Zebrafish are teleosts (bony fish) that share with mammals a common ancestor belonging to the phylum Osteichthyes, from which their endoskeletal systems have been inherited. Indeed, teleosts and mammals have numerous genetically conserved features in terms of skeletal elements, ossification mechanisms, and bone matrix components in common. Yet differences related to bone morphology and function need to be considered when investigating zebrafish in skeletal research. In this review, we focus on zebrafish skeletal architecture with emphasis on the morphology of the vertebral column and associated anatomical structures. We provide an overview of the different ossification types and osseous cells in zebrafish and describe bone matrix composition at the microscopic tissue level with a focus on assessing mineralization. Processes of bone formation also strongly depend on loading in zebrafish, as we elaborate here. Furthermore, we illustrate the high regenerative capacity of zebrafish bones and present some of the technological advantages of using zebrafish as a model. We highlight zebrafish axial and fin skeleton patterning mechanisms, metabolic bone disease such as after immunosuppressive glucocorticoid treatment, as well as osteogenesis imperfecta (OI) and osteopetrosis research in zebrafish. We conclude with a view of why larval zebrafish xenografts are a powerful tool to study bone metastasis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Protein disulphide isomerase A3 (PDIA3) is an ER-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix proteins. PDIA3 is upregulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 in cells decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified “scratch wound” assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wild-type or Pdia3-/- mouse embryo fibroblasts. The conditioned medium of Pdia3-/- fibroblasts was less effective in promoting cell spreading and F-actin organisation or supporting “scratch wound” closure. Similarly, extracellular matrix prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

4Immune mediated diseases (IMDs) arise from a lack of immune tolerance, causing chronic 5 inflammation. Despite their growing prevalence, targeted therapies to treat IMDs are lacking. 6Cytokines and their receptors, which mediate inflammation, have been associated with IMD 7 susceptibility. However, the complex signalling networks and multiple cell-types required to 8 orchestrate inflammatory responses have made it difficult to pinpoint specific cytokines and immune 9 cell-types which drive IMDs. 10In this study, we developed an analytical framework which integrates Mendelian randomisation (MR) 11 and multiple-trait colocalization (moloc) analyses to determine putative cell-specific drivers of IMDs. 12We used MR to determine the likelihood of causal associations between the levels of 10 circulating 13 cytokines/cytokine receptors and 9 IMDs within human populations of European descent. 14 Conservative (single SNP) and liberal (multiple SNP) MR analysis supported a causal role for IL-18 in 15 inflammatory bowel disease (P = 1.17 x 10 -4 ) and eczema/dermatitis (P = 2.81 x 10 -3 ), as well as roles 16 for IL-2rα and IL-6R in several IMDs. 17Where associations between cytokines/cytokine receptors and IMDs were discovered using MR, we 18 undertook moloc analyses. This was to assess the likelihood that cytokine/cytokine receptor protein 19 quantitative trait loci (pQTL) and IMD-associated loci share a causal genetic variant along with 20 expression QTL (eQTL) using data from 3 immune cell-types: monocytes, neutrophils and T cells. We 21 found a monocyte and neutrophil-driven role for IL-18 in IBD pathogenesis, amongst evidence 22 supporting several other cell-specific inflammatory drivers of IMDs. Our study helps to elucidate causal 23

Immune-mediated diseases (IMDs) arise when tolerance is lost and chronic inflammation is targeted towards healthy tissues. Despite their growing prevalence, therapies to treat IMDs are lacking. Cytokines and their receptors orchestrate inflammatory responses by regulating elaborate signalling networks across multiple cell types making it challenging to pinpoint therapeutically relevant drivers of IMDs. We developed an analytical framework that integrates Mendelian randomization (MR) and multiple-trait colocalization (moloc) analyses to highlight putative cell-specific drivers of IMDs. MR evaluated causal associations between the levels of 10 circulating cytokines and 9 IMDs within human populations. Subsequently, we undertook moloc analyses to assess whether IMD trait, cytokine protein and corresponding gene expression are driven by a shared causal variant. Moreover, we leveraged gene expression data from three separate cell types (monocytes, neutrophils and T cells) to discern whether associations may be attributed to cell type-specific drivers of disease. MR analyses supported a causal role for IL-18 in inflammatory bowel disease (IBD) (P = 1.17 × 10−4) and eczema/dermatitis (P = 2.81 × 10−3), as well as associations between IL-2rα and IL-6R with several other IMDs. Moloc strengthened evidence of a causal association for these results, as well as providing evidence of a monocyte and neutrophil-driven role for IL-18 in IBD pathogenesis. In contrast, IL-2rα and IL-6R associations were found to be T cell specific. Our analytical pipeline can help to elucidate putative molecular pathways in the pathogeneses of IMDs, which could be applied to other disease contexts.

Bone homeostasis is a dynamic, multicellular process that is required throughout life to maintain bone integrity, prevent fracture, and respond to skeletal damage. WNT16 has been linked to bone fragility and osteoporosis in human genome wide-association studies, as well as the functional hematopoiesis of leukocytes in vivo. However, the mechanisms by which WNT16 promotes bone health and repair are not fully understood. In this study, CRISPR-Cas9 was used to generate mutant zebrafish lacking Wnt16 (wnt16 −/−) to study its effect on bone dynamically. The wnt16 mutants displayed variable tissue mineral density (TMD) and were susceptible to spontaneous fractures and the accumulation of bone calluses at an early age. Fractures were induced in the lepidotrichia of the caudal fins of wnt16 −/− and WT zebrafish; this model was used to probe the mechanisms by which Wnt16 regulates skeletal and immune cell dynamics in vivo. In WT fins, wnt16 expression increased significantly during the early stages for bone repair. Mineralization of bone during fracture repair was significantly delayed in wnt16 mutants compared with WT zebrafish. Surprisingly, there was no evidence that the recruitment of innate immune cells to fractures or soft callus formation was altered in wnt16 mutants. However, osteoblast recruitment was significantly delayed in wnt16 mutants postfracture, coinciding with precocious activation of the canonical Wnt signaling pathway. In situ hybridization suggests that canonical Wnt-responsive cells within fractures are osteoblast progenitors, and that osteoblast differentiation during bone repair is coordinated by the dynamic expression of runx2a and wnt16. This study highlights zebrafish as an emerging model for functionally validating osteoporosis-associated genes and investigating fracture repair dynamically in vivo. Using this model, it was found that Wnt16 protects against fracture and supports bone repair, likely by modulating canonical Wnt activity via runx2a to facilitate osteoblast differentiation and bone matrix deposition.