Induction of cells expressing vascular endothelium markers from undifferentiated Xenopus presumptive ectoderm by co-treatment with activin and angiopoietin-2

Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegfl7. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as Xl-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.     

Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo

The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.     

Paracrine and autocrine mechanisms of apelin signaling govern embryonic and tumor angiogenesis

Apelin and its G protein-coupled receptor APJ play important roles in blood pressure regulation, body fluid homeostasis, and possibly the modulation of immune responses. Here, we report that apelin-APJ signaling is essential for embryonic angiogenesis and upregulated during tumor angiogenesis. A detailed expression analysis demonstrates that both paracrine and autocrine mechanisms mark areas of embryonic and tumor angiogenesis. Knockdown studies in Xenopus reveal that apelin-APJ signaling is required for intersomitic vessel angiogenesis. Moreover, ectopic expression of apelin but not vascular endothelial growth factor A (VEGFA) is sufficient to trigger premature angiogenesis. In vitro, apelin is non-mitogenic for primary human endothelial cells but promotes chemotaxis. Epistasis studies in Xenopus embryos suggest that apelin-APJ signaling functions downstream of VEGFA. Finally, we show that apelin and APJ expression is highly upregulated in microvascular proliferations of brain tumors such as malignant gliomas. Thus, our results define apelin and APJ as genes of potential diagnostic value and promising targets for the development of a new generation of anti-tumor angiogenic drugs.     

In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.     

Isolation of cardiac cells from E8.5 yolk sac by ALCAM (CD166) expression

It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system.     

Conditions that influence the response to Fgf during otic placode induction

More than a century ago, several embryologists described sites of hematopoietic activity in the vascular wall of mid-gestation vertebrate embryos, and postulated the transient existence of a blood generating endothelium during ontogeny. This hypothesis gained significant attention in the 1970s when orthotopic transplantation experiments between quail and chick embryos revealed specific vascular areas as the site of the origin of definitive hematopoiesis. However, the vascular origin of hematopoietic precursors remained elusive and controversial for decades. Only recently, multiple experimental approaches have clearly documented that during vertebrate development definitive hematopoietic precursors arise from a subset of vascular endothelial cells. Interestingly, this differentiation is promoted by the intravascular fluid mechanical forces generated by the establishment of blood flow upon the initiation of heartbeat, and it is therefore connected with cardiovascular development in several critical aspects. In this review we present our current understanding of the relationship between vascular and definitive hematopoietic development through an historical analysis of the scientific evidence produced in this area of investigation.     

Changes in embryonic cell fate produced by expression of an endodermal transcription factor, Xsox17

Many molecules induce the ectopic expression of tissue-specific genes in Xenopus embryos. Conversely, interfering with their activity disrupts patterns of gene expression, implicating them in normal development. Does this mean that they control cell fate (i.e. position, as well as differentiation)? Xsox17alpha and beta can induce ectopic expression of endodermal markers; inhibiting their function suppresses expression of endodermal marker genes in the developing gut (Cell 91 (1997) 397). Here we show the effect of these manipulations on cell lineage. Expressing Xsox17 in a cells normally fated to become ectoderm causes their descendants either to relocate into the embryonic gut or to die at a late developmental stage. Conversely, disrupting Xsox17 activity in cells normally fated to be endodermal causes them to enter mesodermal and ectodermal lineages.     

YY1 regulates the neural crest-associated slug gene in Xenopus laevis

slug gene expression is associated with the specification and migration of neural crest cells in the African clawed frog Xenopus laevis. We provide evidence that the protein Ying-Yang 1 (YY1) regulates the slug gene expression both indirectly and directly, via a YY1 cis-element in the slug promoter, during Xenopus development. The ability of the YY1 to bind this YY1 cis-element was confirmed by electromobility shift assays and reporter assays. YY1 was detected in the nuclei of ectodermal cells contemporaneously with the process of neural crest specification. The injection of anti-YY1 morpholino, which targeted both YY1alpha and YY1beta gene products, depleted YY1 expression below 20% and was lethal at gastrulation. Sublethal depletion of YY1 reduced the length of the anterior-posterior axis and severely inhibited the expression of the neural marker Nrp1 and of the slug gene. Overexpression of YY1 or mutation of the YY1 cis-element reduced the restricted spatial expression of the slug reporter gene in the neural ectoderm border and provoked its expression in the nonneural ectoderm. Chromatin immunoprecipitation indicated that endogenous YY1 interacts directly with the YY1 cis-element of the endogenous slug gene and with the slug gene reporter sequence injected into embryos. The results suggest that YY1 is essential for Xenopus development; is necessary for neural ectoderm differentiation, a prerequisite for neural crest specification; and restricts which cells can form neural crest mesenchyme through directly blocking slug gene activity.     

Endocardial cells are a distinct endothelial lineage derived from Flk1+ multipotent cardiovascular progenitors

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal–spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.     

The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.