The BulkECexplorer compiles endothelial bulk transcriptomes to predict functional versus leaky transcription.

Transcriptomic data can be mined to understand the molecular activity of cell types. Yet, functional genes may remain undetected in RNA sequencing (RNA-seq) experiments for technical reasons, such as insufficient read depth or gene dropout. Conversely, RNA-seq experiments may detect lowly expressed mRNAs thought to be biologically irrelevant products of leaky transcription. To represent a cell type's functional transcriptome more accurately, we propose compiling many bulk RNA-seq datasets into a compendium and applying established classification models to predict whether detected transcripts are likely products of active or leaky transcription. Here, we present the BulkECexplorer (bulk RNA-seq endothelial cell explorer) compendium of 240 bulk RNA-seq datasets from five vascular endothelial cell subtypes. This resource reports transcript counts for genes of interest and predicts whether detected transcripts are likely the products of active or leaky gene expression. Beyond its usefulness for vascular biology research, this resource provides a blueprint for developing analogous tools for other cell types.

SEMA6A drives GnRH neuron-dependent puberty onset by tuning median eminence vascular permeability.

Innervation of the hypothalamic median eminence by Gonadotropin-Releasing Hormone (GnRH) neurons is vital to ensure puberty onset and successful reproduction. However, the molecular and cellular mechanisms underlying median eminence development and pubertal timing are incompletely understood. Here we show that Semaphorin-6A is strongly expressed by median eminence-resident oligodendrocytes positioned adjacent to GnRH neuron projections and fenestrated capillaries, and that Semaphorin-6A is required for GnRH neuron innervation and puberty onset. In vitro and in vivo experiments reveal an unexpected function for Semaphorin-6A, via its receptor Plexin-A2, in the control of median eminence vascular permeability to maintain neuroendocrine homeostasis. To support the significance of these findings in humans, we identify patients with delayed puberty carrying a novel pathogenic variant of SEMA6A. In all, our data reveal a role for Semaphorin-6A in regulating GnRH neuron patterning by tuning the median eminence vascular barrier and thereby controlling puberty onset.

Emergent mechanical control of vascular morphogenesis.

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.

A Refined Single Cell Landscape of Haematopoiesis in the Mouse Foetal Liver.

During prenatal life, the foetal liver is colonised by several waves of haematopoietic progenitors to act as the main haematopoietic organ. Single cell (sc) RNA-seq has been used to identify foetal liver cell types via their transcriptomic signature and to compare gene expression patterns as haematopoietic development proceeds. To obtain a refined single cell landscape of haematopoiesis in the foetal liver, we have generated a scRNA-seq dataset from a whole mouse E12.5 liver that includes a larger number of cells than prior datasets at this stage and was obtained without cell type preselection to include all liver cell populations. We combined mining of this dataset with that of previously published datasets at other developmental stages to follow transcriptional dynamics as well as the cell cycle state of developing haematopoietic lineages. Our findings corroborate several prior reports on the timing of liver colonisation by haematopoietic progenitors and the emergence of differentiated lineages and provide further molecular characterisation of each cell population. Extending these findings, we demonstrate the existence of a foetal intermediate haemoglobin profile in the mouse, similar to that previously identified in humans, and a previously unidentified population of primitive erythroid cells in the foetal liver.

Enlarged Field of View in Spatially Modulated Selective Volume Illumination Microscopy.

Three-dimensional fluorescence microscopy is a key technology for inspecting biological samples, ranging from single cells to entire organisms. We recently proposed a novel approach called spatially modulated Selective Volume Illumination Microscopy (smSVIM) to suppress illumination artifacts and to reduce the required number of measurements using an LED source. Here, we discuss a new strategy based on smSVIM for imaging large transparent specimens or voluminous chemically cleared tissues. The strategy permits steady mounting of the sample, achieving uniform resolution over a large field of view thanks to the synchronized motion of the illumination lens and the camera rolling shutter. Aided by a tailored deconvolution method for image reconstruction, we demonstrate significant improvement of the resolution at different magnification using samples of varying sizes and spatial features.

Evaluating VEGF-Induced Vascular Leakage Using the Miles Assay.

