Spatial transcriptomic analysis identifies tnni2a.3 as a candidate associated with intermuscular bone formation in zebrafish

Intermuscular bones (IBs) are intramyoseptal ossified structures that negatively affect flesh quality and processing efficiency in many aquaculture fish species. However, the spatial regulatory mechanisms underlying IB formation remain poorly understood. In this study, a spatial transcriptomic atlas of zebrafish IB-associated muscle tissue was constructed using six transverse sections from one adult zebrafish, in which clustering of 1,406 quality-controlled Visium spots identified 11 transcriptionally distinct tissue domains. Of the three tnni2 homologs examined, tnni2a.3 showed the highest global mean expression and was preferentially enriched in muscle- and bone-associated regions adjacent to IB structures. Correlation analysis revealed weak positive associations between tnni2a.3 and type I collagen genes, with domain-specific patterns: positive correlations in the cartilage/bone matrix domain and negative correlations in two fast-muscle domains. qRT-PCR showed that tnni2a.3 was predominantly expressed in IB-containing muscle, with lower expression in vertebral samples and minimal expression in other tissues. Functionally, CRISPR/Cas9-mediated F0 disruption of tnni2a.3 was associated with a reduction in IB number, providing preliminary evidence for its involvement in IB formation. Collectively, these findings provide new insights into the spatial expression patterns associated with IB development and identify tnni2a.3 as a candidate associated with IB formation. Its cellular localization and functional role require further validation before potential applications in aquaculture breeding can be evaluated.