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

Abstract

SUMMARYEndothelial 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.

Flt-1 (Vascular Endothelial Growth Factor Receptor-1) Is Essential for the Vascular Endothelial Growth Factor–Notch Feedback Loop During Angiogenesis

“…We found perturbed intersegmental vessel (ISV) development in Notch-inhibited embryos (Figure 3A-C), similar to previous reports. 30,31 However, in these same embryos, the CVPs were unaffected, as determined by the presence of multiple lumenized vessels conducting blood flow. (Figure 3A-C).…”

“…To test this idea, we analyzed the developing vessels in the zebrafish embryo, an established model of blood vessel formation that occurs in the context of blood flow. 29 Notch manipulations in zebrafish are reported to affect vessel formation in certain scenarios, 30,31 but not all situations of vessel growth. 32 Moreover, the caudal vein plexus (CVP) does not exhibit detectable Notch activation via Notch reporter readout (Wiley et al, in revision).…”

Endothelial cell–cell adhesion during zebrafish vascular development

“…Noteworthy, both the Vegfa ligand and Vegfr-1 (Flt1) have been described to be expressed in the neural tube in zebrafish. 60,61 Therefore, vascular defects observed in glo mutants might be secondary to the severe neuronal abnormalities in this mutant. In addition, it will be important to examine N-cadherin expression in endothelial cells in vivo and determine if it functions cell-autonomously in vascular development.…”

Regulation of blood vessel sprouting

“…In contrast, VEGFR-1 (Flt-1) is positively regulated by Notch signaling [29,30]. VEGFR-1 acts as a competitive inhibitor for the VEGFR-2 receptor [31,32], and its increased expression in stalk cells may restrict the responsiveness of these endothelial cells to VEGF-A [33,34]. Thus Notch signaling appears to regulate tip cell dynamics through its effects on multiple angiogenic factors.…”