Abstract
Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5′-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16–17 hpf, a typical “salt-and-pepper” expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.
Highlights
Cell fate tracing and tissue-specific transgenic imaging have highlighted some significantly conserved processes essential for vertebrate endoderm development and organ formation [1]
The expression pattern recapitulates that of Apo-14 in orange-spotted grouper [15] and in gibel carp embryogenesis[16], indicating that the Apo-14 promoter drives reliably reporter gene expression in an typical manner identical to the endogenous gene
The onset expression of green fluorescent protein (GFP) in Tg(Apo14: GFP) transgenic line is reminiscent of the spatiotemporal expression of ceruloplasmin, the earliest liver differentiation marker described so far in zebrafish, which was reported to be asymmetrically expressed on the left hand side of the endoderm at 16 hpf (14-somite stage), and in the developing liver by 32h pf[20]
Summary
Cell fate tracing and tissue-specific transgenic imaging have highlighted some significantly conserved processes essential for vertebrate endoderm development and organ formation [1]. The gutGFP zebrafish line Tg(gutGFP)s854, using a Xenopus EF-1a promoter to drive GFP expression in endoderm and endoderm-derived organs including liver, gut, and pancreas from about 30 hpf (hours post fertilization) through to adulthood, had already provided significant advantages for investigating morphogenesis of liver and pancreas [2,3] and for revealing functional role of endothelial cells in later stages of the organogenesis[4,5,6]. The intestine-specific transgenic zebrafish that utilizes intestine-type fatty acid binding protein promoter to drive GFP expression was generated, but the GFP-reporter gene expression was not observed until 72 hpf [7]. As far as we know, no any transgenic GFP zebrafish lines that mark liver, intestine, or pancreas development and morphogenesis from the onset expression of early endodermal cells in the gastrula embryos to the complete organ formation in the larvae have been obtained
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