Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

Arterialization requires the timely suppression of cell growth.

The formation of arteries is thought to occur by the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow1,2. The initial steps of arterial specification require both the VEGF and Notch signalling pathways3-5. Here, we combine inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous differentiation and mobilization. We show that endothelial cells with high levels of VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; however, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that increased levels of VEGF and Notch signalling in pre-arterial capillaries suppresses MYC-dependent metabolic and cell-cycle activities, and promotes the incorporation of endothelial cells into arteries. Mosaic lineage-tracing studies showed that endothelial cells that lack the Notch-RBPJ transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when the function of MYC was suppressed. Thus, the development of arteries does not require the direct induction of a Notch-dependent arterial differentiation programme, but instead depends on the timely suppression of endothelial cell-cycle progression and metabolism, a process that precedes arterial mobilization and complete differentiation.

iSuRe-HadCre is an essential tool for effective conditional genetics.

Methods for modifying gene function at high spatiotemporal resolution in mice have revolutionized biomedical research, with Cre-loxP being the most widely used technology. However, the Cre-loxP technology has several drawbacks, including weak activity, leakiness, toxicity, and low reliability of existing Cre-reporters. This is mainly because different genes flanked by loxP sites (floxed) vary widely in their sensitivity to Cre-mediated recombination. Here, we report the generation, validation, and utility of iSuRe-HadCre, a new dual Cre-reporter and deleter mouse line that avoids these drawbacks. iSuRe-HadCre achieves this through a novel inducible dual-recombinase genetic cascade that ensures that cells expressing a fluorescent reporter had only transient Cre activity, that is nonetheless sufficient to effectively delete floxed genes. iSuRe-HadCre worked reliably in all cell types and for the 13 floxed genes tested. This new tool will enable the precise, efficient, and trustworthy analysis of gene function in entire mouse tissues or in single cells.

Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.

BACKGROUND: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood. METHODS: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries. RESULTS: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction. CONCLUSIONS: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.

The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis.

The Notch pathway is a highly conserved signaling system that controls a diversity of growth, differentiation, and patterning processes. In growing blood vessels, sprouting of endothelial tip cells is inhibited by Notch signaling, which is activated by binding of the Notch receptor to its ligand Delta-like 4 (Dll4). Here, we show that the Notch ligand Jagged1 is a potent proangiogenic regulator in mice that antagonizes Dll4-Notch signaling in cells expressing Fringe family glycosyltransferases. Upon glycosylation of Notch, Dll4-Notch signaling is enhanced, whereas Jagged1 has weak signaling capacity and competes with Dll4. Our findings establish that the equilibrium between two Notch ligands with distinct spatial expression patterns and opposing functional roles regulates angiogenesis, a mechanism that might also apply to other Notch-controlled biological processes.

Zebrafish mutants in vegfab can affect endothelial cell proliferation without altering ERK phosphorylation and are phenocopied by loss of PI3K signaling.

The formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa had an impact on endothelial cell (EC) migration and proliferation. Surprisingly, mutations in vegfab more strongly affected EC proliferation in distinct blood vessels, such as intersegmental blood vessels in the zebrafish trunk and central arteries in the head. Analysis of downstream signaling pathways revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutant phenotypes in affected blood vessels. Together, these results suggest that extracellular matrix bound Vegfa might act through PI3K signaling to control EC proliferation in a distinct set of blood vessels during angiogenesis.

Endothelial sprouting, proliferation, or senescence: tipping the balance from physiology to pathology.

