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

Abstract

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.

Fluorescence-activated cell sorting (FACS) of osteoblasts and osteoclasts for RNA sequencing in a medaka, Oryzias latipes (Temming & Schlegel, 1846), osteoporosis model

“…At 11 dpf, no osteoclasts have formed in the vertebral column of medaka. Hence, ctsk :nuGFP transgenic larvae show no GFP expression in the trunk (Figure d; To et al., ; Yu et al., ). These reporter lines were then crossed to fish expressing RANKL under control of a heat‐shock promoter ( RANKL :HSE:CFP; see also To et al., ).…”

“…Osteoporosis model in medaka (see also To et al., ; Yu et al., ). (a) Schematic diagram showing transgenic markers expressed during different stages of osteoblast (top) and osteoclast differentiation (bottom).…”

“…Mineralized matrix is absent from arches and large lesions are present in centra. (h) col10a1 :nu GFP positive osteoblast progenitors accumulate around lesion sites, differentiate into osx ‐positive osteoblasts (see Renn et al., ) and contribute to remineralization of centra and arches (see Yu et al., ). Scale bars: 0.05 mm…”

“…At this stage, the notochordal sheath is fully mineralized and ossification has started around the notochord, and along the hemal and neural arches (Renn et al, 2013). At the same stage, osx:mCherry larvae express mCherry in premature and mature osteoblasts of the cranial and axial skeleton (Figure 1c; Renn & Winkler, 2009 Figure 1d; To et al, 2012;Yu et al, 2016). These reporter lines were then crossed to fish expressing RANKL under control of a heat-shock promoter (RANKL:HSE:CFP; see also To et al, 2012).…”

“…Inducible expression of RANKL in these lines leads to ectopic osteoclast formation and osteoporosis‐like defects (To et al., ). This triggers a rapid repair response by recruiting osteoblast progenitor cells to the lesion sites (Renn et al., ; Yu et al., ). Understanding the transcriptional profiles of distinct bone cells during the different phases of bone resorption and repair will give important insight into the mechanisms of osteoblast recruitment and activation, the cross‐talk between osteoblasts and osteoclasts, and ultimately the molecular basis of bone homeostasis.…”