Periodic Delta-like 4 expression in developing retinal arteries

During vascular development, Notch signalling plays important roles in cell-cell communication and cell fate decisions. We studied expression of Notch 1-4 and its ligand Delta-like 4 (Dll4) in the developing retinal vasculature. Dll4 mRNA is strongly expressed in endothelial cells at the very tips of growing vessels ('tip cells') and also in arteries, where it is expressed in a segmented 'tiger's tail' pattern. This implies that developing retinal arteries contain different types of endothelial cells, Dll4-positive and Dll4-negative. The Dll4-positive stripes do not correspond to any obvious morphological property of the vascular network but correlate to some extent with the distribution of platelet derived growth factor B (PDGF-B) mRNA. However, PDGF-B expression is neither as artery-specific nor as clearly segmented as Dll4. Possible target cells for Dll4 signalling are retinal astrocytes (Notch1 positive), arterial pericytes (Notch3 positive) or arterial endothelial cells themselves (Notch4 positive). However, there is no clear reciprocity of Notch and Dll4 expression that allows identification of the interacting cells. Nevertheless, Dll4 stripes are a novel property of immature arteries, the origin and function of which remain to be explained.     

New pathways and mechanisms regulating and responding to Delta-like ligand 4-Notch signalling in tumour angiogenesis

Notch signalling is a key pathway controlling angiogenesis in normal tissues and tumours. This has become a major focus of development of anticancer therapy, but to develop this appropriately, we need further understanding of the mechanisms of regulation of Dll4 (Delta-like ligand 4), a key endothelial Notch ligand. Dll4 and VEGF (vascular endothelial growth factor) cross-talk, with VEGF up-regulation of Dll4 and Dll4 down-regulating VEGFR (VEGF receptor) signalling. Both are essential for normal angiogenesis, and blockade of one may produce compensatory changes in the other. The present review considers recent developments in the regulation of Dll4 expression and functions, its role as a mechanism of resistance to anti-angiogenic therapy, and methods needed to develop effective therapy against this target.     

Hepatitis B Virus HBx Activates Notch Signaling via Delta-Like 4/Notch1 in Hepatocellular Carcinoma

Hepatitis virus B (HBV) infection is one of the major causes of hepatocellular carcinomas (HCC). HBx protein encoded in HBV genome is one of the key viral factors leading to malignant transformation of infected cells. HBx functions by interfering with cellular functions, causing aberration in cellular behaviour and transformation. Notch signalling is a well-conserved pathway involved in cellular differentiation, cell survival and cell death operating in various types of cells. Aberration in the Notch signalling pathways is linked to various tumors, including HCC. The role of HBx on the Notch signalling in HCC, however, is still controversial. In this study, we reported that HBV genome-containing HCC cell line HepG2 (HepG2.2.15) expressed higher Notch1 and Delta-like 4 (Dll4), compared to the control HepG2 without HBV genome. This upregulation coincided with increased appearance of the cleavage of Notch1, indicating constitutively activated Notch signalling. Silencing of HBx specifically reduced the level of Dll4 and cleaved Notch1. The increase in Dll4 level was confirmed in clinical specimens of HCC lesion, in comparison with non-tumor lesions. Using specific signalling pathway inhibitors, we found that MEK1/2, PI3K/AKT and NF-κB pathways are critical for HBx-mediated Dll4 upregulation. Silencing of HBx clearly decreased the level of phosphorylation of Akt and Erk1/2. Upon silencing of Dll4 in HepG2.2.15, decreased cleaved Notch1, increased apoptosis and cell cycle arrest were observed, suggesting a critical role of HBx-Dll4-Notch1 axis in regulating cell survival in HCC. Furthermore, clonogenic assay confirmed the important role of Dll4 in regulating cell survival of HBV-genome containing HCC cell line. Taken together, we reported a link between HBx and the Notch signalling in HCC that affects cell survival of HCC, which can be a potential target for therapy.     

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.     

The role of extracellular matrix in vascular branching morphogenesis

Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.     

The role of extracellular matrix in vascular branching morphogenesis

Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.     

Expression of Dll4 during mouse embryogenesis suggests multiple developmental roles

Delta-Notch signalling regulates cell-fate decisions in a variety of tissues in diverse organisms, through cell-to-cell interactions. Here, we report the expression pattern of a Delta gene family member, Delta-like 4 (Dll4). Dll4 expression was analyzed in mouse embryos and selected adult organs by monitoring beta-galactosidase (beta-gal) expression from a lacZ reporter cassette inserted downstream of the Dll4 promoter, which allowed for high sensitivity and single cell resolution. Expression was detected in several tissues where Notch signalling is known to control cell-fate decisions, like the vascular system, the nervous system, the gastrointestinal system, and the thymus. Throughout embryonic cardiovascular development, Dll4 expression was seen only on endocardial cells and endothelial cells of the arteries, arterioles, and capillaries, being absent from vascular smooth muscle cells and veins. In the nervous system, expression was detected in the brain, neural tube, retina, and, for the first time, in the olfactory epithelium, vomeronasal organs and para-aortic bodies. Extensive Dll4 expression was also observed in the gut. This detailed expression analysis reveals new clues for both endothelial and non-endothelial Dll4 function in different organs.     

