A new method for regionalization of the vertebral column in salmonids based on radiographic hallmarks.

Current procedures to establish vertebral column regionalization (e.g., histology) in fish are time consuming and difficult to apply. The aim of this study was to develop a more rapid and accurate radiology-based method for Atlantic salmon (Salmo salar). A detailed analysis of 90 animals (4 kg) led to the establishment of region-specific radiographic hallmarks. To elucidate its transferability to other salmonid species, radiography was carried out in brown trout (Salmo trutta), Arctic char (Salvelinus alpinus), rainbow trout (Oncorhynchus mykiss), pink salmon (Oncorhynchus gorbuscha), and Chinook salmon (Oncorhynchus tshawytscha). This method was also evaluated for whole ungutted fish. The vertebral column of Atlantic salmon can be subdivided into five regions (R1-R5) based on anatomy: postcranial (R1, V1, and V2), abdominal (R2, V3-V26), transitional (R3, V27-V36), caudal (R4, V37-V53), and ural (R5, V54-V59). The following specific radiographic hallmarks allow the identification of regions: (i) lack of ribs in R1, (ii) modified parapophysis of the first vertebra of R3, (iii) prominent hemal spine of the first vertebra of R4, and (iv) the separated hemal spine of the most cranial pre-ural vertebra of R5. These hallmarks were all transferable to the other salmonid species assessed. The results include a further description of various region-specific characteristics in Atlantic salmon. The method was found applicable for sedated/whole ungutted fish, verifying it as quick and easy compared to other regionalization methods. The regions defined by radiology in this study agree with the vertebral column regions recently defined for Chinook salmon (O. tshawytscha). Thus, and considering the results of this study on various salmonid species, the currently developed regionalization protocol can be generally used for salmonids.

Elevated Water CO2 Can Prevent Dietary-Induced Osteomalacia in Post-Smolt Atlantic Salmon (Salmo salar, L.).

Expansion of land-based systems in fish farms elevate the content of metabolic carbon dioxide (CO2) in the water. High CO2 is suggested to increase the bone mineral content in Atlantic salmon (Salmo salar, L.). Conversely, low dietary phosphorus (P) halts bone mineralization. This study examines if high CO2 can counteract reduced bone mineralization imposed by low dietary P intake. Atlantic salmon post-seawater transfer (initial weight 207.03 g) were fed diets containing 6.3 g/kg (0.5P), 9.0 g/kg (1P), or 26.8 g/kg (3P) total P for 13 weeks. Atlantic salmon from all dietary P groups were reared in seawater which was not injected with CO2 and contained a regular CO2 level (5 mg/L) or in seawater with injected CO2 thus raising the level to 20 mg/L. Atlantic salmon were analyzed for blood chemistry, bone mineral content, vertebral centra deformities, mechanical properties, bone matrix alterations, expression of bone mineralization, and P metabolism-related genes. High CO2 and high P reduced Atlantic salmon growth and feed intake. High CO2 increased bone mineralization when dietary P was low. Atlantic salmon fed with a low P diet downregulated the fgf23 expression in bone cells indicating an increased renal phosphate reabsorption. The current results suggest that reduced dietary P could be sufficient to maintain bone mineralization under conditions of elevated CO2. This opens up a possibility for lowering the dietary P content under certain farming conditions.

Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish.

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/- mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+ zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b-/- and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

A Baseline for Skeletal Investigations in Medaka (Oryzias latipes): The Effects of Rearing Density on the Postcranial Phenotype.

