The expression patterns of P2075 construct in embryos and larvae of zebrafish. A–C: EGFP expression patterns in whole myotomal region of zebrafish embryo (1 dpf, A, B) and larva (2 dpf, C) injected with the reporter construct of P2075. D-F: EGFP expression patterns in pectoral fin (D) and craniofacial muscles (E, F) in zebrafish larvae at 4 dpf. Arrows indicate muscle fibers expressing EGFP. Scale bars: 100 μm.

The expression patterns of MYH_M743-2:EGFP in stable transgenic line embryos and larvae of zebrafish. A–C: EGFP expression patterns of MYH_M743-2:EGFP in the whole myotomal region of stable transgenic line zebrafish embryo (1 dpf, A, B) and larva (2 dpf, C). D–F: EGFP expression patterns in pectoral fin (D) at 3 dpf and craniofacial muscles (E, F) at 4 dpf in stable transgenic line zebrafish larvae. Arrows indicate muscle fibers expressing EGFP. Scale bars: 100 μm.

EGFP expression was localized to both fast and slow muscle fibers in P2075-injected larvae and only fast muscle fibers in stable transgenic line (MYH_M743-2:EGFP) larvae. A–B: Fast muscle fibers expressing EGFP as reacted with F310 antibody in a P2075-injected larva at 3 dpf (A, lateral view; B, transverse section). C-D, Slow muscle fibers also expressing EGFP as reacted with F59 antibody (C, lateral view; D, transverse section) in a P2075-injected larva at 3 dpf. Some EGFP expressing muscle fibers were stained with F59 antibody (arrows in D). E-J: MYH_M743-2:EGFP transgene in a stable transgenic line larva showed EGFP expression in fast muscle fibers as reacted with F310 antibody (E, F, G; transverse sections) but not in slow muscle fibers as no EGFP expressing muscle fiber reacted with F59 antibody (H, I, J; transverse sections). Scale bars: 20 μm.

Effects of MYH_M743-2 promoter deletions on EGFP expression in the myotomal compartments of zebrafish embryos and larvae. A: Schematic representations of consensus-binding sites within 2075-bp upstream region of torafugu MYH_M743-2, as well as a deletion series in the torafugu MYH_M743-2 promoter region. Vertical lines represent consensus-binding sites for transcriptional factors of MEF2, MyoD and SRF. All promoter deletions (black lines) are linked to EGFP and SV40-polyA sequence in pT2AL200R150G vector. B: Bar graph showing percentages of embryos that express EGFP in the myotomal compartments in microinjection of each deletion construct. The total number of embryos injected with each construct is shown in parentheses. C–G: Embryos injected with a series of distal promoter deletion constructs at one- or two-cell stages were examined for transient EGFP expression at 1–2 dpf using fluorescent microscopy. Reduced EGFP fluorescence typically correlates with a smaller MYH_M743-2 promoter (C–F) and no EGFP expression was observed in P448 (G). An arrowhead indicates a single fiber expressing EGFP in P468 (F). Scale bars: 100 μm. H: 20 nucleotides spanning the region from - 468 to - 449 contain positive cis-elements required for MYH_M743-2 expression. Blue color with bold face in the SRF sequence indicates a central core sequence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Effects of SRF-like binding sites deletions on EGFP expression in the myotomal compartments. A: Schematic representation showing SRF-like binding sites deleted one by one and the percentages of EGFP-expressing embryos (high, medium and low) in each SRF-deletion constructs. The total number of embryos injected with each construct is shown in parentheses. B-D: Lateral view showing high (B), medium (C) and low (D) EGFP expression in myotomal compartments in embryos injected with the P2075ΔSRF1-3 construct. E: Bar graph showing reduced relative EGFP expression in the P2075ΔSRF1-2 and P2075ΔSRF1-3 constructs using real-time PCR analysis. Differences are significant in ANOVA followed by Tukey test at *** P < 0.001.

The - 664 to - 364 bp region in the 52-flanking region of MYH_M743-2 is crucial for its promoter activity. A:. rVISTA plot showing homology (- 664 to - 364 bp region) in pairwise sequence alignments between - 2075 bp of torafugu MYH_M743-2 and the corresponding region of the Tetraodon orthologue. Peaks are shown relative to their positions in MYH_M743-2 and their percent identities (50%–100%) are indicated on the vertical axis. B: EGFP expression was observed in the whole myotomal region in larvae microinjected with the P2075 construct. C: No EGFP expression was observed in embryos injected with the P2075Δ664-364 construct.