Before the endothelial mitogenic activity of the Vascular Endothelial Growth Factor A (VEGF) was described, VEGF had already been identified for its ability to induce vascular leakage. VEGF-induced vascular leakage has been most frequently studied in vivo using the Miles assay, a simple yet invaluable technique that has allowed researchers to unravel the molecular mechanisms underpinning vascular leakage both for VEGF and other permeability inducing agents. In this protocol, a mouse is intravenously injected with Evans Blue dye before VEGF is administered locally via intradermal injection. VEGF promotes vascular leak of serum proteins in the dermis, enabling Evans Blue-labeled albumin extravasation from the circulation and subsequent accumulation in the skin. As the volume of dye extravasation is proportional to the degree of vascular leak, it can be quantified as a proxy measurement of VEGF-induced vascular leakage.

The Embryonic Mouse Hindbrain and Postnatal Retina as In Vivo Models to Study Angiogenesis.

Angiogenesis, the growth of new blood vessels from pre-existing ones, is a fundamental process for organ development, exercise-induced muscle growth, and wound healing, but is also associated with different diseases such as cancer and neovascular eye disease. Accordingly, elucidating the molecular and cellular mechanisms of angiogenesis has the potential to identify new therapeutic targets to stimulate new vessel formation in ischemic tissues or inhibit pathological vessel growth in disease. This chapter describes the mouse embryo hindbrain and postnatal retina as models to study physiological angiogenesis and provides detailed protocols for tissue dissection, sample staining and analysis.

KIT is dispensable for physiological organ vascularisation in the embryo.

Blood vessels form vast networks in all vertebrate organs to sustain tissue growth, repair and homeostatic metabolism, but they also contribute to a range of diseases with neovascularisation. It is, therefore, important to define the molecular mechanisms that underpin blood vessel growth. The receptor tyrosine kinase KIT is required for the normal expansion of hematopoietic progenitors that arise during embryogenesis from hemogenic endothelium in the yolk sac and dorsal aorta. Additionally, KIT has been reported to be expressed in endothelial cells during embryonic brain vascularisation and has been implicated in pathological angiogenesis. However, it is neither known whether KIT expression is widespread in normal organ endothelium nor whether it promotes blood vessel growth in developing organs. Here, we have used single-cell analyses to show that KIT is expressed in endothelial cell subsets of several organs, both in the adult and in the developing embryo. Knockout mouse analyses revealed that KIT is dispensable for vascularisation of growing organs in the midgestation embryo, including the lung, liver and brain. By contrast, vascular changes emerged during late-stage embryogenesis in these organs from KIT-deficient embryos, concurrent with severe erythrocyte deficiency and growth retardation. These findings suggest that KIT is not required for developmental tissue vascularisation in physiological conditions, but that KIT deficiency causes foetal anaemia at late gestation and thereby pathological vascular remodelling.

Quantifying and Characterizing Angiogenesis Using the Postnatal Mouse Retina.

Angiogenesis refers to the expansion of blood vessels from a preexisting vascular plexus, and it is a fundamental process for organ development, the female reproductive system, and wound healing, but it is also a common denominator in several diseases such as cancer and neovascular eye disease. For these reasons, shedding light on the molecular and cellular mechanisms of angiogenesis has the potential to devise new therapeutic strategies to refrain pathological vessel growth or even promote new vessel formation in ischemic conditions and organ grafts. The mouse postnatal retina provides an excellent and widely adopted model to study physiological angiogenesis in vivo, and this chapter outlines a detailed protocol for its dissection, staining, and analysis of the vasculature.

Fetal liver hematopoiesis revisited: a precast hierarchy.

Late fetal liver hematopoiesis was thought to primarily rely on hematopoietic stem cells (HSCs). Using new genetic-tracing tools, a study shows that EVI1-positive HSCs mainly undergo expansion in the fetal liver, while differentiated blood cell production depends on HSC-independent intermediate hematopoietic progenitors.

KIT Is Required for Fetal Liver Hematopoiesis.