VEGF and Notch Signaling

Tubular sprouting in angiogenesis relies on division of labour between the endothelial tip cell, leading and guiding the sprout and their neighbouring stalk cells, which divide and form the vascular lumen. We previously learned how the graded extracellular distribution of heparin-binding Vascular Endothelial Growth Factor (VEGF)-A orchestrates and balances tip and stalk cell behaviour. Recent data now provided insight into the regulation of tip cell numbers, illustrating how Delta-like (Dll)4 – Notch signalling functions to limit the explorative tip cell behaviour induced by VEGF-A. These data also provided a first answer to the question why not all endothelial cells stimulated by VEGF-A turn into tip cells. Here we review this new model and discuss how VEGF-A and Dll4/Notch signalling may interact dynamically at cellular level to control vascular patterning.     

Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway

The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked bygamma-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jkappa-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jkappa, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jkappa-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype.     

sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation

The appearance of molecular differences between arterial and venous endothelial cells before circulation suggests that genetic factors determine these cell types. We find that vascular endothelial growth factor (vegf) acts downstream of sonic hedgehog (shh) and upstream of the Notch pathway to determine arterial cell fate. Loss of Vegf or Shh results in loss of arterial identity, while exogenous expression of these factors causes ectopic expression of arterial markers. Microinjection of vegf mRNA into embryos lacking Shh activity can rescue arterial differentiation. Finally, activation of the Notch pathway in the absence of Vegf signaling can rescue arterial marker gene expression. These studies reveal a complex signaling cascade responsible for establishing arterial cell fate and suggest differential effects of Vegf on developing endothelial cells.     

A cell autonomous role for the Notch ligand Delta-like 3 in αβ T-cell development

Notch signalling is critical to help direct T-cell lineage commitment in early T-cell progenitors and in the development of αβ T-cells. Epithelial and stromal cell populations in the thymus express the Notch DSL (Delta, Serrate and Lag2)ligands Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged 1 and Jagged 2, and induce Notch signalling in thymocytes that express the Notch receptor. At present there is nothing known about the role of the Delta-like 3 (Dll3) ligand in the immune system. Here we describe a novel cell autonomous role for Dll3 in αβ T-cell development. We show that Dll3 cannot activate Notch when expressed in trans but like other Notch ligands it can inhibit Notch signalling when expressed in cis with the receptor. The loss of Dll3 leads to an increase in Hes5 expression in double positive thymocytes and their increased production of mature CD4(+) and CD8(+) T cells. Studies using competitive irradiation chimeras proved that Dll3 acts in a cell autonomous manner to regulate positive selection but not negative selection of autoreactive T cells. Our results indicate that Dll3 has a unique function during T-cell development that is distinct from the role played by the other DSL ligands of Notch and is in keeping with other recent studies indicating that Dll1 and Dll3 ligands have non-overlapping roles during embryonic development.     

Anti-Dll4 therapy: can we block tumour growth by increasing angiogenesis?

Since the early 1970s, the dogma postulating that blocking tumour angiogenesis can inhibit tumour growth has been accepted widely and has resulted in the generation of a variety of successful anti-angiogenic therapies. More recently, new signalling pathways, such as the Dll4-Notch signalling pathway, have been shown to regulate angiogenesis during development. In pathological conditions, such as cancer, Dll4 is up-regulated strongly in the tumour vasculature. Based on this expression pattern, different molecules have been generated to block Dll4 signalling. Unexpectedly, these blocking agents inhibited tumour growth in vivo by triggering excessive but nonfunctional angiogenesis. Altogether, these molecules constitute a new category of pro-angiogenic yet anticancer agents and offer an exciting alternative to previously described vascular targeting molecules.     

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.     

Arterial-venous endothelial cell fate is related to vascular endothelial growth factor and Notch status during human bone mesenchymal stem cell differentiation

Human bone mesenchymal stem cells (hMSCs) can differentiate into endothelial cells (ECs), so we aimed to investigate whether hMSCs could also differentiate into a specific arterial or venous ECs. hMSCs were induced to differentiate into ECs using vascular endothelial growth factor (VEGF). Low VEGF concentration (50ng/ml) upregulated the venous marker gene EphB4, however high concentration (100ng/ml) upregulated the arterial marker genes ephrinB2, Dll4 and Notch4, and downregulated the venous marker genes EphB4 and COUP-TFll. This VEGF dose-dependent induction was largely blocked by inhibition of the Notch pathway in hMSCs treated with gamma-secretase inhibitor. Therefore, differentiation of hMSCs into arterial- or venous-specific ECs depends on VEGF and is regulated by the Notch pathway.