Blood Flow Changes Coincide with Cellular Rearrangements during Blood Vessel Pruning in Zebrafish Embryos

After the initial formation of a highly branched vascular plexus, blood vessel pruning generates a hierarchically structured network with improved flow characteristics. We report here on the cellular events that occur during the pruning of a defined blood vessel in the eye of developing zebrafish embryos. Time-lapse imaging reveals that the connection of a new blood vessel sprout with a previously perfused multicellular endothelial tube leads to the formation of a branched, Y-shaped structure. Subsequently, endothelial cells in parts of the previously perfused branch rearrange from a multicellular into a unicellular tube, followed by blood vessel detachment. This process is accompanied by endothelial cell death. Finally, we show that differences in blood flow between neighboring vessels are important for the completion of the pruning process. Our data suggest that flow induced changes in tubular architecture ensure proper blood vessel pruning.     

Endothelial Cell Self-fusion during Vascular Pruning

During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell–cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.     

The blood flow-klf6a-tagln2 axis drives vessel pruning in zebrafish by regulating endothelial cell rearrangement and actin cytoskeleton dynamics

Recent studies have focused on capillary pruning in various organs and species. However, the way in which large-diameter vessels are pruned remains unclear. Here we show that pruning of the zebrafish caudal vein (CV) from ventral capillaries of the CV plexus in different transgenic embryos is driven by endothelial cell (EC) rearrangement, which involves EC nucleus migration, junction remodeling, and actin cytoskeleton remodeling. Further observation reveals a growing difference in blood flow velocity between the two vessels in CV pruning in zebrafish embryos. With this model, we identify the critical role of Kruppel-like factor 6a (klf6a) in CV pruning. Disruption of klf6a functioning impairs CV pruning in zebrafish. klf6a is required for EC nucleus migration, junction remodeling, and actin cytoskeleton dynamics in zebrafish embryos. Moreover, actin-related protein transgelin 2 (tagln2) is a direct downstream target of klf6a in CV pruning in zebrafish embryos. Together these results demonstrate that the klf6a-tagln2 axis regulates CV pruning by promoting EC rearrangement.     

Visualizing the cell-cycle progression of endothelial cells in zebrafish

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.     

