Collagen10a1 GEN EXPRESSION AND BONE MINERALIZATION IN THE col10a1:nlGFP TRANSGENTIC MEDAKA FISH LARVAE

Abstract

Objective: To assess the expression level of the osteoblast-specific collagen10a1 gene and the degree of bone mineralization during the early larval stages of col10a1:nlGFP transgenic medaka fish to identify a specific developmental time window suitable for conducting bone anabolic effect evaluations of bioactive substances. Methods: Homozygous and hemizygous col10a1:nlGFP fish larval groups at 7, 9, 10, 11, and 14 days post fertilization (dpf) were used in the study (n=…/group). The fish were subjected to live staining with alizarin red S (ARS) dye to visualize their mineralized bone structures. Fluorescent images were captured using a fluorescent stereoscope to document both the GFP and ARS signals in the fish. The density of the GFP signal was utilized to assess the expression level of the collagen10a1 gene in osteoblasts, while the density of the ARS signal was used to evaluate the extent of bone mineralization. ImageJ software was employed for this analysis. Results: The intensity of GFP signal exhibited an upward trend as the fish grew from 7 to 9 dpf, followed by a gradual decline from 9 to 14 dpf. Along with that, the intensity of ARS signal increased as the fish grew from 7 to 10 dpf, followed by a gradual decrease from 10 to 14 dpf. These findings indicate that the optimal developmental window for conducting tests on the fish with bioactive compounds, utilizing a stereo fluorescence microscope, is between 7 and 9 dpf.