title: Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish.
abstract: 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore. dbskorzh@nus.edu.sg
authors.forename: Svetlana
authors.initials: S
authors.lastname: Korzh
authors.forename: Xiufang
authors.initials: X
authors.lastname: Pan
authors.forename: Marta
authors.initials: M
authors.lastname: Garcia-Lecea
authors.forename: Cecilia Lanny
authors.initials: CL
authors.lastname: Winata
authors.forename: Xiaotao
authors.initials: X
authors.lastname: Pan
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
authors.forename: Vladimir
authors.initials: V
authors.lastname: Korzh
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Gong
mesh_terms: 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*
references.citation: Development. 1999 Feb;126(4):827-38
references.pmid: 9895329
references.citation: Development. 2004 Nov;131(22):5753-66
references.pmid: 15509774
references.citation: Development. 1995 Oct;121(10):3141-50
references.pmid: 7588049
references.citation: Dev Biol. 2007 Jul 1;307(1):29-42
references.pmid: 17531218
references.citation: Development. 1996 Dec;123:293-302
references.pmid: 9007249
references.citation: Genes Dev. 2005 Dec 1;19(23):2900-11
references.pmid: 16322560
references.citation: Endocrinology. 1960 Feb;66:222-39
references.pmid: 14437876
references.citation: FEBS Lett. 2003 Mar 13;538(1-3):125-33
references.pmid: 12633865
references.citation: Dev Biol. 1998 May 15;197(2):248-69
references.pmid: 9630750
references.citation: J Biomed Opt. 2007 Jan-Feb;12(1):014034
references.pmid: 17343509
references.citation: Rev Sci Instrum. 2007 May;78(5):053711
references.pmid: 17552829
references.citation: Dev Biol. 2003 Mar 1;255(1):12-29
references.pmid: 12618131
references.citation: Mech Dev. 2003 Jan;120(1):19-33
references.pmid: 12490293
references.citation: BMC Dev Biol. 2007 Oct 19;7:117
references.pmid: 17945029
references.citation: Science. 2001 Oct 19;294(5542):559-63
references.pmid: 11577199
references.citation: Development. 1996 Dec;123:321-8
references.pmid: 9007252
references.citation: Dev Biol. 2002 Aug 15;248(2):307-18
references.pmid: 12167406
references.citation: Mech Dev. 2003 Jan;120(1):5-18
references.pmid: 12490292
references.citation: Dev Biol. 2003 Jan 15;253(2):279-90
references.pmid: 12645931
references.citation: Mech Dev. 2001 May;103(1-2):137-9
references.pmid: 11335121
references.citation: Development. 1996 Dec;123:285-92
references.pmid: 9007248
references.citation: Nat Genet. 2002 May;31(1):106-10
references.pmid: 11967535
references.citation: Nature. 1995 Jul 6;376(6535):62-6
references.pmid: 7596435
references.citation: Dev Biol. 2001 Feb 15;230(2):278-301
references.pmid: 11161578
references.citation: Exp Cell Res. 2006 May 15;312(9):1526-39
references.pmid: 16490192
references.citation: Development. 1997 Jan;124(2):381-9
references.pmid: 9053314
references.citation: Nat Genet. 2007 Mar;39(3):397-402
references.pmid: 17259985