When It Is Better to Regress: Dynamics of Vascular Pruning

Blood vascular networks in vertebrates are essential to tissue survival. Establishment of a fully functional vasculature is complex and requires a number of steps including vasculogenesis and angiogenesis that are followed by differentiation into specialized vascular tissues (i.e., arteries, veins, and lymphatics) and organ-specific differentiation. However, an equally essential step in this process is the pruning of excessive blood vessels. Recent studies have shown that pruning is critical for the effective perfusion of blood into tissues. Despite its significance, vessel pruning is the least understood process in vascular differentiation and development. Two recently published PLOS Biology papers provide important new information about cellular dynamics of vascular regression.Vascular biology is a rapidly emerging field of research. Given the critical role the vasculature frequently plays in a wide range of common and serious diseases such as arteriosclerosis, ischemic diseases, cancer, and chronic inflammatory diseases, a better understanding of the formation, maintenance, and remodeling of blood vessels is of major importance.A mature vascular network is a highly anisotropic, hierarchical, and dynamic structure that has evolved to provide optimal oxygen delivery to tissues under a variety of conditions. Whilst much has been learned about early steps in vascular development such as vasculogenesis and angiogenesis, we still know relatively little about how such anatomical and functional organization is achieved. Furthermore, the dynamic nature of mature vascular networks, with its potential for extensive remodeling and a continuing need for stability and maintenance, is even less understood. The issue of optimal vascular density in tissue is of particular importance as several recent studies demonstrated that excessive vascularity may, in fact, reduce effective perfusion [1–3]. Since all neovascularization processes initially result in the formation of excessive amounts of vasculature, be that capillaries, arterioles, or venules, pruning must occur to return the vascular density to its optimal value in order to achieve effective tissue perfusion.Yet despite its functional importance, little is known about how regression of the once formed vasculature actually happens. While several potential mechanisms have been proposed including apoptosis of endothelial cells, intussusception vascular pruning, and endothelial cell migration away from the regressing vessel, cellular and molecular understanding of how this might happen is conspicuously lacking. Two articles recently published in PLOS Biology describe migration of endothelial cells as the key mechanism of apoptosis-independent vascular pruning and place it in a specific biologic context. This important advance offers not only a new understanding of a poorly understood aspect of vascular biology but may also prove to be of considerable importance in the development of pro- and anti-angiogenic therapies.To put vessel regression in context, it helps to briefly outline the current understanding of vessel formation. During embryonic development, vasculature forms in several distinct steps that begin with vasculogenesis, a step that involves differentiation of stem cells into primitive endothelial cells that then form initial undifferentiated and nonhierarchically organized lumenized vascular structures termed the primary plexus [4]. The primary plexus is then remodeled, by the process termed angiogenesis, into a more mature vascular network [5]. This remodeling event involves both formation of new vessels accomplished either by branching angiogenesis, a process dependent on tip cell-driven formation of new branches [6], or intussusception, a poorly understood process of splitting an existing vessel into two [7]. This incompletely differentiated and still nonhierarchical vasculature then further remodels into a number of distinctly different types of vessels such as capillaries, arteries, and veins. This requires fate specification, differentiation, and incorporation of various mural cells into evolving vascular structures. Finally, additional specialization of the vascular network occur in an organ-specific manner.Once formed, vascular networks require active maintenance as withdrawal of key signals, such as of ongoing fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) stimulation, can lead to a rapid loss of vascular integrity and even changes in endothelial cell fate [8–12]. In addition, mature vessels retain the capacity for extensive remodeling and new growth as can be seen in a number of conditions from cancer to myocardial infarction and wound healing responses, among many others [5].A key issue common to both embryonic and adult vessel remodeling is how an existing lumenized vessel connected to the rest of the vasculature undergoes a change that results in its remodeling into something else. Such a change may involve either a new branch formation or regression of an existing branch, while the patency and integrity of the remaining circulation is maintained. Two types of cellular process leading to branching have been described—sprouting and intussusception. Formation of vascular branches by sprouting involves VEGF-A-induced expression of high levels of delta-like ligand 4 (Dll4) in a subset of endothelial cells at the leading edge of the vascular sprouts that are lying closest to the source of VEGF, thus converting them to a “tip cell” phenotype. Some of the key features of tip cells include the presence of cytoplasmic processes that extend into avascular (or hypoxic) tissue that form nascent branches. Dll4 expressed on tip cells binds Notch-1 receptor in neighboring endothelial cells, thereby activating their downstream Notch signaling. In turn, Notch signaling shuts down the formation of additional filopodia processes, converting these cells to a “stalk cell” phenotype and thereby avoiding excessive branching [13–15]. The bone morphogenetic protein signaling pathway provides further input in determining stalk cell fate [16]. Importantly, tip cells are only partially lumenized; only once they have converted to a stalk phenotype does the lumen extend to what was a tip cell and its sprouts.An alternative mechanism of branching involves intussusception, a process by which a tissue pillar from the surrounding tissue splits the existing endothelial tube into two along its long axis, creating two adjusting vessels. While this process has been described morphologically, virtually nothing is known about its molecular and cellular regulation. In development, angiogenesis by intussusception occurs in vessels previously formed by sprouting angiogenesis [17,18]. Importantly, however, both sprouting angiogenesis and intussusception allow growth and remodeling of vascular network without any integrity compromise, thereby avoiding bleeding and related complications.There are certain parallels between vessel formation and branching and vessel regression. While growth occurs either via sprouting (a process linked to endothelial cell-migration) or intussusception, regression involves either “reverse intussusception,” endothelial migration-dependent regression, or apoptosis. The latter is the primary means of regression of the hyaloid vasculature in the eye and of the vascular loss seen in oxygen-induced retinopathy (OIR). In the case of hyaloid vasculature, secretion of WNT7b by macrophages invading the hyaloid membrane induces apoptosis of hyaloid endothelial cells leading to the regression of the entire hyaloid vasculature [19]. This total apoptosis-induced loss of hyaloid blood vessels contrasts with a less extensive vascular regression seen in the setting of OIR. In this condition, exposure of the developing retinal vasculature to abnormally high oxygen levels leads to vascular damage characterized by capillary pruning [20]. The pruning is the