To confirm that the ventricle-specific expression of the vmhc promoter construct was consistent with endogenous expression, we generated a transgenic line using a pZVMHC-1834 construct and injected NotI-linearized pZVMHC-1834 into fertilized eggs. Ventricle-specific GFP expression was displayed in 25–30% of the surviving embryos. The GFP-expressing embryos were selected and raised to adulthood. After 3 months, the surviving founders were mated with wild-type (WT) zebrafish to find germline-transmitted transgenic zebrafish. A stable ventricle-specific GFP-expressing zebrafish line was established from the 50 founders tested. The transgenic line was mated and produced normal offspring. The GFP-labeled ventricles of both homozygotic and heterozygotic transgenic zebrafish were highly resolved throughout their life span, which confirmed normal atrial and ventricular contractions (Supp. Movie S1). Using in situ hybridization, we identified a difference between endogenous vmhc and transgenic GFP expression by the zebrafish vmhc promoter region (Fig. 2). The transgene was expressed with a same pattern to the endogenous vmhc in the heart region (Fig. 2A,C,E,G,I,K). In trunk muscle, where the endogenous vmhc transcripts were detected, gfp transcript was not observed in transgenic fish (Fig. 2B,D,F,H,J,L). Fluorescence microscopy revealed that the GFP protein from the transgene was expressed in the primitive heart tube, 21.5 and 24 hours postfertilization (hpf), when the cardiac precursors form heart tube (Fig. 2M,N). At 48 hpf, GFP signals were clearly shown at the ventricle of transgenic zebrafish embryo (Fig. 3A–C). Using the monoclonal antibody MF20, which recognizes sarcomeres (Bader et al.,1982) and allows clear visualization of the heart, we confirmed that a green fluorescent signal appeared specifically in the ventricle at 48 hpf (Fig. 3D–F). At 2 months postfertilization, ventricle-specific GFP expression was maintained in heterozygotic F3 progeny (Fig. 3G–I). GFP expression began at 3.5 days postfertilization (dpf) in jaw, eye and trunk muscles, and at 1 month postfertilization, we detected additional GFP signals in muscles of pectoral fin, dorsal fin, anal fin, caudal fin (data not shown).
Figure 2. Identical expression pattern of green fluorescent protein (GFP) reporter gene by vmhc promoter to endogenous vmhc mRNA in the heart region at the early heart-forming stage. A,C,E: Expression patterns of endogenous vmhc mRNA are shown in heart region at 18, 21.5, 24 hours postfertilization (hpf)each. Dorsal views with anterior to the left. B,D,F: Endogenous vmhc transcripts are observed in trunk muscle. Lateral view with anterior to the left. G,I,K: Expression patterns of gfp mRNA are observed in the heart region. Dorsal views with anterior to the left. H,J,L: No gfp transcript is observed in the trunk region. Lateral view with anterior to the left. M,N: GFP fluorescence signals from transgenic zebrafish embryos in heart region at 21.5 and 24 hpf. Dorsal views with anterior to the left. h, heart; tm, trunk muscle.
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Figure 3. Ventricle-specific expression of the green fluorescent protein (GFP) reporter gene using the 5′-flanking region of vmhc as a promoter. Transgenic zebrafish embryo at 48 hours postfertilization (hpf). A: Nomarski image. B: Ventricle-specific green fluorescent signal. C: Overlay. Confocal-microscopic (Zeiss) images of the 48-hpf transgenic embryo immunostained with myocardium-specific MF20 monoclonal antibody followed by the Alexa594 secondary antibody. D–F: Ventral heart view with green fluorescent signal at the ventricular region (D), red (E), overlay (F). G: Isolated heart from 2 months after fertilization using a Leica fluorescence stereomicroscope. H,I: Brightfield, green fluorescent signal in the ventricular region (H), overlay (I). Views of the embryos are lateral and anterior to the left. a, atrium; v, ventricle.
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