Ets1-related protein is a key regulator of vasculogenesis in zebrafish

During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp–morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis.     

Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis

Formation of embryonic vasculature involves vasculogenesis as endothelial cells differentiate and aggregate into vascular cords and angiogenesis which includes branching from the existing vessels. In the zebrafish which has emerged as an advantageous model to study vasculogenesis, cranial vasculature is thought to originate by a combination of vasculogenesis and angiogenesis, but how these processes are coordinated is not well understood. To determine how angioblasts assemble into cranial vasculature, we generated an etsrp:GFP transgenic line in which GFP reporter is expressed under the promoter control of an early regulator of vascular and myeloid development, etsrp/etv2. By utilizing time-lapse imaging we show that cranial vessels originate by angiogenesis from angioblast clusters, which themselves form by the mechanism of vasculogenesis. The two major pairs of bilateral clusters include the rostral organizing center (ROC) which gives rise to the most rostral cranial vessels and the midbrain organizing center (MOC) which gives rise to the posterior cranial vessels and to the myeloid and endocardial lineages. In Etsrp knockdown embryos initial cranial vasculogenesis proceeds normally but endothelial and myeloid progenitors fail to initiate differentiation, migration and angiogenesis. Such angioblast cluster-derived angiogenesis is likely to be involved during vasculature formation in other vertebrate systems as well.     

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish

Background

Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.

Methodology/Principal Findings

In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp ⁺ cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli ⁺ etsrp ⁺ hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp ⁺ cell numbers at the LPM.

Conclusions/Significance

This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.     

Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential,Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C₃(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.     

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.     

Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp^y11 mutant embryos or cardia bifida phenotypes in fau^tm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.     

Different Regulation of Physiological and Tumor Angiogenesis in Zebrafish by Protein Kinase D1 (PKD1)

Protein kinase D isoenzymes (PKDs, Prkds) are serine threonine kinases that belong to the CAMK superfamily. PKD1 is expressed in endothelial cells and is a major mediator of biological responses downstream of the VEGFRs that are relevant for angiogenesis such as endothelial cell migration, proliferation and tubulogenesis in vitro. PKDs also play a critical role in tumor development and progression, including tumor angiogenesis. However, given the plethora of signaling modules that drive angiogenesis, the precise role of PKD1 in both physiological and tumor angiogenesis in vivo has not been worked out so far. This study aimed at dissecting the contribution of PKD1 to physiological blood vessel formation, PKD1 was found to be widely expressed during zebrafish development. As far as physiological angiogenesis was concerned, morpholino-based silencing of PKD1 expression moderately reduced the formation of the intersomitic vessels and the dorsal longitudinal anastomotic vessel in tg(fli1:EGFP) zebrafish. In addition, silencing of PKD1 resulted in reduced formation of the parachordal lymphangioblasts that serves as a precursor for the developing thoracic duct. Interestingly, tumor angiogenesis was completely abolished in PKD1 morphants using the zebrafish/tumor xenograft angiogenesis assay. Our data in zebrafish demonstrate that PKD1 contributes to the regulation of physiological angiogenesis and lymphangiogenesis during zebrafish development and is essential for tumor angiogenesis.     

Ablation of SGK1 Impairs Endothelial Cell Migration and Tube Formation Leading to Decreased Neo-Angiogenesis Following Myocardial Infarction

Serum and glucocorticoid inducible kinase 1 (SGK1) plays a pivotal role in early angiogenesis during embryonic development. In this study, we sought to define the SGK1 downstream signalling pathways in the adult heart and to elucidate their role in cardiac neo-angiogenesis and wound healing after myocardial ischemia. To this end, we employed a viable SGK1 knockout mouse model generated in a 129/SvJ background. Ablation of SGK1 in these mice caused a significant decrease in phosphorylation of SGK1 target protein NDRG1, which correlated with alterations in NF-κB signalling and expression of its downstream target protein, VEGF-A. Disruption of these signalling pathways was accompanied by smaller heart and body size. Moreover, the lack of SGK1 led to defective endothelial cell (ECs) migration and tube formation in vitro, and increased scarring with decreased angiogenesis in vivo after myocardial infarct. This study underscores the importance of SGK1 signalling in cardiac neo-angiogenesis and wound healing after an ischemic insult in vivo.     

GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.     

Syk and Zap-70 function redundantly to promote angioblast migration

Spleen tyrosine kinase (Syk) plays critical roles in B-cell and T-cell development, the maintenance of vascular integrity, and proper partitioning of the blood vascular and lymphatic vascular system. Here, we utilize the zebrafish as an in vivo system to demonstrate novel roles for Syk and the related kinase Zeta associated protein (Zap-70) in promoting angioblast migration. Partial knockdown of either gene results in early angiogenic delay of the intersegmental vessels, dorsal intersegmental vessel patterning defects, and partial loss of the thoracic duct. Higher dose knockdown of both genes results in little to no angiogenic sprouting of the intersegmental vessels, a phenotype which resembles knockdown of vegfa. Di-phosphorylated ERK, an effector of the vegfa pathway, is also downregulated in the aorta of syk:zap double morphants. Over-expression of syk under the control of a blood-specific or vascular-specific promoter rescues sprouting defects after loss of vegfa. Together these results suggest that syk and zap-70 function redundantly in an early progenitor to promote the migration of intersegmental vessel angioblasts and lymphangioblasts that contribute to the thoracic duct, either downstream of, or in parallel to vegfa.     

The Hemodynamically-Regulated Vascular Microenvironment Promotes Migration of the Steroidogenic Tissue during Its Interaction with Chromaffin Cells in the Zebrafish Embryo

Background

While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role.

Methodology and Principal Findings

In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms.

Conclusions and Significance

These results demonstrate that hemodynamically regulated Fn-pFak signaling promotes the migration of steroidogenic cells, ensuring their interaction with chromaffin cells along both sides of the midline during interrenal gland development.     

Hela l-CaD is Implicated in the Migration of Endothelial Cells/Endothelial Progenitor Cells in Human Neoplasms

Caldesmon (CaD) is a major actin-binding protein distributed in a variety of cell types. No functional differences among the isoforms in in vitro studies were found so far. In a previous study we found that the low molecular caldesmon isoform (Hela l-CaD) is expressed in endothelial cells (ECs)/endothelial progenitor cells (EPCs) in tumor vasculature of various human tumors. Activation of cell motility is necessary for the navigation of the tip ECs during angiogenesis, and migration of EPCs from the bone marrow during vasculogenesis. In the present study we searched for features of motility and the intracellular expression sites of Hela l-CaD in ECs/EPCs of various human tumors under histologically preserved microenviroment. We discovered a variety of motility-related cell protrusions like filopodia, microspikes, lamellipodia, podosomes, membrane blebs and membrane ruffles in the activated ECs/EPCs. Hela l-CaD appeared to be invariably expressed in the subregions of these cell protrusions. The findings suggest that Hela l-CaD is implicated in the migration of ECs/EPC in human neoplasms where they contribute to tumor vasculogenesis and angiogenesis.Key words: Hela l-CaD, cell motility, angiogenesis, vasculogenesis, ECs/EPCs