Essential role of RANK-NF-κB signaling pathway in spinal deformities: Insights from largemouth bass and zebrafish models

Spinal deformities, leading to reduced growth and survival rates, pose a significant challenge in fish aquaculture. However, the molecular mechanisms underlying their occurrence remain poorly understood. In this study, we investigated the role of the RANK-NF-κB signaling pathway in spinal deformities using both largemouth bass (Micropterus salmoides) and zebrafish (Danio rerio) models. We found that the expression of nfkbiaa and nfkbiab encoding NF-kappa-B inhibitor alpha and beta respectively was significantly upregulated in the deformed largemouth bass, indicating the involvement of NF-κB signaling in spinal deformities. Using the transgenic reporter zebrafish line Tg(NFkB:EGFP), we observed strong NF-κB signaling activity in tissues surrounding the vertebral column. Inhibition of NF-κB signaling via overexpression of IkBSR (a dominant-negative inhibitor of NF-κB) resulted in ventral curvature of the spine in zebrafish which is similar to the phenotype of the deformed largemouth bass. Moreover, we generated tnfrsf11a mutant zebrafish using CRISPR-Cas9 and observed that these mutants exhibited ventral spinal curvature and a significant reduction in osteoclasts. Additionally, tnfrsf11a mutant zebrafish exhibited structural damage in lateral line canals, suggesting that the morphology of lateral line canals could serve as a non-invasive diagnostic marker for spinal deformities. For the first time, this study demonstrates that dysregulation of the RANK-NF-κB signaling pathway as a critical regulator of spinal deformities in fish, providing key insights into the molecular pathology of vertebral malformations and offering novel targets for prevention and therapeutic intervention.