Biology subjects

Callewaert, B.

Publications and source records attributed to Callewaert, B..

7 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↗

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↗

bioRxiv2022

Transglutaminase mediated asprosin oligomerization allows its tissue storage as fibers

Asprosin, the C-terminal furin cleavage product of profibrillin-1, was reported to act as a hormone that circulates at nanomolar levels and is recruited to the liver where it induces G protein-coupled activation of the cAMP-PKA pathway and stimulates rapid glucose release into the circulation. Although derived upon C-terminal cleavage of fibrillin-1, a multidomain extracellular matrix glycoprotein with a ubiquitous distribution in connective tissues, little is known about the mechanisms controlling the bioavailability of asprosin in tissues. In the current view, asprosin is mainly produced by white adipose tissue from where it is released into the blood in monomeric form. Here, by employing newly generated specific asprosin antibodies we monitored the distribution pattern of asprosin in human and murine connective tissues such as placenta, and muscle. Thereby we detected the presence of asprosin positive extracellular fibers. Further, by screening established cell lines for asprosin synthesis we found that most cells derived from musculoskeletal tissues render asprosin into an oligomerized form. Our analyses show that asprosin already multimerizes intracellularly, but that stable multimerization via covalent bonds is facilitated by transglutaminase activity. Further, asprosin fiber formation requires an intact fibrillin-1 fiber network for proper linear deposition. Our data suggest a new extracellular storage mechanism of asprosin in an oligomerized form which may regulate its cellular bioavailability in tissues.

biochemistry↗

bioRxiv2021

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype, and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A, and recorded up-regulated ISG expression and interferon alpha protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy. SummaryDominant-negative mutations in ATAD3A, a ubiquitously expressed mitochondrial protein, cause mitochondrial DNA-dependent up-regulation of type I interferon signaling in the context of neurological disease and autoimmunity, thereby defining a novel type I interferonopathy.

immunology↗

bioRxiv2020

Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidification in vivo

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

molecular biology↗

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↗