Oryzias latipes is increasingly used as a model in biomedical skeletal research. The standard approach is to generate genetic variants with particular skeletal phenotypes which resemble skeletal diseases in humans. The proper diagnosis of skeletal variation is key for this type of research. However, even laboratory rearing conditions can alter skeletal phenotypes. The subject of this study is the link between skeletal phenotypes and rearing conditions. Thus, wildtype medaka were reared from hatching to an early juvenile stage at low (LD: 5 individuals/L), medium (MD: 15 individuals/L), and high (HD: 45 individuals/L) densities. The objectives of the study are: (I) provide a comprehensive overview of the postcranial skeletal elements in medaka; (II) evaluate the effects of rearing density on specific meristic counts and on the variability in type and incidence of skeletal anomalies; (III) define the best laboratory settings to obtain a skeletal reference for a sound evaluation of future experimental conditions; (IV) contribute to elucidating the structural and cellular changes related to the onset of skeletal anomalies. The results from this study reveal that rearing densities greater than 5 medaka/L reduce the animals' growth. This reduction is related to decreased mineralization of dermal (fin rays) and perichondral (fin supporting elements) bone. Furthermore, high density increases anomalies affecting the caudal fin endoskeleton and dermal rays, and the preural vertebral centra. A series of static observations on Alizarin red S whole mount-stained preural fusions provide insights into the etiology of centra fusion. The fusion of preural centra involves the ectopic formation of bony bridges over the intact intervertebral ligament. An apparent consequence is the degradation of the intervertebral ligaments and the remodeling and reshaping of the fused vertebral centra into a biconoid-shaped centrum. From this study it can be concluded that it is paramount to take into account the rearing conditions, natural variability, skeletal phenotypic plasticity, and the genetic background along with species-specific peculiarities when screening for skeletal phenotypes of mutant or wildtype medaka.

Compression Fractures and Partial Phenotype Rescue With a Low Phosphorus Diet in the Chihuahua Zebrafish Osteogenesis Imperfecta Model.

Osteogenesis imperfecta (OI) is a group of heritable disorders affecting bone and other connective tissues. Dominant OI forms are mainly caused by mutations in collagen type I. Patients suffer from skeletal deformities, fractures of long bones and vertebral compression fractures from early childhood onward. Altered collagen structure and excess mineralisation are the main causes for the bone phenotype. The Chihuahua (Chi/+) zebrafish has become an important model for OI. Given that reduced dietary phosphorus (P) intake reduces the bone mineral content and promotes bone matrix formation in teleosts, including zebrafish, we tested whether a low dietary P (LP) intake mitigates the OI phenotype in the Chi/+ model. To answer this question, we characterised the Chi/+ vertebral column phenotype at a morphological, cellular and subcellular level. We present the first description of vertebral compression fractures in Chi/+ and assess the effects of LP diet on the Chi/+ phenotype (Chi/+LP). Compared to untreated Chi/+, two months of LP dietary treatment decreases vertebral deformities in the abdominal region and reduces shape variation of caudal vertebral bodies to a condition more similar to wild type (WT). At the histological level, the osteoid layer, covering the bone at the vertebral body endplates in WT zebrafish, is absent in Chi/+, but it is partially restored with the LP diet. Whole mount-stained specimens and histological sections show various stages of vertebral compression fractures in Chi/+ and Chi/+LP animals. Both Chi/+ and Chi/+LP show abundant osteoclast activity compared to WT. Finally, the ultrastructure analysis of WT, Chi/+ and Chi/+LP shows Chi/+ and Chi/+LP osteoblasts with enlarged endoplasmic reticulum cisternae and a high protein content, consistent with intracellular retention of mutated collagen. Nevertheless, the secreted collagen in Chi/+LP appears better organised concerning fibre periodicity compared to Chi/+. Our findings suggest that a reduced mineral content of Chi/+ bone could explain the lower frequency of vertebral column deformities and the restored shape of the vertebral bodies in Chi/+LP animals. This, together with the improved quality of the bone extracellular matrix, suggests that two months of reduced dietary P intake can alleviate the severe bone phenotype in Chi/+ zebrafish.

Cells at the Edge: The Dentin-Bone Interface in Zebrafish Teeth.

Bone-producing osteoblasts and dentin-producing odontoblasts are closely related cell types, a result from their shared evolutionary history in the ancient dermal skeleton. In mammals, the two cell types can be distinguished based on histological characters and the cells' position in the pulp cavity or in the tripartite periodontal complex. Different from mammals, teleost fish feature a broad diversity in tooth attachment modes, ranging from fibrous attachment to firm ankylosis to the underlying bone. The connection between dentin and jaw bone is often mediated by a collar of mineralized tissue, a part of the dental unit that has been termed "bone of attachment". Its nature (bone, dentin, or an intermediate tissue type) is still debated. Likewise, there is a debate about the nature of the cells secreting this tissue: osteoblasts, odontoblasts, or yet another (intermediate) type of scleroblast. Here, we use expression of the P/Q rich secretory calcium-binding phosphoprotein 5 (scpp5) to characterize the cells lining the so-called bone of attachment in the zebrafish dentition. scpp5 is expressed in late cytodifferentiation stage odontoblasts but not in the cells depositing the "bone of attachment". nor in bona fide osteoblasts lining the supporting pharyngeal jaw bone. Together with the presence of the osteoblast marker Zns-5, and the absence of covering epithelium, this links the cells depositing the "bone of attachment" to osteoblasts rather than to odontoblasts. The presence of dentinal tubule-like cell extensions and the near absence of osteocytes, nevertheless distinguishes the "bone of attachment" from true bone. These results suggest that the "bone of attachment" in zebrafish has characters intermediate between bone and dentin, and, as a tissue, is better termed "dentinous bone". In other teleosts, the tissue may adopt different properties. The data furthermore support the view that these two tissues are part of a continuum of mineralized tissues. Expression of scpp5 can be a valuable tool to investigate how differentiation pathways diverge between osteoblasts and odontoblasts in teleost models and help resolving the evolutionary history of tooth attachment structures in actinopterygians.

