Eph-ephrin signaling couples endothelial cell sorting and arterial specification.

Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.

Induction of osteogenesis by bone-targeted Notch activation.

Declining bone mass is associated with aging and osteoporosis, a disease characterized by progressive weakening of the skeleton and increased fracture incidence. Growth and lifelong homeostasis of bone rely on interactions between different cell types including vascular cells and mesenchymal stromal cells (MSCs). As these interactions involve Notch signaling, we have explored whether treatment with secreted Notch ligand proteins can enhance osteogenesis in adult mice. We show that a bone-targeting, high affinity version of the ligand Delta-like 4, termed Dll4(E12), induces bone formation in male mice without causing adverse effects in other organs, which are known to rely on intact Notch signaling. Due to lower bone surface and thereby reduced retention of Dll4(E12), the same approach failed to promote osteogenesis in female and ovariectomized mice but strongly enhanced trabecular bone formation in combination with parathyroid hormone. Single cell analysis of stromal cells indicates that Dll4(E12) primarily acts on MSCs and has comparably minor effects on osteoblasts, endothelial cells, or chondrocytes. We propose that activation of Notch signaling by bone-targeted fusion proteins might be therapeutically useful and can avoid detrimental effects in Notch-dependent processes in other organs.

Endothelial EphB4 maintains vascular integrity and transport function in adult heart.

The homeostasis of heart and other organs relies on the appropriate provision of nutrients and functional specialization of the local vasculature. Here, we have used mouse genetics, imaging and cell biology approaches to investigate how homeostasis in the adult heart is controlled by endothelial EphB4 and its ligand ephrin-B2, which are known regulators of vascular morphogenesis and arteriovenous differentiation during development. We show that inducible and endothelial cell-specific inactivation of Ephb4 in adult mice is compatible with survival, but leads to rupturing of cardiac capillaries, cardiomyocyte hypertrophy, and pathological cardiac remodeling. In contrast, EphB4 is not required for integrity and homeostasis of capillaries in skeletal muscle. Our analysis of mutant mice and cultured endothelial cells shows that EphB4 controls the function of caveolae, cell-cell adhesion under mechanical stress and lipid transport. We propose that EphB4 maintains critical functional properties of the adult cardiac vasculature and thereby prevents dilated cardiomyopathy-like defects.

Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier.

RATIONALE: The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/ß-catenin signaling is responsible for the early phases of brain vascularization and BBB differentiation. However, this signal declines after birth, and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown. OBJECTIVE: Sox17 (SRY [sex-determining region Y]-box 17) constitutes a major downstream target of Wnt/ß-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/ß-catenin in inducing BBB differentiation and maintenance. METHODS AND RESULTS: Using reporter mice and nuclear staining of Sox17 and ß-catenin, we report that although ß-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo and progressively declines after birth. In search of Sox17 mechanism of action, RNA sequencing analysis of gene expression of brain endothelial cells has identified members of the Wnt/ß-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/ß-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the ß-catenin destruction complex or expression of a degradation-resistant ß-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17. CONCLUSIONS: Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB, and they underline the strict reciprocal tuning of this transcription factor and Wnt/ß-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.

Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation.

In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction 1 . Defective trabeculation leads to embryonic lethality2-4 or non-compaction cardiomyopathy (NCC) 5 . There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium 6 , whereas in chicks, chamber wall thickening occurs before overt trabeculation 7 . In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs 2 . Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch2,8 and Neuregulin (NRG) 4 . Late disruption of the Notch pathway causes NCC 5 . Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice9,10 and the matrix metalloprotease ADAMTS1 promotes trabecular termination 3 , the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

Dll4 and Notch signalling couples sprouting angiogenesis and artery formation.

Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis.

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin ß1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization.

For a skin wound to successfully heal, the cut epidermal-edge cells have to migrate forward at the interface between scab and healthy granulation tissue. Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back. Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells. Additionally, this signaling leads to the shutdown of actomyosin stress fibers in these same epidermal cells, which may act to release tension within the wound monolayer. If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man.

Arteries are formed by vein-derived endothelial tip cells.

Tissue vascularization entails the formation of a blood vessel plexus, which remodels into arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endothelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to developing arteries when in association with wild-type cells, suggesting collective migration of endothelial cells. Together, our findings reveal specific cell migratory behaviours in the developing blood vessel plexus and uncover a conserved mode of artery formation.

Regulation of signaling interactions and receptor endocytosis in growing blood vessels.

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRß and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.

Direct cell-cell contact with the vascular niche maintains quiescent neural stem cells.