consequence of apoptosis of endothelial cells due to the toxic effect of a combination of high oxygen and low VEGF level. Interestingly, larger vessels and mature capillaries are not sensitive to hyperoxia [21].Intussusception vascular pruning was also described in a low VEGF level context in the chick chorioallantoic membrane. Application of VEGF-releasing hydrogels to the membrane surface results in formation of an excessive vasculature. Removal or degradation of the hydrogel induces an abrupt VEGF withdrawal. In this context, formation of transluminal pillars, similar to the ones seen in intussusception angiogenesis, is observed in vessels undergoing pruning [22]. The same process is observed in the tumor vasculature in the setting of anti-angiogenic therapy [23]. Finally, apoptosis-independent vascular regression, driven by endothelial cell migration, has been described in the mouse retina, yolk vessels of the chick and mouse embryos, branchial arches, and the zebrafish brain [24–28].In all of these cases, only a subset of vessels is designated for pruning, and the selection of these vessels is highly regulated. Yet, factors involved in choosing a particular vascular branch for pruning remain ill-defined. One such factor is low blood flow [27,28]. Another is Notch signaling that has been shown to at least partially control vascular pruning in mouse retina and in intersegmental vessels (ISVs) in zebrafish [24]. Loss of Notch-regulated ankyrin repeat protein (Nrarp), target gene of Notch signaling, leads to an increase in vascular regression in these tissues due to a decrease in Wnt signaling-induced stalk cell proliferation. Similarly, in Dll4 ^+/- mice, developmental retinal vascular regression and OIR-induced vascular pruning are reduced [29], confirming the involvement of the Notch pathway in the control of vascular regression.The two factors may be linked, as low flow can affect endothelial shear stress and lead to a decrease in Notch activation. Such a link is suggested by studies on vascular regression in mice with endothelial expression of dominant negative NFκB pathway inhibitor that demonstrate excessive vascular growth but reduced tissue perfusion [2]. Molecular studies showed inhibition of flow- or cytokine-induced NFκB activation results in decreased Dll4 expression [2].Another important issue is the fate of endothelial cells from vessels undergoing pruning. In PLOS Biology, two groups recently described endothelial cell behavior during vascular pruning in three different models: the mouse retina, the ISVs in zebrafish, and the subintestinal vessel in zebrafish [30,31]. Using a high resolution time-lapse microscopy technique, Lenard and collaborators showed that vascular pruning during the subintestinal vessel formation occurs in two different ways. In type I pruning, the first step is the collapse of the lumen. Once that occurs, endothelial cells migrate and incorporate into the neighboring vessels. In type II pruning, the lumen is maintained. One endothelial cell in the center of the pruning vessel undergoes self-fusion, leading to a unicellular lumenized vessel. At the same time, other endothelial cells migrate away and incorporate into the neighboring vessels. The eventual lumen collapse is the last step after which the remaining single endothelial cell migrates and incorporates into one of the major vessels.Franco and collaborators described a pruning mechanism similar to the type I pruning described by Lenard et al., showing lumen disruption as an initial step in pruning of retinal vasculature in mice and ISVs in zebrafish [31]. By analyzing the first axial polarity map of endothelial cells in these models, they demonstrated that axial orientation predicts endothelial cell migration, and that migration-driven pruning occurs in vessels with low flow. Interestingly, migrating endothelial cells in regressing vessel display a tip cell phenotype with filopodia.The cellular dynamic of vessel pruning described here is the reverse of the cellular dynamic during anastomosis and angiogenesis [32]. Given the crucial role of factors as VEGF for the migration of endothelial cells during angiogenesis, can we go further and propose that other cytokines or cell–cell signaling may be involved in the migration of these endothelial cells? Indeed, low blood flow seems to be the cause of vessel pruning, but how can we explain the direction of endothelial cell migration, moreover with a tip cell morphology? Also, what determines the choice between type I and type II pruning? The collapse of lumen suggests a reorganization of the cytoskeleton, and a loss of polarity and electrostatic repulsion of endothelial cells. Molecular mechanisms leading from low shear stress to loss of endothelial cell polarity need further investigation. As defective vascular pruning could be involved in poor recovery after injury or ischemic accident, a better understanding of the molecular control of this phenomenon appears to have medical consequences. Another question that is still unanswered is the fate of mural cells that surrounded the pruned vessels. Small vessels are covered by pericytes, which have strong interaction with endothelial cells. How and when are these interactions disrupted? Are pericytes integrated into the neighboring vessel, or do they undergo apoptosis? Further studies are needed to understand the molecular and cellular mechanisms by which vasculature can adapt, even at the adult stage, to support the nutrient and oxygen needs of each cell.Overall, taking the results of these studies together with other recent developments in this field, the following picture is emerging (Fig 1). Under conditions of low blood flow in certain vascular tree branches, pruning will occur via endothelial cell migration out of these branches to the neighboring (presumably higher blood flow) vessels. This results in decreased total vascular cross-sectional area and increased average blood flow, thereby terminating further pruning. Importantly, this occurs without the loss of luminal integrity and without reduction in the total endothelial cell mass. At the same time, vessels that suddenly find themselves in a low VEGF environment will regress either by apoptosis of endothelial cells or by intussusception. In both cases, there is a reduction in the total vasculature without an increase in blood flow to this tissue. Thus, the local context determines the mechanism: migratory regression and remodeling in low shear stress versus apoptotic pruning in low VEGF milieu.Open in a separate windowFig 1Vessel regression under low flow versus low VEGF conditions.Vessel regression under low flow conditions proceeds by endothelial cell (EC) migration-driven regression, resulting in a decrease in total vessel areas but an increase in blood flow (left panel). Vessel regression under low VEGF conditions proceeds by EC apoptosis or intussusception regression, resulting in decreased vessel number and decreased flow to tissues subtended by the regressing vasculature (right panel). Image credit: Nicolas Ricard & Michael Simons.This distinction is likely to be of a significant practical importance, in particular in the context of therapies designed to facilitate vessel normalization in tumors after VEGF-targeting treatments and therapies designed to promote vascularization of mildly ischemic tissues as occurs, for example, in the setting of chronic stable angina and other similar conditions. In the former case, a precipitous drop in VEGF levels is likely to induce vascular regression by induction of endothelial apoptosis, and further promotion of apoptosis may facilitate this process. In contrast, in the latter case, low flow in newly formed collateral arteries may induce their regression by stimulating outmigration of endothelial cells, thereby limiting their beneficial functional impact. Therapies designed to inhibit this mechanism, therefore, may promote growth of the new functional vasculature.     