Seadragon genome analysis provides insights into its phenotype and sex determination locus.

The iconic phenotype of seadragons includes leaf-like appendages, a toothless tubular mouth, and male pregnancy involving incubation of fertilized eggs on an open "brood patch." We de novo-sequenced male and female genomes of the common seadragon (Phyllopteryx taeniolatus) and its closely related species, the alligator pipefish (Syngnathoides biaculeatus). Transcription profiles from an evolutionary novelty, the leaf-like appendages, show that a set of genes typically involved in fin development have been co-opted as well as an enrichment of transcripts for potential tissue repair and immune defense genes. The zebrafish mutants for scpp5, which is lost in all syngnathids, were found to lack or have deformed pharyngeal teeth, supporting the hypothesis that the loss of scpp5 has contributed to the loss of teeth in syngnathids. A putative sex-determining locus encoding a male-specific amhr2y gene shared by common seadragon and alligator pipefish was identified.

Morphological and histological characterization of an ectopically mineralized structure in a gilthead sea bream Sparus aurata with opercular deformation.

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro-CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix-rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair.

Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish.

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.

Bone resorption and body reorganization during maturation induce maternal transfer of toxic metals in anguillid eels.

During their once-in-a-lifetime transoceanic spawning migration, anguillid eels do not feed, instead rely on energy stores to fuel the demands of locomotion and reproduction while they reorganize their bodies by depleting body reserves and building up gonadal tissue. Here we show how the European eel (Anguilla anguilla) breaks down its skeleton to redistribute phosphorus and calcium from hard to soft tissues during its sexual development. Using multiple analytical and imaging techniques, we characterize the spatial and temporal degradation of the skeletal framework from initial to final gonadal maturation and use elemental mass ratios in bone, muscle, liver, and gonadal tissue to determine the fluxes and fates of selected minerals and metals in the eels' bodies. We find that bone loss is more pronounced in females than in males and eventually may reach a point at which the mechanical stability of the skeleton is challenged. P and Ca are released and translocated from skeletal tissues to muscle and gonads, leaving both elements in constant proportion in remaining bone structures. The depletion of internal stores from hard and soft tissues during maturation-induced body reorganization is accompanied by the recirculation, translocation, and maternal transfer of potentially toxic metals from bone and muscle to the ovaries in gravid females, which may have direct deleterious effects on health and hinder the reproductive success of individuals of this critically endangered species.

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 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.

Segmentation pattern of zebrafish caudal fin is affected by developmental temperature and defined by multiple fusions between segments.

Caudal-fin lepidotrichia is composed of numerous segments, which are linked to each other by intersegmental joints. During fish growth, lepidotrichia elongate by the addition of new segments at their distal margin, whereas the length of each segment remains constant after it is formed. In the present paper, we examined whether the water temperature affects the segmentation pattern of the juvenile and adult caudal fin. For this purpose, zebrafish (Danio rerio) embryos and larvae were exposed to three different temperature conditions (24°C, 28°C, and 32°C) from the pharyngula stage (1 day postfertilization [dpf]) to metamorphosis, whereas the control temperature (28°C) was applied to all the groups before and after this period. Results demonstrated that water temperature had a significant effect on the length of the segments of each lepidotrichium, at both the juvenile and adult stages. Moreover, at higher temperatures, there was a significant proximal shift of the position of the first bifurcation of the second lepidotrichium of the dorsal lobe. At all the experimental conditions, the length of proximal segment was not constant during fish growth, but it followed a discontinuous saltatory growth. Histological analysis of the proximal lepidotrichia segments revealed that the observed apparent growth of segments is the result of fusions between segments. Fusion occurs not by mineralization of the intersegmental joints, but by bone deposition around the joints.

