Comparative expression analysis of tnni1 paralogs during zebrafish embryonic development

Troponin I 1 (TNNI1) encodes the slow skeletal isoform of troponin I and is essential for the regulation of contraction in slow-twitch skeletal muscle. However, the developmental and tissue-specific expression of TNNI1-related genes across vertebrates remains incompletely characterized. In zebrafish, multiple tnni genes have been identified, including four tnni1-related paralogs (tnni1a, tnni1b, tnni1c, and tnni1d), which share high sequence similarity. While individual tnni genes have been partially characterized, a systematic comparison of the spatial and temporal expression patterns of these tnni1 paralogs during embryogenesis remains lacking. Here, we analyzed and compared the spatial and temporal expression patterns of the four tnni1 paralogs during zebrafish embryogenesis to assess their potential functional divergence. Multiple sequence alignment revealed that proteins encoded by zebrafish Tnni1 paralogs are highly conserved relative to mammalian TNNI1, with paralog-specific divergence primarily in the N-terminal region. Whole-mount in situ hybridization showed that tnni1a is first detected in the embryonic heart and was later expressed in cranial and hypaxial muscles. tnni1b was expressed in bilateral cardiac precursor cells, exhibited the strongest and most sustained cardiac expression among the paralogs, and was subsequently expressed in extraocular, craniofacial, hypaxial, and trunk muscles. In contrast, tnni1c and tnni1d were initially expressed in somites, displayed weaker or transient cardiac expression, and were later broadly expressed in extraocular, craniofacial, and hypaxial muscles. Taken together, these findings indicate that zebrafish tnni1 paralogs exhibit distinct yet partially overlapping spatiotemporal expression profiles, supporting partial subfunctionalization following gene duplication during cardiac and skeletal muscle development.