In the mouse embryo, endothelial cell (EC) progenitors almost concomitantly give rise to the first blood vessels in the yolk sac and the large vessels of the embryo proper. Although the first blood cells form in the yolk sac before blood vessels have assembled, consecutive waves of hematopoietic progenitors subsequently bud from hemogenic endothelium located within the wall of yolk sac and large intraembryonic vessels in a process termed endothelial-to-hematopoietic transition (endoHT). The receptor tyrosine kinase KIT is required for late embryonic erythropoiesis, but KIT is also expressed in hematopoietic progenitors that arise via endoHT from yolk sac hemogenic endothelium to generate early, transient hematopoietic waves. However, it remains unclear whether KIT has essential roles in early hematopoiesis. Here, we have combined single-cell expression studies with the analysis of knockout mice to show that KIT is dispensable for yolk sac endoHT but required for transient definitive hematopoiesis in the fetal liver.

Neuropilin 1 Regulation of Vascular Permeability Signaling.

The vascular endothelium acts as a selective barrier to regulate macromolecule exchange between the blood and tissues. However, the integrity of the endothelium barrier is compromised in an array of pathological settings, including ischemic disease and cancer, which are the leading causes of death worldwide. The resulting vascular hyperpermeability to plasma molecules as well as leukocytes then leads to tissue damaging edema formation and inflammation. The vascular endothelial growth factor A (VEGFA) is a potent permeability factor, and therefore a desirable target for impeding vascular hyperpermeability. However, VEGFA also promotes angiogenesis, the growth of new blood vessels, which is required for reperfusion of ischemic tissues. Moreover, edema increases interstitial pressure in poorly perfused tumors, thereby affecting the delivery of therapeutics, which could be counteracted by stimulating the growth of new functional blood vessels. Thus, targets must be identified to accurately modulate the barrier function of blood vessels without affecting angiogenesis, as well as to develop more effective pro- or anti-angiogenic therapies. Recent studies have shown that the VEGFA co-receptor neuropilin 1 (NRP1) could be playing a fundamental role in steering VEGFA-induced responses of vascular endothelial cells towards angiogenesis or vascular permeability. Moreover, NRP1 is involved in mediating permeability signals induced by ligands other than VEGFA. This review therefore focuses on current knowledge on the role of NRP1 in the regulation of vascular permeability signaling in the endothelium to provide an up-to-date landscape of the current knowledge in this field.

Semaphorin Regulation by the Chromatin Remodeler CHD7: An Emerging Genetic Interaction Shaping Neural Cells and Neural Crest in Development and Cancer.

CHD7 is a chromatin remodeler protein that controls gene expression via the formation of multi-protein complexes with specific transcription factors. During development, CHD7 controls several differentiation programs, mainly by acting on neural progenitors and neural crest (NC) cells. Thus, its roles range from the central nervous system to the peripheral nervous system and the organs colonized by NC cells, including the heart. Accordingly, mutated CHD7 is linked to CHARGE syndrome, which is characterized by several neuronal dysfunctions and by malformations of NC-derived/populated organs. Altered CHD7 has also been associated with different neoplastic transformations. Interestingly, recent evidence revealed that semaphorins, a class of molecules involved in developmental and pathological processes similar to those controlled by CHD7, are regulated by CHD7 in a context-specific manner. In this article, we will review the recent insights that support the existence of genetic interactions between these pathways, both during developmental processes and cancer progression.

PLXNA1 and PLXNA3 cooperate to pattern the nasal axons that guide gonadotropin-releasing hormone neurons.

Gonadotropin-releasing hormone (GnRH) neurons regulate puberty onset and sexual reproduction by secreting GnRH to activate and maintain the hypothalamic-pituitary-gonadal axis. During embryonic development, GnRH neurons migrate along olfactory and vomeronasal axons through the nose into the brain, where they project to the median eminence to release GnRH. The secreted glycoprotein SEMA3A binds its receptors neuropilin (NRP) 1 or NRP2 to position these axons for correct GnRH neuron migration, with an additional role for the NRP co-receptor PLXNA1. Accordingly, mutations in SEMA3A, NRP1, NRP2 and PLXNA1 have been linked to defective GnRH neuron development in mice and inherited GnRH deficiency in humans. Here, we show that only the combined loss of PLXNA1 and PLXNA3 phenocopied the full spectrum of nasal axon and GnRH neuron defects of SEMA3A knockout mice. Together with Plxna1, the human orthologue of Plxna3 should therefore be investigated as a candidate gene for inherited GnRH deficiency.

