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As miR-1 and miR-206 share identical seed sequences, they are commonly speculated to target the same gene. Here, we identify an mRNA encoding seryl-tRNA synthetase (SARS), which is targeted by miR-1, but refractory to miR-206. SARS is increased in miR-1-knockdown embryos, but it remains unchanged in the miR-206 knockdown. Either miR-1 knockdown or sars overexpression results in a failure to develop some blood vessels and a decrease in vascular endothelial growth factor Aa (VegfAa) expression. In contrast, sars knockdown leads to an increase of VegfAa expression and abnormal branching of vessels, similar to the phenotypes of vegfaa-overexpressed embryos, suggesting that miR-1 induces angiogenesis by repressing SARS. Unlike the few endothelial cells observed in the miR-1-knockdown embryos, knockdown of miR-206 leads to abnormal branching of vessels accompanied by an increase in endothelial cells and VegfAa. Therefore, we propose that miR-1 and miR-206 target different genes and thus have opposing roles during embryonic angiogenesis in zebrafish.

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the β-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.

Troponin I (TnnI), a constituent of the troponin complex on the thin filament, providers a calcium-sensitive switch for striated muscle contraction. Cardiac TnnI is, therefore, a highly sensitive and specific marker of myocardial injury in acute coronary syndromes. The TnnI gene, which has been identified in birds and mammals , encodes the isoforms expressed in cardiac muscle fast skeletal muscle and slow skeletal muscle. However, very little is known about the TnnI gene in lower vertebrates. Here, we cloned and characterized the molecular structures and expression patterns of three types of zebrafish tnni genes: tnni1, tnni2, and tnn-HC (Heart and craniofacial). Based on the unrooted radial gene tree analysis of the TnnI gene among vertebrates, the zebrafish Tnni1 and TnnI2 we cloned were homologous of the slow muscle TnnI1 and fast muscle TnnI2 of other vertebrates, respectively. In addition, reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization demonstrated that zebrafish tnni1 and tnni2 transcripts were not detectable in the somites until 16 h post-fertilization (hpf), after which they were identified as slow-and fast muscle-specific, respectively . Interestingly, tnni-HC, a novel tnni isoform of zebrafish was expressed exclusive in heart during early cardiogenesis as 16 hpf, but then extended its expression in craniofacial muscle after 48 hpf. Thus, using zebrafish as our system model, it is suggested that the results, as noted above, may provide more insight into the molecular structure and expression pattens of the lower vertebrate TnnI gene.