Drosophila Cyclin J is a mitotically stable Cdk1 partner without essential functions

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.     

Endothelial cells promote migration and proliferation of enteric neural crest cells via β1 integrin signaling

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.     

Dynamic Endothelial Cell Rearrangements Drive Developmental Vessel Regression

Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.     

Shoot and Root Growth of Lettuce Seedlings Following Root Pruning

Hydroponically-grown lettuce seedlings with 13 to 18 primarylateral roots were root pruned in one of four ways; the rootapices were removed from the main root only (1) or from allthe root membranes (2), or half the total root system was removedwith the remaining apices left intact (3) or removed (4). Duringthe following 8 d the rate of lateral root production on prunedplants increased, decreased, and then increased again relativeto the unpruned control. Conversely, the rate of increase intotal root length decreased, then increased, and if all theroot apices were removed, declined again, prior to increasingon day 8. These changes in the rates of lateral root productionand growth resulted in similar, but less pronounced, patternsof change in the total root length and the total number of lateralroots with time. The changes in total lateral root productionwere related to differences in the rates of primary, secondaryand tertiary root emergence. The shoot d. wt of the most severely root pruned seedlings (treatment4) fell below that of the control 4 d after pruning and remainedlower than the control on day 14, whereas the root d. wt hadrecovered to the control level by day 6. The root: shoot d.wt ratio, which was reduced by root pruning, rose above thatof the control on days 6 and 8. Lactuca sativa L., lettuce, root pruning, root growth, lateral root, nutrient solution     

