NMIHBA results from hypomorphic PRUNE1 variants that lack short-chain exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

The potassium channel Kcne3 is a VEGFA-inducible gene selectively expressed by vascular endothelial tip cells.

Angiogenesis is largely driven by motile endothelial tip-cells capable of invading avascular tissue domains and enabling new vessel formation. Highly responsive to Vascular Endothelial Growth-Factor-A (VEGFA), endothelial tip-cells also suppress angiogenic sprouting in adjacent stalk cells, and thus have been a primary therapeutic focus in addressing neovascular pathologies. Surprisingly, however, there remains a paucity of specific endothelial tip-cell markers. Here, we employ transcriptional profiling and a lacZ reporter allele to identify Kcne3 as an early and selective endothelial tip-cell marker in multiple angiogenic contexts. In development, Kcne3 expression initiates during early phases of angiogenesis (E9) and remains specific to endothelial tip-cells, often adjacent to regions expressing VEGFA. Consistently, Kcne3 activation is highly responsive to exogenous VEGFA but maintains tip-cell specificity throughout normal retinal angiogenesis. We also demonstrate endothelial tip-cell selectivity of Kcne3 in several injury and tumor models. Together, our data show that Kcne3 is a unique marker of sprouting angiogenic tip-cells and offers new opportunities for investigating and targeting this cell type.

A coiled-coil masking domain for selective activation of therapeutic antibodies.

The use of monoclonal antibodies in cancer therapy is limited by their cross-reactivity to healthy tissue. Tumor targeting has been improved by generating masked antibodies that are selectively activated in the tumor microenvironment, but each such antibody necessitates a custom design. Here, we present a generalizable approach for masking the binding domains of antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions. On exposure to tumor-associated proteases, such as matrix metalloproteinases 2 and 9, the coiled-coil peptides are cleaved and antigen binding is restored. We test multiple coiled-coil formats and show that the optimized masking domain is broadly applicable to antibodies of interest. Our approach prevents anti-CD3-associated cytokine release in mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to antibody-drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased anti-tumor efficacy compared with unmasked antibodies in mouse models of cancer.

Infectious Entry of Merkel Cell Polyomavirus.

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

Cysteine Disulfides (Cys-ss-X) as Sensitive Plasma Biomarkers of Oxidative Stress.

We developed a high-throughput mass spectrometry-based method to simultaneously quantify numerous small-molecule thiols and disulfides in blood plasma. Application of this assay to analyze plasma from patients with known oxidative stress (sickle cell disease and sepsis) and from a patient with sickle cell disease treated with the antioxidant N-acetylcysteine suggests that cysteine disulfides, in particular protein-bound cysteine, serve as sensitive plasma biomarkers for the extent of oxidative stress and effectiveness of antioxidant treatment.

Effects of Different Irrigation Solutions on Root Fracture Resistance: An in Vitro Study.

INTRODUCTION: The aim of this in vitro study was to evaluate the effects of sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), chlorhexidine (CHX) and hydroxyethylidene bisphosphonate (HEBP), also known as etidronate, on susceptibility to root fracture resistance (RFR) in human teeth subjected to endodontic preparation. METHODS AND MATERIALS: Seventy extracted single-rooted human teeth were selected, endodontically prepared using the ProTaper Next rotary system (PTN, Dentsply, Maillefer, Ballaigues, Switzerland) and then randomly divided according to the following irrigation regimes (n=10): G1, saline solution (0.9% NaCl); G2, 2.5% NaOCl + 17% EDTA; G3, 2% CHX gel + 17% EDTA; and G4, a mixture of 5% NaOCl + 18% HEBP. After this step, all samples received a final irrigation with distilled water. The samples were subjected to axial forces by mechanical compression testing in a universal testing machine (Dynamometers KRATOS, LTDA, SP, Brazil). Data analyses included the Shapiro-Wilk normality test, analysis of variance (one-way ANOVA) and a subsequent multiple comparison test (Tukey's test). RESULTS: The results indicated that G1 (0.9% NaCl) presented greater resistance to root fracture. No significant differences were observed in G2 (2.5% NaOCl + 17% EDTA) and G3 (2% CHX gel + 17% EDTA). A significant difference was identified in G4 (mixture of 5% NaOCl + 18% HEBP) (P<0.05). CONCLUSION: A mixture of 5% NaOCl + 18% HEBP resulted in a lower fracture resistance when used to irrigate canals during endodontic instrumentation.

