Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence.

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.

Cell behaviors and dynamics during angiogenesis.

Vascular networks are formed and maintained through a multitude of angiogenic processes, such as sprouting, anastomosis and pruning. Only recently has it become possible to study the behavior of the endothelial cells that contribute to these networks at a single-cell level in vivo This Review summarizes what is known about endothelial cell behavior during developmental angiogenesis, focusing on the morphogenetic changes that these cells undergo.

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.

Vascular morphogenesis in the zebrafish embryo.

During embryonic development, the vertebrate vasculature is undergoing vast growth and remodeling. Blood vessels can be formed by a wide spectrum of different morphogenetic mechanisms, such as budding, cord hollowing, cell hollowing, cell wrapping and intussusception. Here, we describe the vascular morphogenesis that occurs in the early zebrafish embryo. We discuss the diversity of morphogenetic mechanisms that contribute to vessel assembly, angiogenic sprouting and tube formation in different blood vessels and how some of these complex cell behaviors are regulated by molecular pathways.

Endothelial cell division in angiogenic sprouts of differing cellular architecture.

The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at detailed cellular level. Reasoning that different tubular architectures may impose discrete mechanistic constraints on endothelial cell division, we analyzed and compared the sequential steps of cell division, namely mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation, in different blood vessels. In particular, we characterized the interplay between cell rearrangement, mitosis and lumen dynamics within unicellular and multicellular tubes. The lumen of unicellular tubes becomes constricted and is ultimately displaced from the plane of cell division, where a de novo junction forms through the recruitment of junctional proteins at the site of abscission. By contrast, the new junctions separating the daughter cells within multicellular tubes form through the alteration of pre-existing junctions, and the lumen is retained throughout mitosis. We also describe variations in the progression of cytokinesis: while membrane furrowing between daughter cells is symmetric in unicellular tubes, we found that it is asymmetric in those multicellular tubes that contained a taut intercellular junction close to the plane of division. Our findings illustrate that during the course of normal development, the cell division machinery can accommodate multiple tube architectures, thereby avoiding disruptions to the vascular network.

Cdh5/VE-cadherin promotes endothelial cell interface elongation via cortical actin polymerization during angiogenic sprouting.

Organ morphogenesis requires the coordination of cell behaviors. Here, we have analyzed dynamic endothelial cell behaviors underlying sprouting angiogenesis in vivo. Two different mechanisms contribute to sprout outgrowth: tip cells show strong migratory behavior, whereas extension of the stalk is dependent upon cell elongation. To investigate the function of Cdh5 in sprout outgrowth, we generated null mutations in the zebrafish cdh5 gene, and we found that junctional remodeling and cell elongation are impaired in mutant embryos. The defects are associated with a disorganization of the actin cytoskeleton and cannot be rescued by expression of a truncated version of Cdh5. Finally, the defects in junctional remodeling can be phenocopied by pharmacological inhibition of actin polymerization, but not by inhibiting actin-myosin contractility. Taken together, our results support a model in which Cdh5 organizes junctional and cortical actin cytoskeletons, as well as provides structural support for polymerizing F-actin cables during endothelial cell elongation.

Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry.

Protein-protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

Fibroblast growth factor signaling affects vascular outgrowth and is required for the maintenance of blood vessel integrity.

Angiogenesis contributes to the development of numerous disorders. Even though fibroblast growth factors (FGFs) were discovered as mediators of angiogenesis more than 30 years ago, their role in developmental angiogenesis still remains elusive. We use a recently described chemical probe, SSR128129E (SSR), that selectively inhibits the action of multiple FGF receptors (FGFRs), in combination with the zebrafish model to examine the role of FGF signaling in vascular development. We observe that while FGFR signaling is less important for vessel guidance, it affects vascular outgrowth and is especially required for the maintenance of blood vessel integrity by ensuring proper cell-cell junctions between endothelial cells. In conclusion, our work illustrates the power of a small molecule probe to reveal insights into blood vessel formation and stabilization and thus of broad interest to the vascular biology community.

Tugging organs into place.

In this issue of Developmental Cell, Weavers and Skaer (2013) show that interplay between two opposing forces generated by tip cell attachment to muscles and migration of kink cells allows for the formation of the looped shape and positioning of renal tubules within the body of a fly larva.

In vivo analysis reveals a highly stereotypic morphogenetic pathway of vascular anastomosis.

Organ formation and growth requires cells to organize into properly patterned three-dimensional architectures. Network formation within the vertebrate vascular system is driven by fusion events between nascent sprouts or between sprouts and pre-existing blood vessels. Here, we describe the cellular activities that occur during blood vessel anastomosis in the cranial vasculature of the zebrafish embryo. We show that the early steps of the fusion process involve endothelial cell recognition, de novo polarization of endothelial cells, and apical membrane invagination and fusion. These processes generate a unicellular tube, which is then transformed into a multicellular tube via cell rearrangements and cell splitting. This stereotypic series of morphogenetic events is typical for anastomosis in perfused sprouts. Vascular endothelial-cadherin plays an important role early in the anastomosis process and is required for filopodial tip cell interactions and efficient formation of a single contact site.

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.

Effects of the protein kinase inhibitor PKC412 on gene expression and link to physiological effects in zebrafish Danio rerio eleuthero-embryos.

To identify molecular effects of the antineoplastic agent protein kinase C inhibitor 412 (PKC412) (midostaurin), we applied gene expression profiling in zebrafish using whole-genome microarrays. Behavioral, developmental, and physiological effects were investigated in order to analyze for correlations between altered gene expression profiles with effects on development and physiology. Zebrafish blastula-stage embryos were exposed for 6 days postfertilization to nominal levels of 2 and 40 µg/l PKC412. Among the 259 and 511 altered transcripts at both concentrations, respectively, the expressions of genes involved in the circadian rhythm were further investigated. Alteration of swimming behavior was not observed. Pathways of interest affected by PKC412 were angiogenesis, apoptosis, DNA damage response, and response to oxidative stress. Angiogenesis was analyzed in double-transgenic zebrafish embryos Tg(fli1a:EGFP)y1;Tg(gata1:dsRed)sd2; no major defects were induced by PKC412 treatment at both concentrations. Apoptosis occurred in olfactory placodes of embryos exposed to 40 µg/l, and DNA damage was induced at both PKC412 concentrations. However, there were no significant effects on reactive oxygen species formation. This study leads to the conclusion that PKC412-induced alterations of gene transcripts are partly paralleled by physiological effects at high, but not at low PKC412 concentrations expected to be of environmental relevance.

Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo.

Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP-tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes. During initial steps of anastomosis, cell junctions that have formed at the initial site of cell contacts expand into rings, generating a cellular interface of apical membrane compartments, as defined by the localization of the apical marker podocalyxin-2 (Pdxl2). During the cord hollowing process, these apical membrane compartments are brought together via cell rearrangements and extensive junctional remodeling, resulting in lumen coalescence and formation of a multicellular tube. Vessel fusion by membrane invagination occurs adjacent to a preexisting lumen in a proximal to distal direction and is blood-flow dependent. Here, the invaginating inner cell membrane undergoes concomitant apicobasal polarization and the vascular lumen is formed by the extension of a transcellular lumen through the EC, which forms a unicellular or seamless tube.