The role of FGF and VEGF in angioblast induction and migration during vascular development.

The embryonic vasculature forms by the processes of vasculogenesis and angiogenesis. Angioblasts (endothelial cell precursors) appear to be induced by fibroblast growth factor 2 (FGF-2). The angioblasts contributing to the dorsal aortae arise by an epithelial to mesenchymal transformation of cells originating from the splanchnic mesoderm. QH-l and vascular endothelial growth factor receptor 2 (VEGFR-2) both appear to label these cells as they adopt a mesenchymal morphology. Since VEGFR-2 is the earliest known VEGF receptor this suggests that VEGF is not involved in angioblast induction. VEGF does appear to be critical, however, for growth and morphogenesis of angioblasts into the initial vascular pattern. Controlled delivery of FGF-2 from beads and aggregates of cells transfected with quail VEGF have been used in our laboratory to study the role of these growth factors in angioblast induction and migration. We have induced cells from the epithelial quail somite to differentiate into angioblasts with FGF-2 both in the embryo and in culture. This is a useful model system to study the origins of endothelial cells that are normally more diffusely induced during gastrulation by an obscure process probably involving signals from the embryonic endoderm. The origins of arterial versus venous endothelial cells is also poorly understood but recent findings on the distribution of ephrins and Eph receptors suggest that molecular differences exist prior to the onset of circulation. Finally, studies on the role of growth factors in such diverse phenomena as stem cell biology, angiogenesis, and molecular medicine in addition to vascular development suggest multiple roles for FGF-2 and VEGF in vascular development.

Angioblast differentiation is influenced by the local environment: FGF-2 induces angioblasts and patterns vessel formation in the quail embryo.

The embryonic vasculature forms by the segregation, migration, and assembly of angioblasts from mesoderm, a process termed vasculogenesis. The initial role of fibroblast growth factor 2 (FGF-2) in vascular development appears to be in the induction of endothelial precursors, angioblasts. Quail somites transplanted into chick embryos will give rise to angioblasts of quail origin. The number of angioblasts present within the chimera is dependent on the host environment. Angioblast induction can be demonstrated in vitro by the addition of FGF-2 to cultures of dissociated somitic mesoderm, as assessed by QH-1 epitope expression. Manipulation of FGF-2 concentration in the quail/chick chimeras by FGF-2 peptide or neutralizing antibody injections increases or decreases angioblast induction in the predicted manner. To better control growth factor release in vivo we have implanted beads that release FGF-2 into the embryonic environment. FGF-2 beads implanted into the somite induce angioblast differentiation in the epithelial somite; whereas, beads lateral to the somitic mesoderm induce the formation of ectopic vessels. These studies suggest that FGF-2 is important for both the induction of angioblasts and the assembly of angioblasts into the initial vasculature pattern.

Developing blood vessels and associated extracellular matrix as substrates for neural crest migration in Japanese quail, Coturnix coturnix japonica.

Japanese quail embryos were used to examine paths of neural crest cell (NC) migration in relationship to the embryonic vasculature. Immunolabeling for NC, angioblasts and the extracellular matrix (ECM) glycoproteins fibronectin (FN), laminin (LN) and tenascin (TN) revealed several instances where spatiotemporal patterns of NC migration coincide with the embryonic vascular pattern and its associated ECM. An in vitro model for angiogenesis was modified to include NC, and associations with "capillary-like" endothelial cell structures were demonstrated. A working hypothesis is that the embryonic vasculature may, in specific instances, be used as a substratum for directed NC migration and that these interactions are mediated primarily through the adhesive interactions of FN. Some members of the TN family of glycoproteins, through their relatively non-adhesive properties, may act to help guide neural crest cells to the FN-rich blood vessel surface.

Endothelial cell origin and migration in embryonic heart and cranial blood vessel development.

Using the QH-1 monoclonal antibody as a marker for quail endothelium, blockage and transplant experiments were carried out to construct fate maps for the embryonic endocardium, to determine whether preendocardial angioblasts are migratory, and, if these cells are migratory, to outline the pathways that they use for directed migration in embryonic blood vessel development. Recent descriptive studies using QH-1 to make immunofluorescent whole mounts have described a sequence of events leading to the establishment of the embryonic heart tube. These reports suggest that the pattern for the endocardium and cranial vasculature is established by migrating angioblasts that form vascular cords which mature into blood vessels. Blockage experiments showed that the ventrolateral edge of the anterior intestinal portal serves as a substrate for the directed migration of pre-endocardial angioblasts and that the pattern of the cranioventral vasculature forms independent of the source of angioblasts. Transplant experiments showed that the origin for endocardial angioblasts lies in mesodermal tissue just anterior to Henson's node, that these cells undergo directed migration to the pericardial area, and that angioblasts are pluripotent with the ability to form different blood vessels. The transplant studies also showed that the embryonic mesoderm may contribute to extraembryonic blood vessels on the embryonic yolksac. These results support the hypothesis that embryonic blood vessels may develop by either the vasculogenesis or by the angiogenesis mechanism, and show that the endocardium of the primitive heart tube forms by vasculogenesis.

Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern.

We are using a monoclonal antibody, QH-1, as a label for angioblasts in quail embryos to study vascular development. Our previous experiments showed that major embryonic blood vessels, such as the dorsal aortae and posterior cardinal veins, develop from angioblasts of mesodermal origin that appear in the body of the embryo proper (Coffin and Poole: Development, 102:735-748, '88). We theorized that there are two separate processes for blood vessel development that occur in quail embryos. One mechanism termed "vasculogenesis" forms blood vessels in place by the aggregation of angioblasts into a cord. The other mechanism, termed "angiogenesis," is the formation of new vessels by sprouting of capillaries from existing vessels. Here we report the results of microsurgical transplantation experiments designed to determine the extent of cell migration taking place during blood vessel formation. Comparison of the chimeras to normal embryos suggests that the vascular pattern develops, in part, from the normally restricted points of entry of angioblasts into the head from the ventral and dorsal aortae. Transplantations of quail mesoderm (1-15 somite stage) into the head of 5-15 somite chick hosts resulted in extensive sprouting and in migration of single and small groups of angioblasts away from the graft sites. Transplantations into the trunk resulted in incorporation of the graft into the normal vascular pattern of the host. Lateral plate mesoderm was incorporated into the dorsal aortae and individual sprouts grew between somites and along the neural tube to contribute to the intersomitic and vertebral arteries, respectively.

Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos.

The development of the embryonic vasculature is examined here using a monoclonal antibody, QH-1, capable of labelling the presumptive endothelial cells of Japanese quail embryos. Antibody labelling is first seen within the embryo proper at the 1-somite stage. Scattered labelling of single cells appears ventral to the somites and at the lateral edges of the anterior intestinal portal. The dorsal aorta soon forms a continuous cord at the ventrolateral edge of the somites and continues into the head to fuse with the ventral aorta forming the first aortic arch by the 6-somite stage. The rudiments of the endocardium fuse at the midline above the anterior intestinal portal by the 3-somite stage and the ventral aorta extends craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior cardinal vein forms from single cells which segregate from somatic mesoderm at the 7-somite stage to form a loose plexus which moves mediad and wraps around the developing Wolffian duct in later stages. These studies suggest two modes of origin of embryonic blood vessels. The dorsal aortae and cardinal veins apparently arise in situ by the local segregation of presumptive endothelial cells from the mesoderm. The intersomitic arteries, vertebral arteries and cephalic vasculature arise by sprouts from these early vessel rudiments. There also seems to be some cell migration in the morphogenesis of endocardium, ventral aorta and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be clarified by microsurgical intervention.

Developmental angiogenesis: quail embryonic vasculature.

We have examined the segregation and early morphogenesis of the embryonic vasculature by using a monoclonal antibody for immunofluorescence and by scanning electron microscopy. This antibody labels the presumptive endothelial cells (PECs) as they segregate from mesoderm. Similar embryos prepared for SEM revealed finer details of how these segregated cells interact to form the rudiments of the major blood vessels. Here we concentrate on the development of the dorsal aortae and the posterior cardinal veins. The dorsal aortae form from single PECs which segregate from the lateral mesoderm and aggregate into a loose cord ventral to the somites. These cells become more closely associated and a lumen forms. The posterior cardinal veins form from a loose plexus of cells segregated from the lateral mesoderm on its dorsal surface. These cells become intimately associated with the Wolffian ducts.

Cell rearrangement and directional migration in pronephric duct development.

