A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Abstract

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.

A Slit/miR-218/Robo regulatory loop is required during heart tube formation in zebrafish

“…Tg(kdrl:ras-mCherry) s896 (Chi et al, 2008), Tg(myl7:GFP) twu26 (Huang et al, 2003), Tg(kdrl:GFP) s843 (Jin et al, 2007), Tg(gata1:dsRED) sd2 (Traver et al, 2003) and Tg(sox17:GFP) s870 (Chung and Stainier, 2008) zebrafish lines were used in these studies. Embryos were injected at the 1-cell stage with 8-12 ng of miR-218 MO 1 , 5-10 ng of miR-218 MO 2 , 4 ng robo1 MO, 1 ng robo4 MO, 2 ng slit2 MO, 3 ng slit3 MO or 2.5 ng vegfa MO.…”

Single cell analysis of endothelial morphogenesis in vivo

“…A variety of recent studies have taken advantage of the optical clarity of zebrafish embryos and larvae to carry out live imaging of tubulogenesis in developing vessels within living animals (Ellertsdottir et al, 2010;Herbert and Stainier, 2011;Herwig et al, 2011;Jin et al, 2005Jin et al, , 2007Kalen et al, 2009;Kamei et al, 2006;Lenard et al, 2013;Wang et al, 2010). These studies have provided in vivo evidence substantiating both the cell hollowing and the cord hollowing models for lumenogenesis.…”

Etiology of intracerebral hemorrhage (ICH): novel insights from Zebrafish embryos

“…In a large-scale and transgeneassisted ENU screen for zebrafish mutants with vascular defects, Jin et al, identified the tomato s805 (tom) mutant (along with several others), which exhibited widespread hemorrhages and, more prominently, an ICH phenotype during development (Jin et al, 2007). The hemorrhages are associated with the loss of EC viability, as evidenced by vascular regression/fragmentation and EC-specific apoptosis .…”

“…In the same ENU screen for genes regulating vascular integrity, Jin et al, isolated the reddish s587 (reh) mutant, which also exhibited a robust ICH phenotype (Jin et al, 2007). Further molecular characterization of the reh strain by Hegarty et al, through positional cloning, revealed a missense mutation in the Ubiad1 gene, which otherwise encodes the UbiA prenyltransferase domain-containing protein 1 (Hegarty et al, 2013).…”