Ultrastructural morphology of the envelope of Dover sole Solea solea eggs from fertilization until hatching with emphasis on sample preparation.

This study is the first to describe the ultrastructural morphology of the envelope of Solea solea eggs from fertilisation until hatching. Defining the ultrastructural morphology of fish eggs is important for species identification and may assist in predicting the effect of external influences on these early life stages. In first instance, various fixation and embedding protocols were assessed to explore the morphology of the egg envelope, whereby the encountered difficulties were highlighted. The successful protocol for SEM proved to be combined fixation with 4% glutaraldehyde in 0.1M cacodylate buffer for minimum 4h with post-fixation of 2h with 1% OsO4 in 0.1M cacodylate buffer. For TEM, puncturing the egg envelope during the first steps of the fixation protocol was necessary to allow the embedding medium to penetrate through the egg envelope. Based on both scanning and transmission electron microscopical examination, three distinct layers were discerned in the egg envelope. During the development of the fish embryo, a change in the outer structure of the egg was observed. Scanning electron microscopical examination of one day post-fertilisation eggs (DPF) revealed a homogeneous outer layer, displaying a large number of pores uniformly distributed on the surface of the egg envelope. Starting from 2 DPF parts of the outermost layer or two outer layers peeled off. The second deeper layer showed larger pores, with less defined edges. In the third innermost layer irregular indentations were noted. On transmission electron microscopy the first outermost layer of 1 DPF eggs clearly folded into the pores. The second layer was more electron dense, had a uniform appearance and did not cover the surface of the pores. The third innermost layer was much thicker and possessed indentations. A total number of 12 undulating zones were discriminated based on different degrees of electron density. Prior to hatching, the compact structure of the innermost layer was distorted by dispersed holes and tears.

A vertebrate-specific and essential role for osterix in osteogenesis revealed by gene knockout in the teleost medaka.

osterix (osx; sp7) encodes a zinc-finger transcription factor that controls osteoblast differentiation in mammals. Although identified in all vertebrate lineages, its role in non-mammalian bone formation remains elusive. Here, we show that an osx mutation in medaka results in severe bone defects and larval lethality. Pre-osteoblasts fail to differentiate leading to severe intramembranous and perichondral ossification defects. The notochord sheath mineralizes normally, supporting the idea of an osteoblast-independent mechanism for teleost vertebral centra formation. This study establishes a key role for Osx for bone formation in a non-mammalian species, and reveals conserved and non-conserved features in vertebrate bone formation.

Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome.

Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease osteogenesis imperfecta (OI). BS is caused by biallelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs crosslinking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and crosslinking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. © 2016 American Society for Bone and Mineral Research.

Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model.

Osteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl) under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs.

An adult osteopetrosis model in medaka reveals the importance of osteoclast function for bone remodeling in teleost fish.

Osteoclasts play important roles during bone growth and in maintaining bone health and bone homeostasis. Dysfunction or lack of osteoclasts leads to increased bone mass and osteopetrosis phenotypes in mouse and human. Here we report a severe osteopetrosis-like phenotype in transgenic medaka fish, in which membrane bound EGFP (mEGFP) was expressed in osteoclasts under control of the cathepsin K promoter (ctsk:mEGFP). In contrast to reporter lines with GFP expression in the cytoplasm of osteoclasts, adult fish of the mEGFP line developed bone defects indicative for an osteoclast dysfunction. Activity of tartrate-resistant acid phosphatase (TRAP) was down-regulated and excess bone was observed in most parts of the skeleton. The osteopetrotic phenotype was particularly obvious at the neural and haemal arches that failed to increase their volume in growing fish. Excess bone caused severe constriction of the spinal cord and the ventral aorta. The continuation of tooth development and the failure to shed teeth resulted in severe hyperdontia. Interestingly, at the vertebral column vertebral body arches displayed a severe osteopetrosis, while vertebral centra had no or only a mild osteopetrotic phenotype. This confirms previous reports from cichlids that, different from the arches, allometric growth of fish vertebral centra initially does not depend on the action of osteoclasts. Independent developmental mechanism that shapes arches and vertebral centra can also lend support to the hypothesis that vertebral centra and arches function as independent developmental modules. Together, this medaka osteopetrosis model confirms the importance of proper osteoclast function during normal skeletal development in teleost fish that requires bone modeling and remodeling.

