Conditional ablation of osteoblasts in medaka

Abstract

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.

Building the backbone: the development and evolution of vertebral patterning

“…When sp7 + cells are genetically ablated in medaka, segmented chordacentra first develop normally, but then fuse at later stages to form an unsegmented rod of bone. Additionally, these fish do not develop vertebral arches, an observation also consistent with an exclusive role for the sclerotome in arch and perichordal centrum formation (Willems et al, 2012).…”

Transcriptome sequencing of Atlantic salmon (Salmo salar L.) notochord prior to development of the vertebrae provides clues to regulation of positional fate, chordoblast lineage and mineralisation

“…Indeed, to ensure an uninterrupted development of the vertebrae, these cells need to be in place before the chordacentrae form. Moreover, in zebrafish, when the osteoblasts surrounding the notochord are abolished, the initial development of chordacentrae proceeds normally, but they continue to grow, so that the spaces separating adjacent centrae become reduced, in some cases finally leading to partial fusion [8]. Hence, osteoblasts that are to form perichordal bone may provide signalling factors that inhibit the notochord epithelium in its secretion of molecules that promote mineralisation of the sheath, arresting further growth of the chordacentrae, and, by extension, defining a boundry with non-mineralised segments that correspond to where prospective intervertebral ligaments are to develop.…”

“…Segmentation and subsequent mineralisation of the notochord comprise the initial morphogenic process in formation of the vertebral column in Atlantic salmon ( Salmo salar L.) [6] and in zebrafish ( Danio rerio ) [2,7]. Segmented mineralisation of the notochord determines the localization of vertebral bodies, and notochord mineralisation is thereby crucial for normal formation of the vertebrae, as the functional study by Willems et al [8] has demonstrated. Furthermore, pathological processes that disrupt notochord mineralisation may lead to malformation of vertebrae [9].…”

Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

“…Species names, dye concentrations, duration of immersion, wash steps, and literature references are indicatedAlizarin compoundConcentrationTime of immersion & washingSpeciesReferencein vivoARS0.003 %2–3 h / rinsing Danio rerio DeLaurier et al 2010 [53]ALC0.005 %O/N / rinsing Oryzias latipes Renn et al 2013 [54]ALC0.005 %n.d. / rinsing Oryzias latipes Inohaya et al 2007 [55]ARS0.005 % + HEPESLarvae 1–2 h; juvenile ON / rinsing Danio rerio Kimmel et al 2010 [56]ALC0.010 %2 h / rinsing Oryzias latipes Willems et al 2012 [57]ALC0.010 %2 h-4 h / 2 h-ON Oryzias latipes To et al 2012 [58]ARS0.020 %10 min / rinsing Danio rerio Tu and Johnson 2011 [38]ALC0.025 %n.d. Danio rerio & Oryzias latipes Chatani et al 2011 [59]ARS0.025 %24 h / rinsed Poecilia reticulata Bashey 2004 [29]ARS0.040 %n.d. / rinsing 10 min Danio rerio Recidoro et al 2014 [60]ARS0.050 %5 min / rinsing Danio rerio Huitema et al 2012 [61]n.d.0.050 %n.d. Danio rerio Fleming et al 2004 [62]n.d.0.300 %n.d. Danio rerio Eames et al 2010 [63]n.d.n.d.n.d. Danio rerio Yan et al 2005 [64]Post mortemALC0.0025 %*6 h / n.d. Theragra chalcogramma Dougherty 2008 [65]ALC0.003 %24 h / n.d. Acanthopagrus butcheri Partridge et al 2009 [66]…”