Therapeutic modulation of vascular cell proliferation and migration is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The general view is that an increase in vascular growth factor levels or mitogenic stimulation is beneficial for angiogenesis, since it leads to an increase in both endothelial proliferation and sprouting. However, several recent studies showed that an increase in mitogenic stimuli can also lead to the arrest of angiogenesis. This is due to the existence of intrinsic signaling feedback loops and cell cycle checkpoints that work in synchrony to maintain a balance between endothelial proliferation and sprouting. This balance is tightly and effectively regulated during tissue growth and is often deregulated or impaired in disease. Most therapeutic strategies used so far to promote vascular growth simply increase mitogenic stimuli, without taking into account its deleterious effects on this balance and on vascular cells. Here, we review the main findings on the mechanisms controlling physiological vascular sprouting, proliferation, and senescence and how those mechanisms are often deregulated in acquired or congenital cardiovascular disease leading to a diverse range of pathologies. We also discuss alternative approaches to increase the effectiveness of pro-angiogenic therapies in cardiovascular regenerative medicine.

Intricacies of conditional genetics in vascular biology.

PURPOSE OF REVIEW: Conditional or inducible recombinase-based genetics is still the gold standard to analyse gene function, given its high specificity, temporal control, limited toxicity and the many available genetic tools. However, it is based on methods that have inherent limitations and shortcomings. The purpose of this review is to summarize and contrast the different available methods used to perform conditional gene function analysis to better inform the community about their particularities and the need to use better methods. RECENT FINDINGS: As any other biomedical field, the vascular biology field has moved from using and analysing standard gene knockout (KO) mice, to use conditional genetics to delete a given gene only at a given time point, cell-type or organ of interest. This is the only way to accurately understand a gene function and avoid other confounding factors. Therefore, nowadays the majority of laboratories working with mice use CreERT2-tamoxifen-inducible genetics. However, this necessary transition from the relatively simple KO genetics to the more sophisticated conditional genetics brought a series of additional methodological issues that are often overlooked or unappreciated. Recent findings from several laboratories have shown how important is to know what to expect from and control for in conditional genetics. Without this a priori knowledge, the quality, robustness, time and costs of conditional genetic experiments can be significantly compromised. SUMMARY: We start this review by discussing the intricacies of the most simple and widely used methods to perform conditional genetics and then extend on the need of novel and more advanced methods to increase the ease, efficiency and reliability of conditional mutagenesis and gene function analysis.

Role of Notch in endothelial biology.

The Notch signalling pathway is one of the main regulators of endothelial biology. In the last 20 years the critical function of Notch has been uncovered in the context of angiogenesis, participating in tip-stalk specification, arterial-venous differentiation, vessel stabilization, and maturation processes. Importantly, pharmacological compounds targeting distinct members of the Notch signalling pathway have been used in the clinics for cancer therapy. However, the underlying mechanisms that support the variety of outcomes triggered by Notch in apparently opposite contexts such as angiogenesis and vascular homeostasis remain unknown. In recent years, advances in -omics technologies together with mosaic analysis and high molecular, cellular and temporal resolution studies have allowed a better understanding of the mechanisms driven by the Notch signalling pathway in different endothelial contexts. In this review we will focus on the main findings that revisit the role of Notch signalling in vascular biology. We will also discuss potential future directions and technologies that will shed light on the puzzling role of Notch during endothelial growth and homeostasis. Addressing these open questions may allow the improvement and development of therapeutic strategies based on modulation of the Notch signalling pathway.

High-throughput identification of small molecules that affect human embryonic vascular development.

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Inhibition of Endothelial NOTCH1 Signaling Attenuates Inflammation by Reducing Cytokine-Mediated Histone Acetylation at Inflammatory Enhancers.

OBJECTIVE: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. APPROACH AND RESULTS: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1ß stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers. CONCLUSIONS: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling.

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.

Identification of a clonally expanding haematopoietic compartment in bone marrow.

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self-renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high-resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48- putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48- cells. Our results identify a previously unrecognized, vessel-associated BM compartment with a specific localization and properties distinct from the marrow cavity.

Notch controls retinal blood vessel maturation and quiescence.

