Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegf(lo). Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing 'Vegf wild-type' visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the 'Vegf wild-type' yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.     

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.     

Retinoic acid signaling in vascular development

Formation of the vasculature is an essential developmental process, delivering oxygen and nutrients to support cellular processes needed for tissue growth and maturation. Retinoic acid (RA) and its downstream signaling pathway is vital for normal pre‐ and post‐natal development, playing key roles in the specification and formation of many organs and tissues. Here, we review the role of RA in blood and lymph vascular development, beginning with embryonic yolk sac vasculogenesis and remodeling and discussing RA's organ‐specific roles in angiogenesis and vessel maturation. In particular, we highlight the multi‐faceted role of RA signaling in CNS vascular development and acquisition of blood–brain barrier properties.     

Defective smooth muscle development in qkI-deficient mice

The qkI gene encodes an RNA binding protein which was identified as a candidate for the classical neurologic mutation, qkv. Although qkI is involved in glial cell differentiation in mice, qkI homologues in other species play important roles in various developmental processes. Here, we show a novel function of qkI in smooth muscle cell differentiation during embryonic blood vessel formation. qkI null embryos died between embryonic day 9.5 and 10.5. Embryonic day 9.5 qkI null embryos showed a lack of large vitelline vessels in the yolk sacs, kinky neural tubes, pericardial effusion, open neural tubes and incomplete embryonic turning. Using X-gal and immunohistochemical staining, qkI is first shown to be expressed in endothelial cells and smooth muscle cells. Analyses of qkI null embryos in vivo and in vitro revealed that the vitelline artery was too thin to connect properly to the yolk sac, thereby preventing remodeling of the yolk sac vasculature, and that the vitelline vessel was deficient in smooth muscle cells. Addition of QKI and platelet-endothelial cell adhesion molecule-1 positive cells to an in vitro para-aortic splanchnopleural culture of qkI null embryos rescued the vascular remodeling deficit. These data suggest that QKI protein has a critical regulatory role in smooth muscle cell development, and that smooth muscle cells play an important role in inducing vascular remodeling.     

Alternatively Spliced Tissue Factor Is Not Sufficient for Embryonic Development

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.     

Platelet-derived growth factor receptors direct vascular development independent of vascular smooth muscle cell function

Complete loss of platelet-derived growth factor (PDGF) receptor signaling results in embryonic lethality around embryonic day 9.5, but the cause of this lethality has not been identified. Because cardiovascular failure often results in embryonic lethality at this time point, we hypothesized that a failure in cardiovascular development could be the cause. To assess the combined role of PDGF receptor α (PDGFRα) and PDGFRβ, we generated embryos that lacked these receptors in cardiomyocytes and vascular smooth muscle cells (VSMC) using conditional gene ablation. Deletion of either PDGFRα or PDGFRβ caused no overt vascular defects, but loss of both receptors using an SM22α-Cre transgenic mouse line led to a disruption in yolk sac blood vessel development. The cell population responsible for this vascular defect was the yolk sac mesothelial cells, not the cardiomyocytes or the VSMC. Coincident with loss of PDGF receptor signaling, we found a reduction in collagen deposition and an increase in MMP-2 activity. Finally, in vitro allantois cultures demonstrated a requirement for PDGF signaling in vessel growth. Together, these data demonstrate that PDGF receptors cooperate in the yolk sac mesothelium to direct blood vessel maturation and suggest that these effects are independent of their role in VSMC development.     

Defective vascular morphogenesis and mid-gestation embryonic death in mice lacking RA-GEF-1

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1(-/-) mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1(-/-) yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1(-/-) embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.     

Vascular remodeling of the mouse yolk sac requires hemodynamic force

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a(-/-)). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.     

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.     

Gastrulation and angiogenesis,not endothelial specification,is sensitive to partial deficiency in vascular endothelial growth factor-a in mice

Mouse embryogenesis is dose sensitive to vascular endothelial growth factor-A (VEGF-A), and mouse embryos partially deficient in VEGF-A die in utero because of severe vascular defects. In this study, we investigate the possible causes that underlie this phenomenon. Although the development of vascular defects in VEGF-A-deficient embryos seems to suggest that endothelial differentiation depends on the presence of a sufficient level of VEGF-A, we were surprised to find that endothelial differentiation per se is insensitive to a significant loss of VEGF-A activity. Instead, the development of the multipotent mesenchymal cells, from which endothelial progenitors arise in the yolk sac, is most highly dependent on VEGF-A. As a result of VEGF-A deficiency, dramatically fewer multipotent mesenchymal cells are generated in the prospective yolk sac. However, among the small number of mesenchymal cells that do enter the prospective yolk sac, endothelial differentiation occurs at a normal frequency. In the embryo proper, vasculogenesis is initiated actively in spite of a significant VEGF-A deficiency, but the subsequent steps of vascular development are defective. We conclude that a full-level VEGF-A activity is not critical for endothelial specification but is important for two distinct processes before and after endothelial specification: the development of the yolk sac mesenchyme and angiogenic sprouting of blood vessels.     

Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells

Zonula occludens (ZO)-1/2/3 are the members of the TJ-MAGUK family of membrane-associated guanylate kinases associated with tight junctions. To investigate the role of ZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1(-/-)) mice were generated by gene targeting. Although heterozygous mice showed normal development and fertility, delayed growth and development were evident from E8.5 onward in Tjp1(-/-) embryos, and no viable Tjp1(-/-) embryos were observed beyond E11.5. Tjp1(-/-) embryos exhibited massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E)9.5. In the yolk sac, the ZO-1 deficiency induced defects in vascular development, with impaired formation of vascular trees, along with defective chorioallantoic fusion. Immunostaining of wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2 were expressed in almost all embryonic cells, showing tight junction-localizing patterns, with or without ZO-3, which was confined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expression without influence on ZO-2/3 expression. In Tjp1(+/+) yolk sac extraembryonic mesoderm, ZO-1 was dominant without ZO-2/3 expression. Thus, ZO-1 deficiency resulted in mesoderms with no ZO-1/2/3, associated with mislocalization of endothelial junctional adhesion molecules. As a result, angiogenesis was defected in Tjp1(-/-) yolk sac, although differentiation of endothelial cells seemed to be normal. In conclusion, ZO-1 may be functionally important for cell remodeling and tissue organization in both the embryonic and extraembryonic regions, thus playing an essential role in embryonic development.     

The notch ligand delta-like 4 regulates multiple stages of early hemato-vascular development

Background

In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis.

Methodology/Principal Findings

Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4^+/− and Dll4^−/− embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis.

Conclusions/Significance

This study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis.     

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.     

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.     

Endoglin, an ancillary TGFbeta receptor, is required for extraembryonic angiogenesis and plays a key role in heart development

Endoglin (CD105) is expressed on the surface of endothelial and haematopoietic cells in mammals and binds TGFbeta isoforms 1 and 3 in combination with the signaling complex of TGFbeta receptors types I and II. Endoglin expression increases during angiogenesis, wound healing, and inflammation, all of which are associated with TGFbeta signaling and alterations in vascular structure. The importance of endoglin for normal vascular architecture is further indicated by the association of mutations in the endoglin gene with the inherited disorder Hereditary Haemorrhagic Telangiectasia Type 1 (HHT1), a disease characterised by bleeding from vascular malformations. In order to study the role of endoglin in vivo in more detail and to work toward developing an animal model of HHT1, we have derived mice that carry a targeted nonsense mutation in the endoglin gene. Studies on these mice have revealed that endoglin is essential for early development. Embryos homozygous for the endoglin mutation fail to progress beyond 10.5 days postcoitum and fail to form mature blood vessels in the yolk sac. This phenotype is remarkably similar to that of the TGFbeta1 and the TGFbeta receptor II knockout mice, indicating that endoglin is needed in vivo for TGFbeta1 signaling during extraembryonic vascular development. In addition, we have observed cardiac defects in homozygous endoglin-deficient embryos, suggesting endoglin also plays a role in cardiogenesis. We anticipate that heterozygous mice will ultimately serve as a useful disease model for HHT1, as some individuals have dilated and fragile blood vessels similar to vascular malformations seen in HHT patients.     

Yolk sac development in lizards (Lacertilia: Scincidae): New perspectives on the egg of amniotes

Embryos of oviparous reptiles develop on the surface of a large mass of yolk, which they metabolize to become relatively large hatchlings. Access to the yolk is provided by tissues growing outward from the embryo to cover the surface of the yolk. A key feature of yolk sac development is a dedicated blood vascular system to communicate with the embryo. The best known model for yolk sac development and function of oviparous amniotes is based on numerous studies of birds, primarily domestic chickens. In this model, the vascular yolk sac forms the perimeter of the large yolk mass and is lined by a specialized epithelium, which takes up, processes and transports yolk nutrients to the yolk sac blood vessels. Studies of lizard yolk sac development, dating to more than 100 years ago, report characteristics inconsistent with this model. We compared development of the yolk sac from oviposition to near hatching in embryonic series of three species of oviparous scincid lizards to consider congruence with the pattern described for birds. Our findings reinforce results of prior studies indicating that squamate reptiles mobilize and metabolize the large yolk reserves in their eggs through a process unknown in other amniotes. Development of the yolk sac of lizards differs from birds in four primary characteristics, migration of mesoderm, proliferation of endoderm, vascular development and cellular diversity within the yolk sac cavity. Notably, all of the yolk is incorporated into cells relatively early in development and endodermal cells within the yolk sac cavity align along blood vessels which course throughout the yolk sac cavity. The pattern of uptake of yolk by endodermal cells indicates that the mechanism of yolk metabolism differs between lizards and birds and that the evolution of a fundamental characteristic of embryonic nutrition diverged in these two lineages. Attributes of the yolk sac of squamates reveal the existence of phylogenetic diversity among amniote lineages and raise new questions concerning the evolution of the amniotic egg. J. Morphol. 278:574–591, 2017. © 2017 Wiley Periodicals, Inc.     

Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.     

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-β superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.