Development and fibronectin signaling requirements of the zebrafish interrenal vessel

Background

The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear.

Methodology and Principal Findings

We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration.

Conclusions and Significance

This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.     

reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins

Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at approximately 35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.     

Endothelial FAK is essential for vascular network stability, cell survival, and lamellipodial formation

Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.5 and 11.5. Capillaries and intercapillary spaces in yolk sacs were dilated before any other detectable abnormalities at e9.5, and explants demonstrate that the defects resulted from the loss of FAK and not from organ failure. Time-lapse microscopy monitoring EC behavior during vascular formation in explants revealed no apparent decrease in proliferation or migration but revealed increases in cell retraction and death leading to reduced vessel growth and increased vessel regression. Consistent with this phenotype, ECs derived from mutant embryos exhibited aberrant lamellipodial extensions, altered actin cytoskeleton, and nonpolarized cell movement. This study reveals that FAK is crucial for vascular morphogenesis and the regulation of EC survival and morphology.     

Physical and chemical response of juvenile Acacia tortilis trees to browsing. Experimental evidence

1. Changes in nutritional value and accessibility of leaves following browsing are important in the dynamics of plant–herbivore interactions because they influence the fitness of the plant attacked and the future utilization of it by the herbivore.
2. Hand pruning of Acacia tortilis , a spinescent tree common in savanna ecosystems of eastern Africa, resulted in higher biomass of spines and new shoots in pruned trees than in unpruned controls.
3. Pruned trees allocated a higher proportion of shoot biomass to spines than unpruned ones, whereas the proportion of leaf biomass in new shoots was slightly reduced. Because increases in spine biomass and density following pruning are coupled with an increase in shoot production, it is concluded that higher production of spines is an inducible response of Acacia tortilis to pruning.
4. No significant changes in the concentration of total phenolics, condensed tannins or leaf nitrogen were induced by pruning.
5. Irrespective of treatment, high foliar concentrations of nitrogen were correlated with an increase in twig production for a given leaf biomass and a reduction in the concentration of secondary substances in leaves. This relation may lead to a conflict between foraging efficiency and nutrition for browsers of A. tortilis.     

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.     

Arterial-venous network formation during brain vascularization involves hemodynamic regulation of chemokine signaling

During angiogenic sprouting, newly forming blood vessels need to connect to the existing vasculature in order to establish a functional circulatory loop. Previous studies have implicated genetic pathways, such as VEGF and Notch signaling, in controlling angiogenesis. We show here that both pathways similarly act during vascularization of the zebrafish central nervous system. In addition, we find that chemokine signaling specifically controls arterial-venous network formation in the brain. Zebrafish mutants for the chemokine receptor cxcr4a or its ligand cxcl12b establish a decreased number of arterial-venous connections, leading to the formation of an unperfused and interconnected blood vessel network. We further find that expression of cxcr4a in newly forming brain capillaries is negatively regulated by blood flow. Accordingly, unperfused vessels continue to express cxcr4a, whereas connection of these vessels to the arterial circulation leads to rapid downregulation of cxcr4a expression and loss of angiogenic characteristics in endothelial cells, such as filopodia formation. Together, our findings indicate that hemodynamics, in addition to genetic pathways, influence vascular morphogenesis by regulating the expression of a proangiogenic factor that is necessary for the correct pathfinding of sprouting brain capillaries.     