Monoallelic BMP2 Variants Predicted to Result in Haploinsufficiency Cause Craniofacial, Skeletal, and Cardiac Features Overlapping Those of 20p12 Deletions.

Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-ß-signaling proteins involved in bone and cartilage biology. Monoallelic deletions of 20p12 are variably associated with cleft palate, short stature, and developmental delay. Here, we report a cranioskeletal phenotype due to monoallelic truncating and frameshift BMP2 variants and deletions in 12 individuals from eight unrelated families that share features of short stature, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. De novo occurrence and autosomal-dominant inheritance of variants, including paternal mosaicism in two affected sisters who inherited a BMP2 splice-altering variant, were observed across all reported families. Additionally, we observed similarity to the human phenotype of short stature and skeletal anomalies in a heterozygous Bmp2-knockout mouse model, suggesting that haploinsufficiency of BMP2 could be the primary phenotypic determinant in individuals with predicted truncating variants and deletions encompassing BMP2. These findings demonstrate the important role of BMP2 in human craniofacial, skeletal, and cardiac development and confirm that individuals heterozygous for BMP2 truncating sequence variants or deletions display a consistent distinct phenotype characterized by short stature and skeletal and cardiac anomalies without neurological deficits.

Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature.

Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPµ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPµ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPµ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPµ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPµ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.

Manifestaciones orales en pacientes reumatológicos: una revisión de los conocimientos / Oral manifestations of rheumatologic patients: a knowledge review

La cavidad oral a través de sus estructuras, dientes y mucosas, desempeñan diversas funciones orgánicas. Actúan en el proceso masticatorio, en la percepción de los sabores, además de servir de vía respiratoria. La relación entre alteraciones sistémicas y manifestaciones orales está bien relatada en la literatura. Observándose en numerosos estudios, palabras claves de morbilidades primariamente bucales, seguidas del diagnóstico de trastornos sistémicos. Las enfermedades reumáticas pueden manifestarse con alteraciones de la condición bucal, además de las presentaciones clínicas peculiares a cada morbilidad. Para la realización de este trabajo fueron investigados en la literatura estudios que evidencien la presencia de manifestaciones orales en enfermedades reumáticas a través de la búsqueda en las bases de datos Lilacs, PubMed y Bireme, incluidos en el año 2009-2015. Las evidencias existentes en la literatura nos revelan la importancia de la inclusión de la evaluación clínica de la boca y sus estructuras anexas, como elemento importante en el diagnóstico auxiliar y orientación para el manejo adecuado de las quejas de estos pacientes.

The oral cavity has several physiological functions through their structures. Such structures act in the chewing process, the perception of flavors, besides serving airway. The relationship between systemic diseases and oral manifestations is well reported in the literature. It is observed in numerous studies, morbidity descriptions primarily mouth followed by a diagnosis of systemic disorders. Rheumatic diseases exemplify that these can manifest with changes in oral health in addition to the peculiar clinical presentations to each morbidity. For this work we investigated in the literature studies that provided evidence of the presence of oral manifestations in rheumatic diseases by searching Lilacs, PubMed and Bireme databases, included in the year 2009­2015. The available evidence in the literature, reveal the importance of including clinical evaluation of the mouth and its associated structures as an important element for the diagnosis and guidance for the proper handling of complaints of these patients.

Loss of SFRP4 Alters Body Size, Food Intake, and Energy Expenditure in Diet-Induced Obese Male Mice.

Secreted frizzled-related protein 4 (SFRP4) is an extracellular regulator of the wingless-type mouse mammary tumor virus integration site family (WNT) pathway. SFRP4 has been implicated in adipocyte dysfunction, obesity, insulin resistance, and impaired insulin secretion in patients with type 2 diabetes. However, the exact role of SFRP4 in regulating whole-body metabolism and glucose homeostasis is unknown. We show here that male Sfrp4(-/-) mice have increased spine length and gain more weight when fed a high-fat diet. The body composition and body mass per spine length of diet-induced obese Sfrp4(-/-) mice is similar to wild-type littermates, suggesting that the increase in body weight can be accounted for by their longer body size. The diet-induced obese Sfrp4(-/-) mice have reduced energy expenditure, food intake, and bone mineral density. Sfrp4(-/-) mice have normal glucose and insulin tolerance and ß-cell mass. Diet-induced obese Sfrp4(-/-) and control mice show similar impairments of glucose tolerance and a 5-fold compensatory expansion of their ß-cell mass. In summary, our data suggest that loss of SFRP4 alters body length and bone mineral density as well as energy expenditure and food intake. However, SFRP4 does not control glucose homeostasis and ß-cell mass in mice.

