Biology subjects

Coucke, P.

Publications and source records attributed to Coucke, P..

7 recordsLinked to original sources

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↗

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↗

bioRxiv2020

Novel insights into the structure and transport mechanisms of TAPT1

Transmembrane anterior-posterior transformation protein 1 (TAPT1), encoded by the TAPT1 gene expressed in the basal ciliary body, plays a crucial role in cilia formation as well as axial skeletal patterning. Mutations in this gene have been reported to cause several ciliopathies and osteo-related diseases. Unfortunately, the cellular and molecular pathogenic mechanisms are still unclear also due to the lack of X-ray crystallographic structure and further characterization of TAPT1 protein. In this study, we attempted to characterize this protein by in silico techniques. A 3D structure of TAPT1 was generated by the ab initio method, which was further used for the analysis of the substrate-binding site, to determine pore size and for the prediction of the possible substrate(s). Validation by using different software packages revealed a reliable 3D model of TAPT1. Topology modeling revealed that TAPT1 has eight transmembrane helices with a total number of 27 helices in secondary structure. The amino acid residues H235, R323, K443, N446, S447, L450, K453, S454, Y457, K511, N513, D533, K535, D536, and T538 were found to form the pore surface as well as involved in the binding interaction with the substrate(s). This study predicted flavonoids as the possible substrate for TAPT1, which could further be confirmed by ingenuity pathway analysis. Moreover, our analysis indicated that TAPT1 might localize in the mitochondrial membrane in addition to the ciliary basal body. Our study gives novel insights for TAPT1 structure and its function.

biochemistry↗

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

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↗