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

Abstract

BackgroundEarly 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.ResultsTo 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 ~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.ConclusionThere 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.

Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos

“…To label liver cells, plasmid pLFABP-rfp was injected into 1-cell stage embryos and transgenic progeny were screened for four generations, resulting in a liver-red (Lr) transgenic line (Zeng and Hong, unpublished). This construct contains the 2.8-kb liver-specific promoter from the zebrafish liver fatty acid binding protein and drives RFP expression specifically in embryonic and adult liver [31]. Similarly, in Lr medaka, transgenic RFP expression occurs strongly and specifically in the developing and adult liver (Zeng and Hong, unpublished).…”

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy

“…Mechanical signals such as shear stress induced by blood flow is essential for normal development of organs involved in circulation (Hove et al, 2003;Korzh et al, 2008). Currently, several approaches have been implemented to measure flow velocities in small animal vessels, e.g.…”

“…Confocal laser scanning microscopy generates highresolution images of up to 100 microns depth within tissue and twophoton excitation can further increase the penetration depth to several hundreds of microns (Helmchen and Denk, 2005). As a result, some animal models, such as nematode (Caenorhabditis elegans), fruit fly (Drosophila melanogaster), zebrafish (Danio rerio), and medaka (Oryzias latipes) can be directly studied using imaging based cell biological methods due to their small size and/or semitransparent body tissues (Beis and Stainier, 2006;Lis, 2007;Garcia-Lecea et al, 2008;Korzh et al, 2008). However, imaging based techniques have limited spatial resolution and events that happen on the protein level could not be seen directly.…”

“…Working with transparent animals helps to alleviate this problem too. Recently, it has been shown that FCS can be used efficiently to measure blood flow velocities in live zebrafish embryos (Pan et al, 2007b;Korzh et al, 2008) and even to quantify protein-protein interactions .…”

Line-scan focal modulation microscopy

“…To demonstrate its potential, we conducted preliminary imaging experiments to visualize the constitutive expression of DsRed fluorescent protein in the liver of live 5-day-old zebrafish larvae of strain Tg (fabp10a:DsRed;elaA:egfp), a two-color transgenic zebrafish line (LiPan). 29,30 Zebrafish larvae were maintained in the fish facilities at the Department of Biological Sciences, National University of Singapore (NUS) according to the established protocols 31 and in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines. Before imaging, a zebrafish was placed on a #0 glass-bottomed dish (MatTek, P35G-0-10-C) and positioned in the dorsal orientation.…”