Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish

Background: Early events in vertebrate liver development have been the major focus in previous studies, however, late events of liver organogenesis remain poorly understood. Liver vasculogenesis in vertebrates occurs through the interaction of endoderm-derived liver epithelium and mesoderm-derived endothelial cells (ECs). In zebrafish, although it has been found that ECs are not required for liver budding, how and when the spatio-temporal pattern of liver growth is coordinated with ECs remains to be elucidated. Results: To study the process of liver development and vasculogenesis in vivo, a two-color transgenic zebrafish line Tg(lfabf: dsRed; elaA: EGFP) was generated and named LiPan for liver-specific expression of DsRed RFP and exocrine pancreas-specific expression of GFP. Using the LiPan line, we first followed the dynamic development of liver from live embryos to adult and showed the formation of three distinct yet connected liver lobes during development. The LiPan line was then crossed with Tg(fli1: EGFP)(y1) and vascular development in the liver was traced in vivo. Liver vasculogenesis started at 55-58 hpf when ECs first surrounded hepatocytes from the liver bud surface and then invaded the liver to form sinusoids and later the vascular network. Using a novel non-invasive and label-free fluorescence correction spectroscopy, we detected blood circulation in the liver starting at similar to 72 hpf. To analyze the roles of ECs and blood circulation in liver development, both cloche mutants (lacking ECs) and Tnnt2 morphants (no blood circulation) were employed. We found that until 70 hpf liver growth and morphogenesis depended on ECs and nascent sinusoids. After 72 hpf, a functional sinusoidal network was essential for continued liver growth. An absence of blood circulation in Tnnt2 morphants caused defects in liver vasculature and small liver. Conclusion: There are two phases of liver development in zebrafish, budding and growth. In the growth phase, there are three distinct stages: avascular growth between 50-55 hpf, where ECs are not required; endothelium-dependent growth, where ECs or sinusoids are required for liver growth between 55-72 hpf before blood circulation in liver sinusoids; and circulation-dependent growth, where the circulation is essential to maintain vascular network and to support continued liver growth after 72 hpf.