Osteoclasts in bone modeling, as revealed by in vivo imaging, are essential for organogenesis in fish

Abstract

Bone modeling is the central system controlling the formation of bone including bone growth and shape in early development, in which bone is continuously resorbed by osteoclasts and formed by osteoblasts. However, this system has not been well documented, because it is difficult to trace osteoclasts and osteoblasts in vivo during development. Here we showed the important role of osteoclasts in organogenesis by establishing osteoclast-specific transgenic medaka lines and by using a zebrafish osteoclast-deficient line. Using in vivo imaging of osteoclasts in the transgenic medaka carrying an enhanced GFP (EGFP) or DsRed reporter gene driven by the medaka TRAP (Tartrate-Resistant Acid Phosphatase) or Cathepsin K promoter, respectively, we examined the maturation and migration of osteoclasts. Our results showed that mononuclear or multinucleated osteoclasts in the vertebral body were specifically localized at the inside of the neural and hemal arches, but not at the vertebral centrum. Furthermore, transmission electron microscopic (TEM) analyses revealed that osteoclasts were flat-shaped multinucleated cells, suggesting that osteoclasts initially differentiate from TRAP-positive mononuclear cells residing around bone. The zebrafish panther mutant lacks a functional c-fms (receptor for macrophage colony-stimulating factor) gene crucial for osteoclast proliferation and differentiation and thus has a low number of osteoclasts. Analysis of this mutant revealed deformities in both its neural and hemal arches, which resulted in abnormal development of the neural tube and blood vessels located inside these arches. Our results provide the first demonstration that bone resorption during bone modeling is essential for proper development of neural and vascular systems associated with fish vertebrae.