Erythro-myeloid progenitors contribute endothelial cells to blood vessels.

The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors.

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes.

Context: Self-limited delayed puberty (DP) segregates in an autosomal-dominant pattern, but the genetic basis is largely unknown. Although DP is sometimes seen in relatives of patients with hypogonadotropic hypogonadism (HH), mutations in genes known to cause HH that segregate with the trait of familial self-limited DP have not yet been identified. Objective: To assess the contribution of mutations in genes known to cause HH to the phenotype of self-limited DP. Design, Patients, and Setting: We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited DP, validated the pathogenicity of the identified gene variant in vitro, and examined the tissue expression and functional requirement of the mouse homolog in vivo. Results: A potentially pathogenic gene variant segregating with DP was identified in 1 of 28 known HH genes examined. This pathogenic variant occurred in HS6ST1 in one pedigree and segregated with the trait in the six affected members with heterozygous transmission (P = 3.01 × 10-5). Biochemical analysis showed that this mutation reduced sulfotransferase activity in vitro. Hs6st1 mRNA was expressed in peripubertal wild-type mouse hypothalamus. GnRH neuron counts were similar in Hs6st1+/- and Hs6st1+/+ mice, but vaginal opening was delayed in Hs6st1+/- mice despite normal postnatal growth. Conclusions: We have linked a deleterious mutation in HS6ST1 to familial self-limited DP and show that heterozygous Hs6st1 loss causes DP in mice. In this study, the observed overlap in potentially pathogenic mutations contributing to the phenotypes of self-limited DP and HH was limited to this one gene.

VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation.

The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth.

A Small Molecule Inhibitor of PDK1/PLCγ1 Interaction Blocks Breast and Melanoma Cancer Cell Invasion.

Strong evidence suggests that phospholipase Cγ1 (PLCγ1) is a suitable target to counteract tumourigenesis and metastasis dissemination. We recently identified a novel signalling pathway required for PLCγ1 activation which involves formation of a protein complex with 3-phosphoinositide-dependent protein kinase 1 (PDK1). In an effort to define novel strategies to inhibit PLCγ1-dependent signals we tested here whether a newly identified and highly specific PDK1 inhibitor, 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5), could affect PDK1/PLCγ1 interaction and impair PLCγ1-dependent cellular functions in cancer cells. Here, we demonstrate that 2-O-Bn-InsP5 interacts specifically with the pleckstrin homology domain of PDK1 and impairs formation of a PDK1/PLCγ1 complex. 2-O-Bn-InsP5 is able to inhibit the epidermal growth factor-induced PLCγ1 phosphorylation and activity, ultimately resulting in impaired cancer cell migration and invasion. Importantly, we report that 2-O-Bn-InsP5 inhibits cancer cell dissemination in zebrafish xenotransplants. This work demonstrates that the PDK1/PLCγ1 complex is a potential therapeutic target to prevent metastasis and it identifies 2-O-Bn-InsP5 as a leading compound for development of anti-metastatic drugs.

2- and 6-O-sulfated proteoglycans have distinct and complementary roles in cranial axon guidance and motor neuron migration.

The correct migration and axon extension of neurons in the developing nervous system is essential for the appropriate wiring and function of neural networks. Here, we report that O-sulfotransferases, a class of enzymes that modify heparan sulfate proteoglycans (HSPGs), are essential to regulate neuronal migration and axon development. We show that the 6-O-sulfotransferases HS6ST1 and HS6ST2 are essential for cranial axon patterning, whilst the 2-O-sulfotransferase HS2ST (also known as HS2ST1) is important to regulate the migration of facial branchiomotor (FBM) neurons in the hindbrain. We have also investigated how HS2ST interacts with other signals in the hindbrain and show that fibroblast growth factor (FGF) signalling regulates FBM neuron migration in an HS2ST-dependent manner.