Conditionals by inversion provide a universal method for the generation of conditional alleles.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.

Discovery of new antimalarial chemotypes through chemical methodology and library development.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.

The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis.

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.

Vascular endothelial tyrosine phosphatase (VE-PTP)-null mice undergo vasculogenesis but die embryonically because of defects in angiogenesis.

Development of the vascular system depends on the highly coordinated actions of a variety of angiogenic regulators. Several of these regulators are members of the tyrosine kinase superfamily, including VEGF receptors and angiopoietin receptors, Tie1 and Tie2. Tyrosine kinase signaling is counter-regulated by the activity of tyrosine phosphatases, including vascular endothelial protein tyrosine phosphatase (VE-PTP), which has previously been shown to modulate Tie2 activity. We generated mice in which VE-PTP is replaced with a reporter gene. We confirm that VE-PTP is expressed in endothelium and also show that VE-PTP is highly expressed in the developing outflow tract of the heart and later is expressed in developing heart valves. Vasculogenesis occurs normally in mice lacking VE-PTP; however, angiogenesis is abnormal. Angiogenic defects in VE-PTP-null mice were most pronounced in the yolk sac and include a complete failure to elaborate the primitive vascular scaffold into higher-order branched arteries, veins, and capillaries. VE-PTP continues to be expressed into adulthood in the vasculature and heart valves, suggesting later roles in vascular development or homeostasis. VE-PTP is also expressed in the vasculature of growing tumors, suggesting that VE-PTP may be a new potential target for angiogenic therapies.

Immobilized hydrogels for screening of molecular interactions.

Spatially arrayed, high-density microarrays enable the rapid assessment of biological recognition events, and this information is of widespread interest for those working in basic research laboratories as well as in the clinic. Today, one can find DNA, protein, or small molecule arrays. Limitations with these systems include covalent modification of the target complement to the array substrate, array- and target-dependent setup conditions, multiple steps, and loss of hydration at the surface. To overcome these limitations, we have designed, prepared, and evaluated immobilized hydrogels as general screening chambers for small molecule-protein, protein-protein, and nucleic acid-nucleic acid interactions. This biomaterial-based approach is facile, rapid, requires only one setup protocol, and physically entraps the target complement within the polymer network and thus offers advantages over the conventional chips.

Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1.

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell-stage embryos efficiently yields F0 generation mice that are fully ES cell-derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.

A spiking neuron model of cortical correlates of sensorineural hearing loss: Spontaneous firing, synchrony, and tinnitus.

Hearing loss due to peripheral damage is associated with cochlear hair cell damage or loss and some retrograde degeneration of auditory nerve fibers. Surviving auditory nerve fibers in the impaired region exhibit elevated and broadened frequency tuning, and the cochleotopic representation of broadband stimuli such as speech is distorted. In impaired cortical regions, increased tuning to frequencies near the edge of the hearing loss coupled with increased spontaneous and synchronous firing is observed. Tinnitus, an auditory percept in the absence of sensory input, may arise under these circumstances as a result of plastic reorganization in the auditory cortex. We present a spiking neuron model of auditory cortex that captures several key features of cortical organization. A key assumption in the model is that in response to reduced afferent excitatory input in the damaged region, a compensatory change in the connection strengths of lateral excitatory and inhibitory connections occurs. These changes allow the model to capture some of the cortical correlates of sensorineural hearing loss, including changes in spontaneous firing and synchrony; these phenomena may explain central tinnitus. This model may also be useful for evaluating procedures designed to segregate synchronous activity underlying tinnitus and for evaluating adaptive hearing devices that compensate for selective hearing loss.

Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development.

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro- and anti-angiogenic therapies.