Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation

Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.     

Mesodermal retinoic acid signaling regulates endothelial cell coalescence in caudal pharyngeal arch artery vasculogenesis

Disruption of retinoic acid signaling causes a variety of pharyngeal arch artery and great vessel defects, as well as malformations in many other tissues, including those derived from the pharyngeal endoderm. Previous studies implied that arch artery defects in the context of defective RA signaling occur secondary to pharyngeal pouch segmentation defects, although this model has never been experimentally verified. In this study, we examined arch artery morphogenesis during mouse development, and the role of RA in this process. We show in normal embryos that the arch arteries form by vasculogenic differentiation of pharyngeal mesoderm. Using various genetic backgrounds and tissue-specific mutation approaches, we segregate pharyngeal arch artery and pharyngeal pouch defects in RA receptor mutants, and show that RA signal transduction only in pharyngeal mesoderm is required for arch artery formation. RA does not control pharyngeal mesodermal differentiation to endothelium, but instead promotes the aggregation of endothelial cells into nascent vessels. Expression of VE-cadherin was substantially reduced in RAR mutants, and this deficiency may underlie the arch artery defects. The consequences of disrupted mesodermal and endodermal RA signaling were restricted to the 4th and 6th arch arteries and to the 4th pharyngeal pouch, respectively, suggesting that different regulatory mechanisms control the formation of the more anterior arch arteries and pouches.     

Tbx1 expression in pharyngeal epithelia is necessary for pharyngeal arch artery development

During embryonic life, the initially paired pharyngeal arch arteries (PAAs) follow a precisely orchestrated program of persistence and regression that leads to the formation of the mature aortic arch and great vessels. When this program fails, specific cardiovascular defects arise that may be life threatening or mild, according to the identity of the affected artery. Fourth PAA-derived cardiovascular defects occur commonly in DiGeorge syndrome and velocardiofacial syndrome (22q11DS), and in Tbx1(+/-) mice that model the 22q11DS cardiovascular phenotype. Tbx1 is expressed in pharyngeal mesoderm, endoderm and ectoderm, and, in addition, we show that it is expressed in precursors of the endothelial cells that line the PAAs, thus expanding the number of tissues in which Tbx1 is potentially required for fourth PAA development. In this study, we have used cell fate mapping and tissue-specific gene deletion, driven by six different Cre lines, to explore Tbx1 gene-dosage requirements in the embryonic pharynx for fourth PAA development. Through this approach, we have resolved the spatial requirements for Tbx1 in this process, and we show pharyngeal epithelia to be a critical tissue. We also thereby demonstrate conclusively that the role of Tbx1 in fourth PAA development is cell non-autonomous.     

PDGF signaling from pharyngeal pouches promotes arch artery morphogenesis

The great vessels of the heart originate from the pharyngeal arch arteries(PAAs).Anomalies of the PAAs often occur together with pharyngeal pouch malfo rmations,but the reasons for this phenomenon are not fully understood.In the current study,we show that platelet-derived growth factor(PDGF) signaling derived from the pharyngeal pouches plays an important function in PAA vasculogenesis,During PAA development in zebrafish embryos,pdgfaa and pdgfab are expressed in the developing pharyngeal pouches.Results from loss-of-function experiments revealed a critical role of these genes in PAA formation.We found that nitroreductase(NTR)-mediated pouch ablation distinctly decreased PDGF receptor tyrosine phosphorylation,yielding a severe loss of PAAs.Importantly,pouch-specific overexpression of pdgfaa in pdgfaa~(-/-);pdgfab~(-/-)mutants significantly relieved the PAA defects,which indicated a primary role of pharyngeal pouch-expressed PDGF ligands in signal activation and PAA morphogenesis.Our findings further showed that PDGF signaling was indispensable for the proliferation of PAA angioblasts.Together,these results established a role for PDGFaa-and PDGFab-mediated tissuetissue interaction during PAA development.     

