Transgenic Assay of Osteoclast-Specific Acp5b Gene Promoter and Observing the Distribution of Osteoclasts

Bone remodeling will occur to rebuild and repair bone when it encounter mechanical stress or hormonal influence. The procedures of bone remodeling involve bone destruction, absorption, and bone regeneration. During bone remodeling, osteoblasts on the bone surface will release some signal factors for promoting osteoclasts to migrate to the site where needed to be re-built, and forming mature multinucleated osteoclasts. Matured osteoclasts dissolve bone by secreting acid and enzymes that degrade the organic bone matrix in this acidic environment. Before osteoclasts break down, osteoblasts replace them when receiving signals and produce new bon matrix. So the coupling between osteoclasts and osteoblasts play a cruial role in the process of bone resorption and formation. For the purpose of degrading bone matrix, absorbing osteoclasts release some enzymes like tartrate resistant acid phosphatase (TRAP), cysteine proteases (for example, cathepsin K) and so on. Among them, TRAP, abundant in osteoclasts, can be used as an osteoclast marker. My research goal is to do transgenic assay of osteoclast-specific Acp5b (= TRAP) gene promoter, using red and green fluorescent proteins as reporter genes to label osteoclasts and to track their development. To this aim, I use zebrafish as a model animal and make a construct harboring 1.3kb zebrafish Acp5b promoter and red- and green-fluorescent protein reporter genes to label osteoclasts. Then, these constructs were microinjected into one-cell stage of zebrafish embryos. The expressions of EGFP and DsRed were observed. My results reveal that the fluorescence signals are visible in zebrafish eye and around the bones in transient assays (F0). Then I screened F0 and obtained F1 stable lines. I got 17 stable lines in which DsRed can be observed in the eyes. However, only two lines also express DsRed reporter gene in osteoclasts. I found that DsRed-labelled osteoclasts were broadly distributed in the skull, gill cover, pectoral fin, tail fin, dorsal fin, anal fin and upper and lower edges of dorsal and ventral spines. In addition, to monitor the expression of Acp5b gene in osteoclasts during fin regeneration, I employed fin amputation-regeneration measures in the stable zebrafish line F1 (No. 7 and 16, about 2 month dpf). However, fluorescent signals were not detected in osteoclasts after amputation. Presumably, the enhancers to control TRAP expression in zebrafish osteoclasts during tail fin regeneration may be located in another TRAP gene, Acp5a. My experiments record the distribution of zebrafish osteoclasts in early developmental stages. In the future, we can apply cytotoxicity using a bacterial nitroreductase to convert a prodrug, Metronidazole, into a cytotoxic metabolite to ablate osteoclasts and observe the bone morphology in the absence of osteoclasts during zebrafish bone growth. By this way, we can evaluate the cell function of osteoclast during bone growth and regeneration