The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish.

During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton.

Power and challenges of using zebrafish as a model for skeletal tissue imaging.

The zebrafish (Danio rerio) is now a widely used model organism in biomedical research. The species is also increasingly used for studying skeletal development and regeneration and for understanding human skeletal diseases. The small size of this model organism is an advantage and an extreme challenge for visualizing and diagnosing the animals' skeleton. This applies especially to early stages of skeletal development. Similar challenges arise for the analysis of the skeleton of other small fish species, such as medaka (Oryzias latipes). High quality histological preparations and knowledge about the special quality of the zebrafish skeleton remain prerequisites for a correct analysis. In addition, new methods for fast and high-resolution 2D and 3D skeletal tissue screening are required for a maximal understanding of skeletal development. We, in this study, review advantages and limitations of adapting current visualization techniques for zebrafish skeletal research. We discuss the methods for in toto visualization, such as X-raying, micro-CT, Alizarin red staining and optical projection tomography. Techniques for in vivo imaging, such as second harmonic generation microscopy and two-photon excitation fluorescence, are also discussed. Finally, we explore the possibilities of light-sheet microscopy for the analysis of the zebrafish skeleton.

Distinct patterns of notochord mineralization in zebrafish coincide with the localization of Osteocalcin isoform 1 during early vertebral centra formation.

BACKGROUND: In chondrichthyans, basal osteichthyans and tetrapods, vertebral bodies have cartilaginous anlagen that subsequently mineralize (chondrichthyans) or ossify (osteichthyans). Chondrocytes that form the vertebral centra derive from somites. In teleost fish, vertebral centrum formation starts in the absence of cartilage, through direct mineralization of the notochord sheath. In a second step, the notochord is surrounded by somite-derived intramembranous bone. In several small teleost species, including zebrafish (Danio rerio), even haemal and neural arches form directly as intramembranous bone and only modified caudalmost arches remain cartilaginous. This study compares initial patterns of mineralization in different regions of the vertebral column in zebrafish. We ask if the absence or presence of cartilaginous arches influences the pattern of notochord sheath mineralization. RESULTS: To reveal which cells are involved in mineralization of the notochord sheath we identify proliferating cells, we trace mineralization on the histological level and we analyze cell ultrastructure by TEM. Moreover, we localize proteins and genes that are typically expressed by skeletogenic cells such as Collagen type II, Alkaline phosphatase (ALP) and Osteocalcin (Oc). Mineralization of abdominal and caudal vertebrae starts with a complete ring within the notochord sheath and prior to the formation of the bony arches. In contrast, notochord mineralization of caudal fin centra starts with a broad ventral mineral deposition, associated with the bases of the modified cartilaginous arches. Similar, arch-related, patterns of mineralization occur in teleosts that maintain cartilaginous arches throughout the spine.Throughout the entire vertebral column, we were able to co-localize ALP-positive signal with chordacentrum mineralization sites, as well as Collagen II and Oc protein accumulation in the mineralizing notochord sheath. In the caudal fin region, ALP and Oc signals were clearly produced both by the notochord epithelium and cells outside the notochord, the cartilaginous arches. Based on immunostaining, real time PCR and oc2:gfp transgenic fish, we identify Oc in the mineralizing notochord sheath as osteocalcin isoform 1 (Oc1). CONCLUSIONS: If notochord mineralization occurs prior to arch formation, mineralization of the notochord sheath is ring-shaped. If notochord mineralization occurs after cartilaginous arch formation, mineralization of the notochord sheath starts at the insertion point of the arches, with a basiventral origin. The presence of ALP and Oc1, not only in cells outside the notochord, but also in the notochord epithelium, suggests an active role of the notochord in the mineralization process. The same may apply to Col II-positive chondrocytes of the caudalmost haemal arches that show ALP activity and Oc1 accumulation, since these chondrocytes do not mineralize their own cartilage matrix. Even without cartilaginous preformed vertebral centra, the cartilaginous arches may have an inductive role in vertebral centrum formation, possibly contributing to the distinct mineralization patterns of zebrafish vertebral column and caudal fin vertebral fusion.