The morphology of the directed migration of the pronephric duct rudiment of three vertebrates, the salamander, chick and sturgeon, has been examined by scanning electron microscopy. Of particular interest in this paper are the morphology of the duct tip, the role of cell rearrangement, and the relation of duct extension to somite segmentation. The duct rudiments of all three species have motile cell processes (lamellipodia and filopodia) largely confined to their posterior tips. The salamander and sturgeon embryos extend their duct rudiments by extensive cell rearrangements. A short, wide rudiment is elongated to form a long, thin one. The chick duct rudiment stays about the same width and apparently gains volume by cell proliferation. The salamander duct rudiment's posterior tip is always two somites behind the last formed somite. Both the sturgeon and chick embryo's duct rudiments lie well posterior of the last segmented somite adjacent to segmental plate mesoderm. There is still a close coupling, however, between the posterior progression of the duct rudiments and the advancing wave of somite segmentation.

MB1, a quail leukocyte-endothelium antigen: partial characterization of the cell surface and secreted forms in cultured endothelial cells.

We describe here conditions allowing the selective growth in culture of embryonic capillary endothelial cells from quail yolk sac. Such cultures were set up to characterize an antigen present on the endothelial cell surface and to study whether it was secreted in the culture medium. This antigen, MB1, was previously evidenced by a monoclonal antibody raised to quail IgM heavy chain. It is present at the surface of all endothelial and hemopoietic cells (except mature erythrocytes) starting from the hemangioblast, the early mesodermal precursor of blood and vascular endothelial cells. The MB1 epitope is also found on quail plasma molecules of 80 and 125-200 kDa. By immunoprecipitation of either surface or metabolically labeled endothelial cellular material, we have chemically characterized MB1-bearing components as glycoproteins of apparent molecular mass ranging from 80 to 200 kDa and provided evidence for their release into the culture medium. This is consistent with the hypothesis that, in the quail, vascular endothelium participates in the secretion of the alpha-MB1-positive plasmatic components.

How do the migratory and adhesive properties of the neural crest govern ganglia formation in the avian peripheral nervous system?

The peripheral nervous system derives mainly from the neural crest both in the head and trunk. Using markers such as fibronectin (FN), neural cell-adhesion molecule (NCAM), the nucleolar marker for quail cells in chimaeric embryos, and NC-1, a monoclonal antibody specific to crest cells and their neural derivatives, we have attempted to reconstruct the processes that lead to the formation of peripheral ganglia. Our observations allow us to propose a model of the formation of ganglia based on morphogenetic movements and on variations of crest cell adhesiveness. In most cases, crest cells migrate in morphologically defined and transient pathways that lead them to their final site of arrest; these pathways are always associated with FN, which appears necessary for crest cell attachment and movement in vitro. The directionality of crest cell migration is probably dictated by the cells' motile properties and population pressure in restricted areas suitable for cell movement. The disappearance of the pathways and of the substrate necessary for migration while the population is rapidly dividing may be responsible for the aggregation of crest cells in the case of the sensory ganglia. To the contrary, the aggregation of crest cells into autonomic ganglia (sympathetic, enteric, and ciliary ganglia) does not seem to obey the same rules, no disappearance of the substratum or of the pathways being obvious; rather, their formation seems correlated with the de novo synthesis of adhesive molecules such as NCAM.

Biologically active synthetic peptides as probes of embryonic development: a competitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos.

We describe a new method for analyzing embryonic events dependent on a specific peptide recognition signal. A short, specific amino acid sequence in fibronectin has been implicated as a recognition site in fibronectin-mediated interactions. Fibroblast adhesion to fibronectin is competitively inhibited by certain synthetic peptides, including the decapeptide Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, which appears to contain the cell recognition sequence. We found that this peptide inhibited both amphibian gastrulation and avian neural crest cell migration in vivo, as well as the attachment and migration of neural crest cells in vitro. These processes are major cell migratory events previously suggested to involve fibronectin. Negative controls included another conserved fibronectin peptide from the collagen-binding region containing the sequence Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr and another peptide. Our results demonstrate the feasibility of using synthetic peptides directed at recognition sites in extracellular proteins as probes of morphogenetic processes, and they provide further support for the hypothesis that fibronectin is involved in gastrulation and neural crest cell migration.

Different modes of pronephric duct origin among vertebrates.

It is possible to distinguish differences in pronephric duct morphogenesis by using scanning electron microscopy to observe the results of blocking, marking and grafting experiments as well as the normal course of development. Here we compare the mode of pronephric duct development in embryos representing three orders of vertebrates: birds (class, Aves; order, Gallus); frogs (class, Amphibia; order, Anura); and salamanders (class, Amphibia; order, Urodela). The axolotl (a urodele) pronephric duct is formed by the caudal extension of a solid stream of cells segregated below somites 2 through 7. During its migration, cells are rearranged so that a short, wide rudiment is extended to form a long, thin one of similar volume. The pronephric duct rudiment of Xenopus laevis (an anuran) shows no evidence of caudal migration. Rather, pronephric duct cells are segregated out in situ by the formation of a fissure which separates them from the lateral plate mesoderm over ten somite widths. The chick pronephric duct forms a part of the intermediate mesoblast that extends by a caudal migration which does not, however, involve extensive cell rearrangements. Instead, cells near the tip of the duct rudiment in the chick proliferate, extending the duct by true growth as well as by active cell locomotion.

