Significance: Type 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 future 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.

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.

Genome‐wide 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 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 (microCT) 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 to 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.

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-mediatedb3galt6knock-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.

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.