Artery/Vein Specification Is Governed by Opposing Phosphatidylinositol-3 Kinase and MAP Kinase/ERK Signaling

Abstract

Angioblasts are multipotent progenitor cells that give rise to arteries or veins . Genetic disruption of the gridlock gene perturbs the artery/vein balance, resulting in generation of insufficient numbers of arterial cells . However, within angioblasts the precise biochemical signals that determine the artery/vein cell-fate decision are poorly understood. We have identified by chemical screening two classes of compounds that compensate for a mutation in the gridlock gene . Both target the VEGF signaling pathway and reveal two downstream branches emanating from the VEGF receptor with opposing effects on arterial specification. We show that activation of ERK (p42/44 MAP kinase) is a specific marker of early arterial progenitors and is among the earliest known determinants of arterial specification. In embryos, cells fated to contribute to arteries express high levels of activated ERK, whereas cells fated to contribute to veins do not. Inhibiting the phosphatidylinositol-3 kinase (PI3K) branch with GS4898 or known PI3K inhibitors, or by expression of a dominant-negative form of AKT promotes arterial specification. Conversely, inhibition of the ERK branch blocks arterial specification, and expression of constitutively active AKT promotes venous specification. In summary, chemical genetic analysis has uncovered unanticipated opposing roles of PI3K and ERK in artery/vein specification.

RhoA prevents apoptosis during zebrafish embryogenesis through activation of Mek/Erk pathway

“…In addition to rhoA mRNA rescue, ectopic expression of Mek mRNA not only restored normal development in 28.9% of the embryos (Figures 4d and k, n ¼ 161, at Po0.05), but also prevented apoptosis in 93.6% phenotypically corrected embryos (n ¼ 47, Figure 4i). To further confirm that Mek/Erk acts as downstream of RhoA to promote cell survival, the Mek inhibitor (U0126; Hong et al, 2006) was used to block the activation of Mek/Erk in the embryos which were injected with rhoA MO1 alone, or co-injected with rhoA MO1 and mRNA encoding either Mek or rhoA. As shown in Supplementary Figure S2a, rhoA mRNA rescue could be inhibited by U0126 in a dose-dependent manner, while no inhibition of such rescue was seen in embryos treated with JNK inhibitor (SP600125; Supplementary Figure S2b).…”

Optimizing methods for the study of intravascular lipid metabolism in zebrafish

“…Future studies may focus on the development of lower vertebrate models of human diseases, for example mutant zebrafish carrying mutations in the lipid metabolism pathway (29)(30)(31). The effects of expressional changes in genes associated with regulation of intravascular lipids induced by Morpholinoor messenger RNA injections may be investigated using Oil Red O staining or breeding water containing CholEsteryl BODIPY 542/563 C11.…”

Simple vertebrate models for chemical genetics and drug discovery screens: Lessons from zebrafish and Xenopus

“…These include screens to identify compounds affecting dorsoventral patterning (Yu et al, 2008b), hematopoiesis (North et al, 2007), erythropoiesis (Shafizadeh et al, 2004), vasculature (Tran et al, 2007), pigmentation (Jung et al, 2005;North et al, 2007), fin regeneration (Mathew et al, 2007), and morphogenesis of the brain, eyes, and other tissues and organs easily identified by visual inspection of compoundtreated embryos (Khersonsky et al, 2003;Sachidanandan et al, 2008). Finally, screens to identify modifiers or suppressors of zebrafish mutant phenotypes have been developed Stern et al, 2005;Hong et al, 2006). We will now review the specifics of these screens in greater detail to highlight key lessons that can be learned from the careful analysis of the published literature.…”