Epidermal regulation of bone morphogenesis through the development and regeneration of osteoblasts in the zebrafish scale

Bone is a connective tissue composed of many cell types, including osteoblasts. How bones acquire their unique size and shape during development remains poorly understood. Herein we investigated the molecular and cellular mechanisms of bone morphogenesis in the zebrafish scale by using transgenic lines to enable visualization of specific types of osteoblasts. We demonstrate that the zebrafish scale contains three distinct types of osteoblasts: (i) a monolayer of central osteoblasts along the inner surface of scales; (ii) marginal osteoblasts elongated along the scale edge; and (iii) submarginal osteoblasts located between the central and marginal osteoblast populations. The size of the central osteoblasts increases progressively during development, suggesting that scale growth is mediated primarily by cell growth rather than the recruitment of new osteoblasts. In addition, the total number of central osteoblasts increases in regenerated scales and is correlated with scale size, possibly allowing for the rapid growth of regenerating scales. Moreover, osteoblast proliferation is not detected during regeneration, suggesting that the osteoblasts originate from post-mitotic precursor cells. Sonic hedgehog a (shha) is expressed in the epidermal cells that make contact with the marginal osteoblasts. Pharmacological inhibition of Hedgehog (Hh) signaling during regeneration reduces the number of marginal osteoblasts and interferes with scale growth, indicating that epidermis-derived Shh regulates scale regeneration. Finally, genetic inhibition of Wnt/planar cell polarity (PCP) signaling in the epidermis results in misorientation of scales with regard to the body axis. These results reveal a novel role for the epidermis in the regulation of bone patterning, namely the regeneration of osteoblasts and directional bone growth.