Small molecule screen for compounds that affect vascular development in the zebrafish retina

Blood vessel formation in the vertebrate eye is a precisely regulated process. In the human retina, both an excess and a deficiency of blood vessels may lead to a loss of vision. To gain insight into the molecular basis of vessel formation in the vertebrate retina and to develop pharmacological means of manipulating this process in a living organism, we further characterized the embryonic zebrafish eye vasculature, and performed a small molecule screen for compounds that affect blood vessel morphogenesis. The screening of approximately 2000 compounds revealed four small molecules that at specific concentrations affect retinal vessel morphology but do not produce obvious changes in trunk vessels, or in the neuronal architecture of the retina. Of these, two induce a pronounced widening of vessel diameter without a substantial loss of vessel number, one compound produces a loss of retinal blood vessels accompanied by a mild increase of their diameter, and finally one other generates a severe loss of retinal vessels. This work demonstrates the utility of zebrafish as a screening tool for small molecules that affect eye vasculature and presents several compounds of potential therapeutic importance.     

Tenascin-C mRNA is expressed in cranial neural crest cells,in some placodal derivatives,and in discrete domains of the embryonic zebrafish brain

A partial zebrafish tenascin-C cDNA clone was isolated from an embryonic zebrafish cDNA library on the basis of homology to mouse tenascin-C. The expression pattern in the head of embryonic zebrafish was analyzed by in situ hybridization. Tenascin-C mRNA was detected in neural crest cells during the period of their migration and differentiation. Expression also occurred in differentiating placodal tissues and in mesodermal cells. In the developing brain, tenascin-C mRNA was expressed in specific domains. In the hindbrain the pattern of the domains was dynamic. At 18 to 22 h postfertilization, expression was widespread in rhombomeres 3, 5, and 6, confined to periventricular cells in rhombomere 2, and not detectable in rhombomere 4. At 32 h postfertilization, tenascin-C was expressed at the rhombomere boundaries. In contrast to the hindbrain, the pattern in the forebrain and midbrain did not show any major changes between 22 and 32 h postfertilization. Domains expressing tenascin-C alternated with regions devoid of it. The most anterior domain of expression was observed at the telencephalic-diencephalic border, surrounding the optic recess. A second domain, at the border between the diencephalon and the midbrain, and a third domain, in the caudal midbrain tegmentum, appeared restricted to the basal plate. Additionally, expression of tenascin-C mRNA was detected in the hypothalamus and in the developing epiphysis. These expression patterns suggest that tenascin-C may play a role in neural crest cell migration and during the differentiation of neural crest, placodal, and mesodermal derivatives. In the developing brain, tenascin-C may be involved in the consolidation of different regional identities. © 1995 John Wiley & Sons, Inc.     

In vivo imaging of embryonic vascular development using transgenic zebrafish

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.     

Notochord-derived BMP antagonists inhibit endothelial cell generation and network formation

Embryonic blood vessel formation is initially mediated through the sequential differentiation, migration, and assembly of endothelial cells (ECs). While many molecular signals that promote vascular development have been identified, little is known about suppressors of this process. In higher vertebrates, including birds and mammals, the vascular network forms throughout the embryonic disk with the exception of a region along the midline. We have previously shown that the notochord is responsible for the generation and maintenance of the avascular midline and that BMP antagonists expressed by this embryonic tissue, including Noggin and Chordin, can mimic this inhibitory role. Here we report that the notochord suppresses the generation of ECs from the mesoderm both in vivo and in vitro. We also report that the notochord diminishes the ability of mature ECs to organize into a primitive plexus. Furthermore, Noggin mimics notochord-based inhibition by preventing mesodermal EC generation and mature EC network formation. These findings suggest that the mesoderm surrounding the midline is competent to give rise to ECs and to form blood vessels, but that notochord derived-BMP antagonists suppress EC differentiation and maturation processes leading to inhibition of midline vessel formation.     

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5–6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10–25 days pf), and by 25–30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain. This study was supported by the US-Israel Bi-national Agricultural Research and Development (BARD) Foundation (grant 3428-03).