Oliver A. Stone

Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis

Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function. DOI: http://dx.doi.org/10.7554/eLife.06489.001

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Neurotrophin-3 Is a Novel Angiogenic Factor Capable of Therapeutic Neovascularization in a Mouse Model of Limb Ischemia

Objective—To investigate the novel hypothesis that neurotrophin-3 (NT-3), an established neurotrophic factor that participates in embryonic heart development, promotes blood vessel growth. Methods and Results—We evaluated the proangiogenic capacity of recombinant NT-3 in vitro and of NT-3 gene transfer in vivo (rat mesenteric angiogenesis assay and mouse normoperfused adductor muscle). Then, we studied whether either transgenic or endogenous NT-3 mediates postischemic neovascularization in a mouse model of limb ischemia. In vitro, NT-3 stimulated endothelial cell survival, proliferation, migration, and network formation on the basement membrane matrix Matrigel. In the mesenteric assay, NT-3 increased the number and size of functional vessels, including vessels covered with mural cells. Consistently, NT-3 overexpression increased muscular capillary and arteriolar densities in either the absence or the presence of ischemia and improved postischemic blood flow recovery in mouse hind limbs. NT-3–induced microvascular responses were accompanied by tropomyosin receptor kinase C (an NT-3 high-affinity receptor) phosphorylation and involved the phosphatidylinositol 3-kinase–Akt kinase–endothelial nitric oxide synthase pathway. Finally, endogenous NT-3 was shown to be essential in native postischemic neovascularization, as demonstrated by using a soluble tropomyosin receptor kinase C receptor domain that neutralizes NT-3. Conclusion—Our results provide the first insight into the proangiogenic capacity of NT-3 and propose NT-3 as a novel potential agent for the treatment of ischemic disease.

Arteriolar Genesis and Angiogenesis Induced by Endothelial Nitric Oxide Synthase Overexpression Results in a Mature Vasculature

Background—Generation of physiologically active vascular beds by delivery of combinations of growth factors offers promise for vascular gene therapy. Methods and Results—In a mesenteric model of physiological angiogenesis, combining endothelial nitric oxide synthase (eNOS) (and hence NO production) with VEGF and angiopoietin-1 overexpression resulted in a more functional vascular phenotype than growth factor administration alone. eNOS gene delivery upregulated eNOS, VEGF, and Ang-1 to similar levels as gene transfer with VEGF or Ang-1. eNOS overexpression resulted in neovascularization to a similar extent as VEGF and Ang-1 combined, but not by sprouting angiogenesis. Whereas combining Ang-1 and VEGF increased both exchange vessels and conduit vessels, neither growth factor nor eNOS alone resulted in vessels with smooth muscle cell (SMC) coverage. In contrast, combining all three generated microvessels with SMCs (arteriolar genesis) and further increased functional vessels. Use of a vasodilator, prazosin, in combination with Ang1 and VEGF, but not alone, also generated SMC-positive vessels. Conclusion—Coexpression of eNOS, VEGF, and Ang-1 results in a more mature vascularization of connective tissue, and generates new arterioles as well as new capillaries, and provides a more physiological therapeutic approach than single growth factor administration, by combining hemodynamic forces with growth factors.

CCR7 mediates directed growth of melanomas towards lymphatics

Please cite this paper as: Emmett, Lanati, Dunn, Stone and Bates (2011). CCR7 Mediates Directed Growth of Melanomas Towards Lymphatics. Microcirculation 18(3), 172–182.

Frameshift indels introduced by genome editing can lead to in-frame exon skipping

The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of “multiple of three nucleotides” exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a “multiple of three nucleotides” exon of LGALS8 that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.

Differential regulation of blood flow induced neovascularisation and mural cell recruitment by VEGF and angiopoietin signalling

Combining nitric oxide (NO)‐mediated increased blood flow with angiopoietin‐1–Tie2 receptor signalling induces arteriolargenesis – the formation of arterioles from capillaries – in a model of physiological angiogenesis. This NO–Tie‐mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO–Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful.

Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling

Combining nitric oxide (NO)‐mediated increased blood flow with angiopoietin‐1–Tie2 receptor signalling induces arteriolargenesis – the formation of arterioles from capillaries – in a model of physiological angiogenesis. This NO–Tie‐mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO–Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful.

Distinct myocardial lineages break atrial symmetry during cardiogenesis in zebrafish

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry.

CNS-resident progenitors direct the vascularization of neighboring tissues

Significance The formation of the vascular network surrounding the central nervous system (CNS) is important for its subsequent vascularization. Previous work has suggested that the developing neural tube provides signals to recruit the endothelial cells that form the peri-neural vascular plexus (PNVP), a vascular network that surrounds the CNS. Here, we identify a critical role for CNS-resident progenitors in directing multistep angiogenic processes required for PNVP formation. Genetic ablation of CNS-resident progenitors in zebrafish affects the formation of the vascular network in tissues adjacent to the spinal cord. Genetic inactivation of Vegfab/Vegfr2 signaling phenocopies these vascular defects, and CNS-resident progenitors serve as a source of Vegfab. Mosaic overexpression of Vegfab in these progenitors induces ectopic blood vessels directed toward their endfeet. Organ growth requires the coordinated invasion and expansion of blood vessel networks directed by tissue-resident cells and morphogenetic cues. A striking example of this intercellular communication is the vascularization of the central nervous system (CNS), which is driven by neuronal progenitors, including neuroepithelial cells and radial glia. Although the importance of neuronal progenitors in vascular development within the CNS is well recognized, how these progenitors regulate the vasculature outside the CNS remains largely unknown. Here we show that CNS-resident radial glia direct the vascularization of neighboring tissues during development. We find that genetic ablation of radial glia in zebrafish larvae leads to a complete loss of the bilateral vertebral arteries (VTAs) that extend along the ventrolateral sides of the spinal cord. Importantly, VTA formation is not affected by ablation of other CNS cell types, and radial glia ablation also compromises the subsequent formation of the peri-neural vascular plexus (PNVP), a vascular network that surrounds the CNS and is critical for CNS angiogenesis. Mechanistically, we find that radial glia control these processes via Vegfab/Vegfr2 signaling: vegfab is expressed by radial glia, and genetic or pharmacological inhibition of Vegfab/Vegfr2 signaling blocks the formation of the VTAs and subsequently of the PNVP. Moreover, mosaic overexpression of Vegfab in radial glia is sufficient to partially rescue the VTA formation defect in vegfab mutants. Thus, our findings identify a critical function for CNS-resident progenitors in the regulation of vascularization outside the CNS, serving as a paradigm for cross-tissue coordination of vascular morphogenesis and growth.