Hedgehog is required for murine yolk sac angiogenesis.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.     

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.     

Extraembryonic origin of circulating endothelial cells

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.     

Defective vascular development in connexin 45-deficient mice

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45(+/)(-) mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45(-)(/)(-) embryos could be caused by defective TGFbeta signalling, as the amount of TGF beta1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45(-)(/)(-) embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45(-)(/)(-) embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).     

Defective paracrine signalling by TGFbeta in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder in humans that is characterised by multisystemic vascular dyplasia and recurrent haemorrhage. Germline mutations in one of two different genes, endoglin or ALK1 can cause HHT. Both are members of the transforming growth factor (TGF) beta receptor family of proteins, and are expressed primarily on the surface of endothelial cells (ECs). Mice that lack endoglin or activin receptor like kinase (ALK) 1 die at mid-gestation as a result of defects in the yolk sac vasculature. Here, we have analyzed TGFbeta signalling in yolk sacs from endoglin knockout mice and from mice with endothelial-specific deletion of the TGFbeta type II receptor (TbetaRII) or ALK5. We show that TGFbeta/ALK5 signalling from endothelial cells to adjacent mesothelial cells is defective in these mice, as evidenced by reduced phosphorylation of Smad2. This results in the failure of vascular smooth muscle cells to differentiate and associate with endothelial cells so that blood vessels remain fragile and become dilated. Phosphorylation of Smad2 and differentiation of smooth muscle can be rescued by culture of the yolk sac with exogenous TGFbeta1. Our data show that disruption of TGFbeta signalling in vascular endothelial cells results in reduced availability of TGFbeta1 protein to promote recruitment and differentiation of smooth muscle cells, and provide a possible explanation for weak vessel walls associated with HHT.     

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.     

Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5

The transforming growth factor-beta (TGF-beta) signals are mediated by a family of at least nine SMAD proteins, of which SMAD5 is thought to relay signals of the bone morphogenetic protein (BMP) pathway. To investigate the role of SMAD5 during vertebrate development and tumorigenesis, we disrupted the Smad5 gene by homologous recombination. We showed that Smad5 was expressed predominantly in mesenchyme and somites during embryogenesis, and in many tissues of the adult. Mice homozygous for the mutation died between days 10.5 and 11.5 of gestation due to defects in angiogenesis. The mutant yolk sacs lacked normal vasculature and had irregularly distributed blood cells, although they contained hematopoietic precursors capable of erythroid differentiation. Smad5 mutant embryos had enlarged blood vessels surrounded by decreased numbers of vascular smooth muscle cells, suffered massive apoptosis of mesenchymal cells, and were unable to direct angiogenesis in vitro. These data suggest that SMAD5 may regulate endothelium-mesenchyme interactions during angiogenesis and that it is essential for mesenchymal survival.     

Visceral Endoderm Expression of Yin-Yang1 (YY1) Is Required for VEGFA Maintenance and Yolk Sac Development

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.     

Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

Cell-autonomous requirement for beta1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice

beta1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of beta1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1(-/-) embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day (e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate that beta1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting beta1 integrins may effectively impair neovascularization.     

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.