Using reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the temporal expression patterns of zebrafish tnni genes during the early development. Total RNA was isolated from different developmental stages (hours of post-fertilization; hpf) as indicated. Specific primers were designed for detecting the existence of tnni1, tnni2, and tnni-HC transcripts. The expected molecular size of each DNA fragment after RT-PCR amplification was indicated on the right. Three types of tnni genes started to be transcribed in 16 hpf. The β-actin transcript was used as an internal control.

The expression pattern of tnni1 transcript during the development of zebrafish embryos. Embryos at different stages as indicated were collected and hybridized with tnni1 using whole-mount in situ hybridization. Panels A, B, C, D, F and H were lateral views and anterior of embryo to the left; panel E and G were dorsal view and anterior of embryo to the top. tnni1 was expressed in the somite at 20–48 hpf, but it was expressed in craniofacial muscle and fin at 72–96 hpf. Panel D and D′ were whole-mount in situ hybridization and transverse section (red line) at 48 hpf Embryos. Panel D′ was indicated that tnni1 gene was slow-muscle-specific. Panels I and J were hybridized with MyoD, specific marker for craniofacial muscles and truck muscle at 72 hpf Embryos. am, adductor mandibulae; lap, levator arcus palatini; ao, adductor operculi; ah, adductor hyoideus; ih, interhyoideus; hh, hyohyoideus; s, somite; f, fin bud; tv 1–5, transversus ventralis 1–5.

The expression pattern of tnni2 transcript during the development of zebrafish embryos. Embryos at different stages as indicated were collected and hybridized with tnni2 using whole-mount in situ hybridization. Panels A, B, C, D, F and H were lateral views and anterior of embryo to the left; and panel E and G were dorsal view and anterior of embryo to the top. tnni2 was expressed in the somite at 20–48 hpf, then it was expressed in craniofacial muscle and fin at 72–96 hpf. Panel D and D′ were whole-mount in situ hybridization and transverse section (red line) at 48 hpf Embryos. Panel D′ was indicated that tnni2 gene was fast-muscle-specific. am, adductor mandibulae; lap, levator arcus palatini; ao, adductor operculi; hm, hapaxial muscles; sh, sternohyoideus; s, somite; f, fin bud.

The expression pattern of tnni-HC transcript during the development of zebrafish embryos. Embryos at different stages as indicated were collected and hybridized with tnni-HC using whole-mount in situ hybridization. Panels A, B, C, D and F were dorsal view, anterior of embryo to the top; and panels E and G were lateral views, anterior of embryo to the left. tnni-HC was expressed in the heart exclusively during early cardiogenesis at 16–48 hpf, but it was not only in the craniofacial muscle but also in the heart after 48 hpf. so, superior oblique; io, inferior oblique; sr, superior rectus; ir, inferior rectus; lr, lateral rectus; mr, medial rectus; sh, sternohyoideus; lap, levator arcus palatini; ao, adductor hyoideus; imp, intermandibularis posterior; ima, intermandibularis anterior; ih, interhyoideus; hh, hyohyoideus; hm, hapaxial muscles; V, ventricle; A, atrium.

The tnni-HC transcript was expressed in the adult heart of zebrafish. Embryos at 48 hpf were collected and hybridized with either antisense tnni-HC probe or sense tnni-HC probe using whole-mount in situ hybridization. Strong signal was observed in the heart of 48-hpf embryos when antisense probe was used (A), but no signal was observed for using sense probe (B). Adult heart was sectioned and also hybridized with either antisense probe or sense probe of tnni-HC. Like tnni-HC expression in embryos, positive signal was observed in the adult heart when antisense probe was used (C), but no signal was observed for using sense probe (D).