenic mechanisms remains a daunting and resource-intensive task. In this study, we evaluated the feasibility of a semi-high throughput zebrafish screening platform for rapid validation and in vivo functional testing and validation of candidate disease-causing genes for a wide range of heritable FBDs. Six genes associated with severe recessive forms of Osteogenesis Imperfecta (OI) and four genes associated with bone mineral density (BMD), a key osteoporosis indicator, identified through genome-wide association studies (GWAS) were selected. The crispant screening approach, based on CRISPR/Cas9 technology, was used to phenotype directly in F0 mosaic founder zebrafish. Next-Generation Sequencing (NGS) analysis revealed a mean indel efficiency of 88% across ten different crispants, indicating a high proportion of knock-out alleles and thus resembling stable knock-out models. We applied multiple techniques to evaluate skeletal characteristics at 7, 14 and 90 days post-fertilization (dpf), including microscopy for osteoblast reporter visualization and mineralization by Alizarin Red S staining, and microCT for quantitative skeletal analysis. While larval crispants exhibited variable differences in osteoblast-positive and mineralized surface areas, adult-stage crispants displayed more pronounced and consistent skeletal phenotypes. Notably, all crispants developed malformed neural and haemal arches, with a majority presenting vertebral fractures and fusions, and some showing significant alterations in vertebral bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR analysis of osteoblast differentiation and bone formation markers at larval stages indicated differential expression of osteogenic markers bglap and col1a1a in a substantial portion of the crispants, hinting at their utility as biomarkers for FBD crispant screening. In summary, our findings demonstrate that crispant screening in zebrafish offers a viable and efficient strategy for the functional assessment of FBD genes. We advocate for a novel comprehensive approach that integrates various techniques and evaluates distinct skeletal and molecular profiles across different developmental and adult stages. This methodology has the potential to provide new insights into the role of these genes in skeletal biology.

genetics↗

bioRxiv2024

Early mechanisms of aortic failure in a zebrafish model for thoracic aortic dissection and rupture

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.

developmental biology↗

bioRxiv2021

Spatial proteogenomics reveals distinct and evolutionarily-conserved hepatic macrophage niches

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here, we present a spatial proteogenomic atlas of the healthy human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and bile-duct macrophages. We also uncover the respective spatially-resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that bile-duct macrophages are induced by local lipid exposure, while Kupffer cells crucially depend on their crosstalk with hepatic stellate cells via the evolutionarily-conserved ALK1-BMP9/10 axis.

immunology↗

bioRxiv2021

Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme

Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.

genetics↗

bioRxiv2020

b3galt6 knock-out zebrafish recapitulate β3GalT6-deficiency disorders in human and reveal a trisaccharide proteoglycan linkage region

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human {beta}3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

genetics↗

bioRxiv2019

SLC2A knockout mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicate GLUT10 in transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations do not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in rodents. Gulo;Slc2a10 knock-out mice show mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 knock-out mice do not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. TGF{beta} signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

genetics↗

bioRxiv2018

Deep Phenotyping Enhances Somatic Mutant Detection and Mosaic Pattern Recognition in the Zebrafish Skeleton

Genetic mosaicism manifests as spatially variable phenotypes, whose detection and interpretation remains challenging. This study identifies biological factors influencing spatial phenotypic patterns in the skeletons of somatic mutant zebrafish, and tests methods for their analysis using deep phenotyping. We explore characteristics of loss-of-function clusters in the skeleton of CRISPR-edited G0 (\"crispant\") zebrafish, and identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. Using microCT-based phenomics, we describe diverse phenotypic manifestations in somatic mutants for genes implicated in monogenic (plod2 and bmp1a) and polygenic (wnt16) bone diseases, each showing convergence with germline mutant phenomes. Finally, we describe statistical frameworks for phenomic analysis which confers heightened sensitivity in discriminating somatic mutant populations, and quantifies spatial phenotypic variation. Our studies provide strategies for decoding spatially variable phenotypes which, paired with CRISPR-based screens, can identify genes contributing to skeletal disease.

genetics↗

bioRxiv2018

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of Osteogenesis Imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and the first zebrafish model of human EDS, which harbors characteristic defects in the soft connective tissues. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen related disease models. Our studies reveal a high degree of inter-genotype variability in phenotypic expressivity that closely correlates with associated OI severity. Further, we demonstrate the potential for select mutations to give rise to variable phenotypic penetrance, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention\n\nSIGNIFICANCE STATEMENTType I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders.

genetics↗

bioRxiv2017

microCT-Based Skeletal Phenomics in Zebrafish Reveals Virtues of Deep Phenotyping at the Whole-Organism Scale

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of measures comprised of morphological and densitometric traits from a large number sites in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ. Analyzing phenotypic patterns may increase productivity in genetic screens, and could facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

genetics↗