Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Abstract

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss-and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1 BAC :yfp) ϫ Tg(kdrl:ras-cherry) s916 embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

miR-30a Regulates Endothelial Tip Cell Formation and Arteriolar Branching

“…8,24 In human umbilical vein EC, loss of miR-30a augments DLL4 protein levels and activates NOTCH receptor signaling pathways. In developing sprouts, angiogenic cell behavior largely depends on the Notch signaling status.…”

“…Tg(fli1a:EGFP) y1 , Tg(fli1a:nEGFP) y7 , Tg(kdrl:hras-mcherry) s896 ; Tg(flt1 BAC :yfp), transgenic zebrafish lines were used as described. [20][21][22][23][24] To knockdown gene expression in zebrafish embryos, we used a morpholino (small antisense molecules capable of blocking mRNA translation or splicing) approach. Morpholinos (GeneTools; sequences in Table S1 in the online-only Data Supplement), and miRNA precursors (Ambion) were injected at the 1 to 2 cell stage.…”

“…In the trunk of the zebrafish embryo, intersegmental vessels (ISVs) branch from the aorta in a highly stereotyped manner involving tip cell-mediated guidance of the developing ISV sprouts. 24 These vessels express arterial identity markers, will carry arteriolar flow, and are, therefore, considered arterioles during early stages of development. Because in zebrafish, dll4 gain-of-function restricts angiogenic cell behavior, we specifically monitored the development of the intersegmental arterioles that sprout/branch from the aorta in the trunk region of the miR-30 morphants ( Figure S2A and S2B).…”

Uterine natural killer cells: supervisors of vasculature construction in early decidua basalis

“…Along with VEGF family signaling, NOTCH family signaling has multiple roles in both normal vascular development and pathological angiogenesis, including regulation of VEGFR1 (Jakobsson et al 2009, Outtz et al 2010, Krueger et al 2011. NOTCH is involved in the differentiation of endothelial tip cells and vascular smooth muscle cells and regulates cell-fate decisions in arteriovenous differentiation (Gridley 2010).…”

Notch Signaling in Ocular Vasculature Development and Diseases

“…The leading tip cells express Dll4 and activate the Notch signal in the adjacent stalk cells that express Jag1 and attenuate the effects of VEGF on stalk cells. This result is achieved primarily through differential regulation of VEGFRs, namely downregulating VEGFR2 and upregulating VEGFR1 (87,91). VEGFR1 can be expressed as a truncated version, resulting in the formation of soluble VEGFR1 (sVEGFR1), which is a potent paracrine inhibitor of VEGF signaling and plays an essential role in keeping the cornea transparent (56).…”