Transcriptomic profiling and RANKL/RANK/OPG-mediated osteoclastogenesis in zebrafish larvae under simulated microgravity conditions

Introduction Microgravity is one type of external stimulus that affects bone homeostasis and bone development. This study investigates the molecular drivers of these effects in order to more fully understand the cellular communication network between bone cells when bone homeostasis is perturbed. Methods The transcriptional responses of bone-related genes in zebrafish larvae ( Danio rerio ) when exposed to simulated microgravity (SMG) using a Random Positioning Machine were analysed. Larvae were initially analyzed at 6, 12, 18, and 24 h post-exposure via RT-qPCR with a focus on the RANKL/RANK/OPG pathway. Results Short exposures (6–12 h) produced minimal changes, whereas 18–24 h SMG triggered a two-phase response: initial suppression of osteoblast markers ( bglap, sp7, alpl , collagens) followed by activation of osteoclast-associated genes ( tnfsf11 /RANKL, tnfrsf11b /OPG, tnfrsf11a /RANK, nfatc1, ctsk ) and stress-adaptive pathways ( hsp family). We then conducted a transcriptomic analysis at 18 and 24 h. Transcriptomic and gene–protein interaction network analyses revealed distinct regulatory clusters encompassing extracellular matrix and osteoclast signaling genes, highlighting the coordinated modulation of bone formation and resorption. Functional enrichment analyses confirmed the involvement of WNT, BMP, HIPPO, and MAPK signaling pathways in skeletal regulation under SMG, and activated stress-adaptive pathways while concurrently downregulating apoptosis-related genes reflecting a complex interplay among developmental, metabolic, and disease-associated bone processes. Discussion This data highlights a developmental stage-specific protective response. Collectively, these results demonstrate that SMG disrupts the balance between osteoblast and osteoclast activity, promoting bone resorption via the RANKL/RANK/OPG pathway while suppressing matrix deposition. These findings lay the groundwork for designing targeted interventions to mitigate bone loss during spaceflight and in osteoporotic conditions.