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 approximately 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.


CONCLUSIONS
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.
Korzh
Svetlana
S
Department of Biological Sciences, National University of Singapore, Singapore. dbskorzh@nus.edu.sg
Pan
Xiufang
X
Garcia-Lecea
Marta
M
Winata
Cecilia Lanny
CL
Pan
Xiaotao
X
Wohland
Thorsten
T
Korzh
Vladimir
V
Gong
Zhiyuan
Z
D000818:Animals; D030801:Animals, Genetically Modified; D004707:Endoderm / Q000098:blood supply / Q000196:embryology / Q000254:growth & development; D042783:Endothelial Cells / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000196:embryology / Q000254:growth & development; D008099:Liver / Q000098:blood supply* / Q000196:embryology / Q000254:growth & development*; D008648:Mesoderm / Q000098:blood supply / Q000196:embryology / Q000254:growth & development; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology*; D015027:Zebrafish / Q000033:anatomy & histology* / Q000196:embryology / Q000235:genetics / Q000502:physiology*
Development. 1999 Feb;126(4):827-38
9895329
Development. 2004 Nov;131(22):5753-66
15509774
Development. 1995 Oct;121(10):3141-50
7588049
Dev Biol. 2007 Jul 1;307(1):29-42
17531218
Development. 1996 Dec;123:293-302
9007249
Genes Dev. 2005 Dec 1;19(23):2900-11
16322560
Endocrinology. 1960 Feb;66:222-39
14437876
FEBS Lett. 2003 Mar 13;538(1-3):125-33
12633865
Dev Biol. 1998 May 15;197(2):248-69
9630750
J Biomed Opt. 2007 Jan-Feb;12(1):014034
17343509
Rev Sci Instrum. 2007 May;78(5):053711
17552829
Dev Biol. 2003 Mar 1;255(1):12-29
12618131
Mech Dev. 2003 Jan;120(1):19-33
12490293
BMC Dev Biol. 2007 Oct 19;7:117
17945029
Science. 2001 Oct 19;294(5542):559-63
11577199
Development. 1996 Dec;123:321-8
9007252
Dev Biol. 2002 Aug 15;248(2):307-18
12167406
Mech Dev. 2003 Jan;120(1):5-18
12490292
Dev Biol. 2003 Jan 15;253(2):279-90
12645931
Mech Dev. 2001 May;103(1-2):137-9
11335121
Development. 1996 Dec;123:285-92
9007248
Nat Genet. 2002 May;31(1):106-10
11967535
Nature. 1995 Jul 6;376(6535):62-6
7596435
Dev Biol. 2001 Feb 15;230(2):278-301
11161578
Exp Cell Res. 2006 May 15;312(9):1526-39
16490192
Development. 1997 Jan;124(2):381-9
9053314
Nat Genet. 2007 Mar;39(3):397-402
17259985