Reduced maternal vitamin A status increases the incidence of normal aortic arch variants

While common in the general population, the developmental origins of “normal” anatomic variants of the aortic arch remain unknown. Aortic arch development begins with the establishment of the second heart field (SHF) that contributes to the pharyngeal arch arteries (PAAs). The PAAs remodel during subsequent development to form the mature aortic arch and arch vessels. Retinoic acid signaling involving the biologically active metabolite of vitamin A, plays a key role in multiple steps of this process. Recent work from our laboratory indicates that the E3 ubiquitin ligase Hectd1 is required for full activation of retinoic acid signaling during cardiac development. Furthermore, our study suggested that mild alterations in retinoic acid signaling combined with reduced gene dosage of Hectd1, results in a benign aortic arch variant where the transverse aortic arch is shortened between the brachiocephalic and left common carotid arteries. These abnormalities are preceded by hypoplasia of the fourth PAA. To further explore this interaction, we investigate whether reduced maternal dietary vitamin A intake can similarly influence aortic arch development. Our findings indicate that the incidence of hypoplastic fourth PAAs, as well as the incidence of shortened transverse arch are increased with reduced maternal vitamin A intake during pregnancy. These studies provide new insights as to the developmental origins of these benign aortic arch variants.     

Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants

Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell‐specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms. genesis 48:146–150, 2010. © 2010 Wiley‐Liss, Inc.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

meis1 regulates the development of endothelial cells in zebrafish

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor (vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino (meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.     

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.     

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1^Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures.     

Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling

Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk^−/− and SLP-76^−/− mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling.     

Notch1 Is Pan-Endothelial at the Onset of Flow and Regulated by Flow

Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm²). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.     

tgfβ3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest

Zebrafish tgfβ3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfβ3 in head skeletal formation, we knocked down tgfβ3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfβ3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfβ3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfβ3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfβ3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfβ3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfβ3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfβ3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfβ3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfβ3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfβ3 protein levels in zebrafish.     

<Emphasis Type="Italic">Hoxa3</Emphasis> and signaling molecules involved in aortic arch patterning and remodeling

Anomalies of the aortic arch have long been of anatomicoclinical interest. Recent studies on gene-targeted mice have identified the candidate genes that are involved in the patterning and remodeling of the pharyngeal arch arteries. In this review, we discuss our present knowledge with regard to the signaling molecules that regulate specific aspects of arch artery development. We focus first on Hoxa3, because it plays a critical role in the regulation of the differentiation of the third pharyngeal arch. Hoxa3 is expressed by the neural crest cells that originate from the rhombomeres, viz., (r)5, r6, and r7, and populate the third pharyngeal arch; it is also expressed in the third pharyngeal pouch. In Hoxa3 homozygous null mutant mice, the third arch artery degenerates bilaterally at embryonic day 11.5, resulting in the malformation of the carotid artery system. Complex combinatorial signals among the neural crest cells, pharyngeal mesoderm, ectoderm, and pouch endoderm are required for the proper development of the arch arterial system. Therefore, we highlight the numerous signaling pathways and individual genes expressed by the ectomesenchymal neural crest cells and also by the other epithelial and mesodermal cells of the pharynx. Defects in these genes result in malformations of the arch artery derivatives. This review should deepen our understanding of congenital human syndromes with abnormal patterns of pharyngeal arch arteries.     

Hes1 and Hes5 regulate vascular remodeling and arterial specification of endothelial cells in brain vascular development

The vascular system is the first organ to form in the developing mammalian embryo. The Notch signaling pathway is an evolutionarily conserved signaling mechanism essential for proper embryonic development in almost all vertebrate organs. The analysis of targeted mouse mutants has demonstrated essential roles of the Notch signaling pathway in embryonic vascular development. However, Notch signaling-deficient mice have so far not been examined in detail in the head region. The bHLH genes Hes1 and Hes5 are essential effectors for Notch signaling, which regulate the maintenance of progenitor cells and the timing of their differentiation in various tissues and organs. Here, we report that endothelial-specific Hes1 and Hes5 mutant embryos exhibited defective vascular remodeling in the brain. In addition, arterial identity of endothelial cells was partially lost in the brain of these mutant mice. These data suggest that Hes1 and Hes5 regulate vascular remodeling and arterial fate specification of endothelial cells in the development of the brain. Hes1 and Hes5 represent critical transducers of Notch signals in brain vascular development.