The vasculature is a prominent component of the subventricular zone neural stem cell niche. Although quiescent neural stem cells physically contact blood vessels at specialized endfeet, the significance of this interaction is not understood. In contrast, it is well established that vasculature-secreted soluble factors promote lineage progression of committed progenitors. Here we specifically investigated the role of cell-cell contact-dependent signalling in the vascular niche. Unexpectedly, we find that direct cell-cell interactions with endothelial cells enforce quiescence and promote stem cell identity. Mechanistically, endothelial ephrinB2 and Jagged1 mediate these effects by suppressing cell-cycle entry downstream of mitogens and inducing stemness genes to jointly inhibit differentiation. In vivo, endothelial-specific ablation of either of the genes which encode these proteins, Efnb2 and Jag1 respectively, aberrantly activates quiescent stem cells, resulting in depletion. Thus, we identify the vasculature as a critical niche compartment for stem cell maintenance, furthering our understanding of how anchorage to the niche maintains stem cells within a pro-differentiative microenvironment.

Eph/ephrin molecules--a hub for signaling and endocytosis.

The development, homeostasis, and regeneration of complex organ systems require extensive cell-cell communication to ensure that different cells proliferate, migrate, differentiate, assemble, and function in a coordinated and timely fashion. Eph receptor tyrosine kinases and their ephrin ligands are critical regulators of cell contact-dependent signaling and patterning. Eph/ephrin binding can lead to very diverse biological readouts such as adhesion versus repulsion, or increased versus decreased motility. Accordingly, depending on cell type and context, a limited and conserved set of receptor-ligand interactions is translated into a large variety of downstream signaling processes. Recent evidence indicates that the endocytosis of Eph/ephrin molecules, together with the internalization of various associated tissue-specific effectors, might be one of the key principles responsible for such highly diverse and adaptable biological roles. Here, we summarize recent insights into Eph/ephrin signaling and endocytosis in three biological systems; i.e., the brain, intestine, and vasculature.

Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.

The retina is a powerful experimental system for the analysis of angiogenic blood vessel growth in the postnatal organisms. The three-dimensional architecture of the vessel network and processes as diverse as endothelial cell (EC) proliferation, sprouting, perivascular cell recruitment, vessel remodeling or maturation can be investigated at high resolution. The characterization of physiological and pathological angiogenic processes in mice has been greatly facilitated by inducible and cell type-specific loss-of-function and gain-of-function genetics. In this paper, we provide a detailed protocol for tamoxifen-inducible gene deletion in neonatal mice, as well as for retina dissection, whole-mount immunostaining and the quantitation of EC sprouting and proliferation. These methods have been optimized by our laboratory and yield reliable results. The entire protocol takes approximately 10 d to complete.

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.

The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis. Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A). The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding. Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2DeltaV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.

TIPT2 and geminin interact with basal transcription factors to synergize in transcriptional regulation.

BACKGROUND: The re-replication inhibitor Geminin binds to several transcription factors including homeodomain proteins, and to members of the polycomb and the SWI/SNF complexes. RESULTS: Here we describe the TATA-binding protein-like factor-interacting protein (TIPT) isoform 2, as a strong binding partner of Geminin. TIPT2 is widely expressed in mouse embryonic and adult tissues, residing both in cyto- and nucleoplasma, and enriched in the nucleolus. Like Geminin, also TIPT2 interacts with several polycomb factors, with the general transcription factor TBP (TATA box binding protein), and with the related protein TBPL1 (TRF2). TIPT2 synergizes with geminin and TBP in the activation of TATA box-containing promoters, and with TBPL1 and geminin in the activation of the TATA-less NF1 promoter. Geminin and TIPT2 were detected in the chromatin near TBP/TBPL1 binding sites. CONCLUSION: Together, our study introduces a novel transcriptional regulator and its function in cooperation with chromatin associated factors and the basal transcription machinery.

MicroRNAs in organogenesis and disease.

Large numbers and quantities of different, small RNA molecules are present in the cytoplasm of animal and plant cells. One subclass of these molecules is represented by the noncoding microRNAs. Since their discovery in the 1990s a multitude of basic information has accumulated, which has identified their function in post-transcriptional control, either via degradation or translational inhibition of target mRNAs. This function is in most of the cases a finetuning of gene expression, working in parallel with transcriptional regulatory processes. MicroRNA expression profiles are highly dynamic during embryonic development and in adulthood. Misexpression of microRNAs can perturb embryogenesis, organogenesis, tissue homeostasis and the cell cycle. Evidence from gain- and loss-of function studies indicates roles for microRNAs in pathophysiologic states including cardiac hypertrophy, muscle dystrophy, hepatitis infection, diabetes, Parkinson syndrome, hematological malignancies and other types of cancer. In this review, we focus on studies addressing the role of various microRNAs in heart, muscle, liver, pancreas, central nervous system, and hematopoiesis.