The role of miR-199a-3p in a benzo[a]pyrene-induced cross-generational bone phenotype in Japanese medaka ( <i>Oryzias latipes</i> )

Benzo[a]pyrene (BaP), a ubiquitous environmental pollutant and known carcinogen, has been shown to disrupt transgenerational bone development via epigenetic mechanisms, including microRNAs (miRNAs). Among these, miR-199a-3p has emerged as a quintessential regulator in transcriptomic analyses of bone tissue from ancestrally BaP-exposed F3 generation medaka fish, with significant enrichment in bone formation and oxidative stress. To investigate the functional role of miR-199a-3p in osteogenesis, wild-type and transgenic Japanese medaka (Oryzias latipes) lines (twist: dsRed/col10a1: GFP and col10a1: GFP/osx: mCherry) were microinjected with 700 pg of miR-199a-3p agonists or antagonists at 3-days post-fertilization (dpf) and assessed through 30 dpf for changes in vertebral calcification, cellular differentiation, and gene expression. In parallel, parental (F0) twist: dsRed/col10a1: GFP adults were exposed to 1 μg/L BaP for 21 days, and vertebral compression and osteoblast subpopulations were evaluated in their F1 progeny. Alizarin red staining revealed that agonist treatment resulted in significantly reduced calcified area and bone thickness, while injection of the antagonist increased both parameters between 24 and 30 dpf. Fluorescent imaging and quantification indicated reduced spatial distribution and cellular density of twist1 + mesenchymal-derived cells and col10a1 + osteochondroprogenitors in agonist-treated fish. qPCR profiling at 5 and 25 dpf suggests that miR-199a-3p may modulate the expression of multiple osteogenesis-related genes, evidencing its epigenetic regulatory role. These results introduce miR-199a-3p as a critical modulator of osteoblast lineage specification, bone mineralization, and vertebral maturation in medaka. This study demonstrated the developmental importance of miRNA-mediated regulation during critical windows for skeletal formation and suggests that miR-199a-3p levels could serve as a potential biomarker for environmentally induced bone disorders, including those associated with early-life BaP exposure and later-life osteoporosis.