In vivo imaging of bone collagen dynamics in zebrafish

Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone collagen formation, particularly the principles governing its orientation, remain unknown due to the lack of a method enabling continuous in vivo observation. To address this challenge, we constructed a method to visualize bone collagen by tagging with GFP in zebrafish and observed the interactions between the osteoblasts and collagen fiber during bone formation in vivo. When Col1a2-GFP was expressed under the control of the osteoblast-specific promoters osx or osc in zebrafish, bone collagen could be observed clearly enough to identify their localization, but collagen from other organs did not. Therefore, we determined that this method was of sufficient quality for detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin ray, and opercular bones was observed in detail in zebrafish, when bone formation is more active. High magnification imaging showed Col1a2-GFP can visualize collagen enough for analyzing collagen fiber orientation and microstructure of bones.Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of expression promoter. Both promoters (osx and osc) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within bone are exclusive, with osx region localizing mainly the outer edge of bone and osc region localizing the central area of bone. This suggests the existence of distinct subpopulations of osteoblasts with various gene expression profiles, each of which may play a unique role in the osteogenic process.These findings would contribute to a better understanding of the mechanisms governing bone collagen formation by osteoblasts.