The generation of new bone requires activation of specific signaling cascades in a temporally and spatially distinct manner. However, until recently, a direct comparison between the dynamics of these signaling events and the formation of mineralizing bone has been precluded by the lack of strategies to simultaneously visualize these relationships in vivo. The optical clarity of the zebrafish caudal fin allows for high-content imaging of cell signaling in real time using fluorescent reporter strains. Previously, we demonstrated the potential to measure crystalline mineral accrual during fin regeneration using quantitative birefringence imaging (Rotopol microscopy [1]). Building on these findings, in this study, we developed a high-content, multi-modal system for tandem imaging of fluorescent transgenic reporters and bone mineralization within the same tissue. The system consisted of a motorized rotating polarizer integrated into a fully motorized Zeiss Axio Imager.M2 high-content fluorescence microscope, with custom software enabling interfacing between systems. We applied this imaging strategy to directly examine the relationships between canonical Wnt signaling and sp7 (osterix) expression (using the Tg(7xTCF-Xla.Siam:GFP)ia4 and Tg(sp7:EGFP) reporter fish, respectively) with changes in cell metabolism (indicated by NADH autofluorescence) and mineralization (via Rotopol acquired birefringence) during bone formation and maturation in the regenerating zebrafish fin.
A custom mapping approach was developed to register images from different imaging days onto one another by creating landmarks of fixed pixel distances along each ray. Mapped images permitted the analysis of signaling events at any location in the regenerate, even before bony tissue was detectable. During the initial outgrowth phase, we find that acute activation of NADH is followed by Wnt signaling and subsequent expression of sp7. Interestingly, step-wise increases in mineralization coincide with second and third peaks in NADH autofluorescence. Furthermore, we find that mineralization is not complete even at 26 dpa in some fish. Using Rotopol imaging in concert with high resolution fluorescent imaging for the first time, we demonstrate that this system can be used to examine relationships between core bone formation events and maturation into fully mineralized bone.
[1] Recidoro AM, et al., Kwon RY. JBMR, 2014.