Blood vessels form a hierarchically organized network of arteries, capillaries and veins, which develops through a series of growth, pruning and maturation processes. In contrast to the rapidly increasing insight into the processes controlling vascular growth and, in particular, endothelial sprouting and proliferation, the conversion of immature vessels into a fully functional, quiescent vasculature remains little understood. Here we used inducible, cell type-specific genetic approaches to show that endothelial Notch signaling is crucial for the remodeling of veins and the perivenous capillary plexus, which occurs after the completion of the initial angiogenic growth phase in the retina of adolescent mice. Mutant vessels showed ectopic proliferation and sprouting, defective recruitment of supporting mural cells, and failed to downregulate the expression of VEGF receptors. Surprisingly, by contrast Notch was dispensable in the endothelium of remodeling postnatal arteries. Taken together, our results identify key processes contributing to vessel remodeling, maturation and the acquisition of a quiescent phenotype in the final stage of developmental angiogenesis.

Esm1 modulates endothelial tip cell behavior and vascular permeability by enhancing VEGF bioavailability.

RATIONALE: Endothelial cell-specific molecule 1 (Esm1) is a secreted protein thought to play a role in angiogenesis and inflammation. However, there is currently no direct in vivo evidence supporting a function of Esm1 in either of these processes. OBJECTIVE: To determine the role of Esm1 in vivo and the underlying molecular mechanisms. METHODS AND RESULTS: We generated and analyzed Esm1 knockout (Esm1(KO)) mice to study its role in angiogenesis and inflammation. Esm1 expression is induced by the vascular endothelial growth factor A (VEGF-A) in endothelial tip cells of the mouse retina. Esm1(KO) mice showed delayed vascular outgrowth and reduced filopodia extension, which are both VEGF-A-dependent processes. Impairment of Esm1 function led to a decrease in phosphorylated Erk1/2 (extracellular-signal regulated kinases 1/2) in sprouting vessels. We also found that Esm1(KO) mice displayed a 40% decrease in leukocyte transmigration. Moreover, VEGF-induced vascular permeability was decreased by 30% in Esm1(KO) mice and specifically on stimulation with VEGF-A165 but not VEGF-A121. Accordingly, cerebral edema attributable to ischemic stroke-induced vascular permeability was reduced by 50% in the absence of Esm1. Mechanistically, we show that Esm1 binds directly to fibronectin and thereby displaces fibronectin-bound VEGF-A165 leading to increased bioavailability of VEGF-A165 and subsequently enhanced levels of VEGF-A signaling. CONCLUSIONS: Esm1 is simultaneously a target and modulator of VEGF signaling in endothelial cells, playing a role in angiogenesis, inflammation, and vascular permeability, which might be of potential interest for therapeutic applications.

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

Notch-mediated cellular interactions between vascular cells.

Vessel formation and differentiation to a proper hierarchical vasculature requires a coordinated effort from endothelial and mural cells. Over the last decade Notch was identified as a key player in this process by promoting vascular arterialization and modulating endothelial tip-stalk phenotypes. Recent work has identified that Notch fine-tunes the diverse endothelial phenotypes through regulation of canonical cell-cycle and metabolism regulators, such as ERK and Myc. During arterialization, Notch signaling inhibits the cell-cycle and metabolism of endothelial cells which coincides with the acquisition of arterial identity. During angiogenesis, the same molecular machinery prevents the hypermitogenic arrest and excessive sprouting of vessels. Notch also signals in pericytes and smooth muscle cells promoting vascular coverage and maturation. Here, we will review the latest findings on how Notch signals regulate the differentiation and interactions among vascular cells during organ development and homeostasis.

Incongruence between transcriptional and vascular pathophysiological cell states.

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

BMPing up endocardial angiogenesis to generate coronary vessels.

Understanding how coronary vessels develop is important for designing better strategies to repair ischemic hearts. In this issue of Developmental Cell, D'Amato et al. report that BMP2 and CXCL12/CXCR4 act sequentially on endocardial cells to drive coronary angiogenesis and artery morphogenesis.

Apelin-driven endothelial cell migration sustains intestinal progenitor cells and tumor growth.

Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.