Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.

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.

Excessive skeletal deformations and brittle fractures in the vast majority of patients suffering from osteogenesis imperfecta (OI) are a result of substantially reduced bone quality. Because the mechanical competence of bone is dependent on the tissue characteristics at small length scales, it is of crucial importance to assess how OI manifests at the micro- and nanoscale of bone. In this context, the Chihuahua (Chi/+) zebrafish, carrying a heterozygous glycine substitution in the α1 chain of collagen type I, has recently been proposed as a suitable animal model of classical dominant OI, showing skeletal deformities, altered mineralization patterns, and a smaller body size. This study assessed the bone quality properties of Chi/+ at multiple length scales using micro-computed tomography (micro-CT), histomorphometry, quantitative back-scattered electron imaging, Fourier-transform infrared spectroscopy, nanoindentation, and X-ray microscopy. At the skeletal level, the Chi/+ displays smaller body size, deformities, and fracture calli in the ribs. Morphological changes at the whole bone level showed that the vertebrae in Chi/+ had a smaller size, smaller thickness, and distorted shape. At the tissue level, Chi/+ displayed a higher degree of mineralization, lower collagen maturity, lower mineral maturity, altered osteoblast morphology, and lower osteocyte lacunar density compared to wild-type zebrafish. The alterations in the cellular, compositional, and structural properties of Chi/+ bones bear an explanation for the impaired local mechanical properties, which promote an increase in overall bone fragility in Chi/+. The quantitative assessment of bone quality in Chi/+ thus further validates this mutant as an important model reflecting osseous characteristics associated with human classical dominant OI. © 2018 American Society for Bone and Mineral Research.

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases.

The limited accessibility of bone and its mineralized nature have restricted deep investigation of its biology. Recent breakthroughs in identification of mutant proteins affecting bone tissue homeostasis in rare skeletal diseases have revealed novel pathways involved in skeletal development and maintenance. The characterization of new dominant, recessive and X‐linked forms of the rare brittle bone disease osteogenesis imperfecta (OI) and other OI‐related bone fragility disorders was a key player in this advance. The development of in vitro models for these diseases along with the generation and characterization of murine and zebrafish models contributed to dissecting previously unknown pathways. Here, we describe the most recent advances in the understanding of processes involved in abnormal bone mineralization, collagen processing and osteoblast function, as illustrated by the characterization of new causative genes for OI and OI‐related fragility syndromes. The coordinated role of the integral membrane protein BRIL and of the secreted protein PEDF in modulating bone mineralization as well as the function and cross‐talk of the collagen‐specific chaperones HSP47 and FKBP65 in collagen processing and secretion are discussed. We address the significance of WNT ligand, the importance of maintaining endoplasmic reticulum membrane potential and of regulating intramembrane proteolysis in osteoblast homeostasis. Moreover, we also examine the relevance of the cytoskeletal protein plastin‐3 and of the nucleotidyltransferase FAM46A. Thanks to these advances, new targets for the development of novel therapies for currently incurable rare bone diseases have been and, likely, will be identified, supporting the important role of basic science for translational approaches.

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Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation‐positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient‐derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation‐positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR‐Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD‐RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Osteogenesis imperfecta (OI) type XIV is a rare recessive bone disorder characterized by variable degree of severity associated to osteopenia. It is caused by mutations in TMEM38B encoding for the trimeric intracellular cation channel TRIC-B, specific for potassium and ubiquitously present in the endoplasmic reticulum (ER) membrane. OI type XIV molecular basis is largely unknown and, due to the rarity of the disease, the availability of patients’ osteoblasts is challenging. Thus, CRISPR/Cas9 was used to knock out (KO) TMEM38B in the human Foetal Osteoblast hFOB 1.19 to obtain an OI type XIV model. CRISPR/Cas9 is a powerful technology to generate in vitro and in vivo models for heritable disorders. Its limited cost and ease of use make this technique widely applicable in most laboratories. Nevertheless, to fully take advantage of this approach, it is important to be aware of its strengths and limitations. Three gRNAs were used and several KO clones lacking the expression of TRIC-B were obtained. Few clones were validated as good models for the disease since they reproduce the altered ER calcium flux, collagen I structure and impaired secretion and osteoblastic markers expression detected in patients’ cells. Impaired proliferation and mineralization in KO clones unveiled the relevance of TRIC-B in osteoblasts functionality.