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Rare monogenic disorders often exhibit significant phenotypic variability among individuals sharing identical genetic mutations. Bruck syndrome (BS), a prime example, is characterized by bone fragility and congenital contractures, although with a pronounced variability among family members. BS arises from recessive biallelic mutations in FKBP10 or PLOD2. FKBP65, the protein encoded by FKBP10, collaborates with the LH2 enzyme (PLOD2) in type I collagen telopeptide lysine hydroxylation, crucial for collagen cross-linking. To identify potential modifier genes and to investigate the mechanistic role of FKBP10 in BS pathogenesis, we established an fkbp10a knockout zebrafish model. Mass-spectrometry analysis in fkbp10a-/- mutants revealed a generally decreased type I collagen lysyl hydroxylation, paralleled by a wide skeletal variability similar to human patients. Ultrastructural examination of the skeleton in severely affected mutants showed enlarged type I collagen fibrils and disturbed elastin layers. Whole-exome sequencing of 7 mildly and 7 severely affected mutant zebrafish siblings, followed by SNP-based linkage analysis, indicated a linked region on chromosome 13, which segregates with phenotypic severity. Transcriptome analysis identified six differentially expressed genes (DEGs) between mildly and severely affected mutants. The convergence of genes within the linked region and DEGs highlighted bmpr1aa as a potential modifier gene, as its reduced expression correlates with increased skeletal severity. In summary, our study provides deeper insights into the role of FKBP10 in BS pathogenesis. Additionally, we identified a pivotal gene that influences phenotypic severity in a zebrafish model of BS. These findings hold promise for novel treatments in the field of bone diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.

Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines and 3D tumor models. To address this goal, a well-characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G>T (p.(Gly12Val)) was used. Fluorescently labeled tumor cells were injected into the perivitelline space of 2 days' post-fertilization zebrafish embryos. At 1 day post-injection, xenografts were assessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib and trametinib + luminespib. Subsequently, xenografts were euthanized and analyzed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses ( n = 84) in vivo, with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.

SNARE proteins comprise a conserved protein family responsible for catalyzing membrane fusion during vesicle traffic. Syntaxin18 (STX18) is a poorly characterized endoplasmic reticulum (ER)-resident t-SNARE. Recently, together with TANGO1 and SLY1, its involvement was shown in ER to Golgi transport of collagen II during chondrogenesis. We report a fetus with a severe osteochondrodysplasia in whom we identified a homozygous substitution of the highly conserved p.Arg10 to Pro of STX18. CRISPR/Cas9-mediated Stx18 deficiency in zebrafish reveals a crucial role for Stx18 in cartilage and bone development. Furthermore, increased expression of multiple components of the Stx18 SNARE complex and of COPI and COPII proteins suggests that Stx18 deficiency impairs antero- and retrograde vesicular transport in the crispant stx18 zebrafish. Taken together, our studies highlight a new candidate gene for a recessive form of osteochondrodysplasia, thereby possibly broadening the SNAREopathy phenotypic spectrum and opening new doors toward future research avenues. © 2023 American Society for Bone and Mineral Research (ASBMR)., (© 2023 American Society for Bone and Mineral Research (ASBMR).)

Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede., (© 2020 The Fisheries Society of the British Isles.)

The popularity of zebrafish in both basic biological and biomedical research has led to an increased need for understanding their behavior. Locomotor behavior is an important outcome of different factors, such as specific genotypes or external stimuli that influence the nervous and musculoskeletal system. Locomotion can be studied by forced swimming in a swim tunnel, a device capable of generating a laminar water flow at different speeds in a chamber where zebrafish can be placed. However, commercially available swim tunnels are relatively expensive and in-house built systems are mostly presented without clear building instructions or proper validation procedures. In this study, we developed an alternative, cheap (<250 euro), and user-friendly, but customizable benchtop swim tunnel, called the " Zebrafish exercise-tunnel " (ZE-Tunnel). Detailed step-by-step instructions on how to construct the tunnel components, including the frame, mechanical, and electric components are given. The ZE-Tunnel was reliably used to exercise fish for prolonged periods and its performance was successfully validated by replicating previously published experiments on critical speed testing in zebrafish. Finally, implementation of behavioral video analysis using freely available motion-tracking software showed differences in swimming dynamics in the Chihuahua skeletal zebrafish mutant.

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 β3GalT6-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., Competing Interests: PS was employed by the company Bruker MicroCT. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Delbaere, De Clercq, Mizumoto, Noborn, Bek, Alluyn, Gistelinck, Syx, Salmon, Coucke, Larson, Yamada, Willaert and Malfait.)

Animal models are essential tools for addressing fundamental scientific questions about skeletal diseases and for the development of new therapeutic approaches. Traditionally, mice have been the most common model organism in biomedical research, but their use is hampered by several limitations including complex generation, demanding investigation of early developmental stages, regulatory restrictions on breeding, and high maintenance cost. The zebrafish has been used as an efficient alternative vertebrate model for the study of human skeletal diseases, thanks to its easy genetic manipulation, high fecundity, external fertilization, transparency of rapidly developing embryos, and low maintenance cost. Furthermore, zebrafish share similar skeletal cells and ossification types with mammals. In the last decades, the use of both forward and new reverse genetics techniques has resulted in the generation of many mutant lines carrying skeletal phenotypes associated with human diseases. In addition, transgenic lines expressing fluorescent proteins under bone cell- or pathway- specific promoters enable in vivo imaging of differentiation and signaling at the cellular level. Despite the small size of the zebrafish, many traditional techniques for skeletal phenotyping, such as x-ray and microCT imaging and histological approaches, can be applied using the appropriate equipment and custom protocols. The ability of adult zebrafish to remodel skeletal tissues can be exploited as a unique tool to investigate bone formation and repair. Finally, the permeability of embryos to chemicals dissolved in water, together with the availability of large numbers of small-sized animals makes zebrafish a perfect model for high-throughput bone anabolic drug screening. This review aims to discuss the techniques that make zebrafish a powerful model to investigate the molecular and physiological basis of skeletal disorders., (Copyright © 2020 Tonelli, Bek, Besio, De Clercq, Leoni, Salmon, Coucke, Willaert and Forlino.)

Zebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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 a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. 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 intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future 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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)