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.

Conditional ablation of osteoblasts in medaka.

Different from tetrapods, teleost vertebral centra form without prior establishment of a cartilaginous scaffold, in two steps: First, mineralization of the notochord sheath establishes the vertebral centra. Second, sclerotome derived mesenchymal cells migrate around the notochord sheath. These cells differentiate into osteoblasts and deposit bone onto the mineralized notochord sheath in a process of intramembranous bone formation. In contrast, most skeletal elements of the cranial skeleton arise by chondral bone formation, with remarkably similar mechanisms in fish and tetrapods. To further investigate the role of osteoblasts during formation of the cranial and axial skeleton, we generated a transgenic osx:CFP-NTR medaka line which enables conditional ablation of osterix expressing osteoblasts. By expressing a bacterial nitroreductase (NTR) fused to Cyan Fluorescent Protein (CFP) under control of the osterix promoter these cells become sensitive towards Metronidazole (Mtz). Mtz treatment of stable osx:CFP-NTR transgenic medaka for several consecutive days led to significant loss of osteoblasts by apoptosis. Live staining of mineralized bone matrix revealed reduced ossification in head skeletal elements such as cleithrum and operculum, as well as in the vertebral arches. Interestingly in Mtz treated larvae, intervertebral spaces were missing and the notochord sheath was often continuously mineralized resulting in the fusion of centra. We therefore propose a dual role for osx-positive osteoblasts in fish. Besides a role in bone deposition, we suggest an additional border function during mineralization of the chordal centra. After termination of Mtz treatment, osteoblasts gradually reappeared, indicating regenerative properties in this cell lineage. Taken together, the osx:CFP-NTR medaka line represents a valuable tool to study osteoblast function and regeneration at different stages of development in whole vertebrate specimens in vivo.

Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model.

Osteoclasts are macrophage-related bone resorbing cells of hematopoietic origin. Factors that regulate osteoclastogenesis are of great interest for investigating the pathology and treatment of bone diseases such as osteoporosis. In mammals, receptor activator of NF-κB ligand (Rankl) is a regulator of osteoclast formation and activation: its misexpression causes osteoclast stimulation and osteoporotic bone loss. Here, we report an osteoporotic phenotype that is induced by overexpression of Rankl in the medaka model. We generated transgenic medaka lines that express GFP under control of the cathepsin K promoter in osteoclasts starting at 12 days post-fertilization (dpf), or Rankl together with CFP under control of a bi-directional heat-shock promoter. Using long-term confocal time-lapse imaging of double and triple transgenic larvae, we monitored in vivo formation and activation of osteoclasts, as well as their interaction with osteoblasts. Upon Rankl induction, GFP-positive osteoclasts are first observed in the intervertebral regions and then quickly migrate to the surface of mineralized neural and haemal arches, as well as to the centra of the vertebral bodies. These osteoclasts are TRAP (tartrate-resistant acid phosphatase) and cathepsin K positive, mononuclear and highly mobile with dynamically extending protrusions. They are exclusively found in tight contact with mineralized matrix. Rankl-induced osteoclast formation resulted in severe degradation of the mineralized matrix in vertebral bodies and arches. In conclusion, our in vivo imaging approach confirms a conserved role of Rankl in osteoclastogenesis in teleost fish and provides new insight into the cellular interactions during bone resorption in an animal model that is useful for genetic and chemical screening.

Combined in vivo imaging and omics approaches reveal metabolism of icaritin and its glycosides in zebrafish larvae.

Flavonoids isolated from Herba Epimedii such as icaritin, icariin and epimedin C have been suggested as potential bone anabolic compounds. However, the "specific localized effects" of these flavonoids in bone, in vivo, and the metabolism of these flavonoids in zebrafish larvae have never been demonstrated. In this study, we used multiple methods including in vivo imaging, drug metabolites profiling, transcriptomic and proteomic approaches to determine the mechanisms involved in the distribution and metabolism of the flavonoids in zebrafish larvae by measuring the fluorescence emission, in vivo, of icaritin and its glycoside derivatives. The fluorescence emission mechanism of icaritin in vitro was identified by spectrophotometric analysis, and the fluorescent property of icaritin was used as a probe to visualize the metabolism and distribution of icaritin and its glycoside derivatives in zebrafish larvae. Phase I and phase II metabolism of icaritin and its derivatives were identified in zebrafish by mass spectrometry. The combined transcriptomics and proteomics demonstrate a high degree of conservation of phase I and phase II drug metabolic enzymes between zebrafish larvae and mammals. Icaritin and its glycoside derivatives were demonstrated using combined approaches of in vivo imaging, drug metabolites identification, and transcriptomic and proteomic profiling to illustrate phase I and phase II metabolism of the flavonoids and their distribution in bone of zebrafish larvae. This study provides a new methodological model for use of the zebrafish larvae to examine drug metabolism.

Osterix-mCherry transgenic medaka for in vivo imaging of bone formation.

Intramembranous and chondral bone formation by osteoblasts is found in all vertebrates. The genetic network controlling osteoblast differentiation is highly conserved and regulated by a small number of key factors, including the zinc-finger transcription factor Osterix. Expression analysis of osterix in the teleost model medaka revealed a highly restricted expression in skeletal regions. For in vivo imaging, we generated transgenic medaka expressing mCherry under control of the osterix promoter. We show that the transgene becomes expressed in early osteoblasts, which have not yet mineralized bone matrix, and remains high in matured and mineralizing osteoblasts. Life imaging of transgenic larvae provided insight into the appearance and behavior of early osteoblasts during development of the teleost cranium, vertebrae, and caudal fin. In summary, osterix-mCherry transgenic medaka enable us to analyze osteoblasts during different maturation phases in vivo and represent a unique tool to study osteoblast behavior in vertebrate embryos and adults.

Dynamic expression of sparc precedes formation of skeletal elements in the Medaka (Oryzias latipes).

Sparc is a secreted calcium-binding glycoprotein that regulates mineralization of bone tissues in mammals. In other vertebrates, its function remains largely unclear. Here, we describe the isolation, genomic organization and expression of the sparc gene in the teleost Medaka (Oryzias latipes), an established vertebrate model for developmental studies. During earliest stages of Medaka embryogenesis, sparc is expressed in the sclerotome compartment of the somites that gives rise to precursor cells of the axial skeleton. Importantly, in this area its expression precedes that of twist-1, which is a crucial regulator of osteoblast formation. Dynamic expression is also found in the floor plate of the neural tube and the notochord. Both structures are passed by migrating skeletal precursors shortly before they differentiate and form the vertebrae. In general, sparc is expressed before the formation and mineralization of bone elements and expression of bone markers like collagen type 1a in the fins and axial skeleton of Medaka embryos. It is also expressed in several non-skeletal tissues of embryos and adult fish, suggesting possible other functions not related to bone mineralization. Taken together, the Medaka sparc gene represents an excellent marker for early sclerotome development. Its restricted and highly dynamic expression suggests a novel function during migration of sclerotome cells and their differentiation into early vertebrae. This marker thus allows the analysis of early skeletal development and formation of extracellular bone matrix in this vertebrate model.