Stimulation of rat peritoneal mast cell migration by tumor-derived peptides.

The accumulation of mast cells is characteristic of a number of pathological states. We demonstrate here the directional motility of mast cells in vitro in response to tumor-derived peptides. Rat peritoneal mast cells were isolated on Percoll gradients and maintained in serum-free medium containing transferrin, albumin, soybean lipid, and cholesterol. The isolated mast cells migrated under agarose in response to medium conditioned by any of eight tumor cell lines but not to medium conditioned by any of a variety of nontumorigenic cell types. The tumor-derived activity is dialyzable (cutoff, Mr 3500), stable to trypsin treatment and to heating at 56 degrees, but destroyed by heating to 100 degrees or by treatment with Streptomyces griseus protease or carboxypeptidase A. Ultrafiltration suggests a molecular weight of 300 to 1000. Two tripeptides, glycylhistidyllysine and N-formylmethionylleucylphenylalanine, were also found to be potent chemoattractants for mast cells. N-Formylmethionylphenylalanine and valylglycylserylglutamic acid (eosinophil chemotactic Factor A) had significantly less chemoattractant activity over the same range of concentrations. Several peptide analogues of glycylhistidyllysine were tested and found to have no activity. The growth of capillary blood vessels toward a growing tumor is generally preceded by an accumulation of mast cells at the tumor site. Based on the results presented here and previous data from our laboratory on mast cell stimulation of capillary endothelial cell migration, we propose an hypothesis that the chemoattraction of mast cells by tumor-derived peptides may be an important early event in tumor neovascularization.

Strategies for specifying form and pattern: adhesion-guided multicellular assembly.

We define a material pattern as a particular arrangement of material elements in space. We then make an effort to categorize the developmental strategies that underlie the emergence of multicellular patterns. These strategies are divided into three broad categories according to whether cell position influences or is influenced by cell fate. In that category of strategies in which cell fate influences cells to move to particular positions, we focus our attention upon morphogenetic and patterning phenomena that appear to be determined by adhesion-mediated interactions of cells with each other and with their surroundings. The differential adhesion hypothesis details how cellular adhesive properties can guide tissue movements and specify patterns of cell association. Motile, adhesive cells will naturally tend to group so as to maximize their adhesive interactions (minimize interfacial free energy). A homogeneous population of uniformly adhesive (isotropic) cells will tend toward spherical form. Cell surface adhesive anisotropies can determine other most-stable (equilibrium) configurations of the population, such as cell sheets, tubes and vesicles. Heterogeneous cell populations may preferentially either intermix or sort out, depending upon the balance of adhesive forces between like and unlike elements. The precise configuration adopted will depend upon the particular adhesive relationships that prevail. Both this end state and the approach toward it arise from the adhesive relationships among the interacting cells. Such morphogenetic phenomena as tissue spreading and the segregation of organ primordia are probably brought about in this way. We outline here some results of our recent experiments on the morphogenesis of the salamander pronephric duct. These illustrate the reality of emergent adhesion-generated tissue immiscibility as a cause of organ segregation and point toward a craniocaudally travelling adhesion gradient as the information that guides the migrating pronephric duct to the cloaca.

Amphibian pronephric duct morphogenesis: segregation, cell rearrangement and directed migration of the Ambystoma duct rudiment.

The axolotl pronephric duct rudiment is readily accessible to both SEM observation and surgical manipulation. The rudiment segregates from the dorsal part of the lateral mesoderm and then extends caudally along the ventrolateral border of the segmenting comites, eventually contacting the cloacal wall. The marked thinning of the rudiment which accompanies this migration is paralleled by a corresponding reduction in cell number across the duct's diameter and by caudad translocation and elongation of vital dye marks applied to the duct mesoderm. Duct extension thus involves appreciable cell rearrangement. The morphology of duct mesoderm and its substratum (somite and lateral mesoderm) suggests that active locomotion of cells near its tip marshals the duct's caudad elongation. Filopodia and small focal areas of intercellular contact may mediate the adhesions between the cells which must be broken and reformed as the cells rearrange.