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.

CXCL12 promotes the crossing of retinal ganglion cell axons at the optic chiasm.

Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development.

Early human lung immune cell development and its role in epithelial cell fate.

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

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.

Tamoxifen exacerbates morbidity and mortality in male mice receiving medetomidine anaesthesia.

Tamoxifen-induced CreER-LoxP recombination is often used to induce spatiotemporally controlled gene deletion in genetically modified mice. Prior work has shown that tamoxifen and tamoxifen-induced CreER activation can have off-target effects that should be controlled. However, it has not yet been reported whether tamoxifen administration, independently of CreER expression, interacts adversely with commonly used anaesthetic drugs such as medetomidine or its enantiomer dexmedetomidine in laboratory mice (Mus musculus). Here, we report a high incidence of urinary plug formation and morbidity in male mice on a mixed C57Bl6/J6 and 129/SvEv background when tamoxifen treatment was followed by ketamine-medetomidine anaesthesia. Medetomidine is therefore contra-indicated for male mice after tamoxifen treatment. As dexmedetomidine causes morbidity and mortality in male mice at higher rates than medetomidine even without tamoxifen treatment, our findings suggest that dexmedetomidine is not a suitable alternative for anaesthesia of male mice after tamoxifen treatment. We conclude that the choice of anaesthetic drug needs to be carefully evaluated in studies using male mice that have undergone tamoxifen treatment for inducing CreER-LoxP recombination.

Endothelial PlexinD1 signaling instructs spinal cord vascularization and motor neuron development.

How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.

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.

The Embryonic Mouse Hindbrain Model to Study Sprouting Angiogenesis In Vivo.

Blood vessel growth is a fundamental process for organ development and wound healing but is also associated with ischemic diseases and cancer. The growth of new blood vessels from preexisting vasculature, termed sprouting angiogenesis, is the predominant mode of blood vessel growth in central nervous system vascularization and pathological vessel growth. Accordingly, studying the molecular and cellular mechanisms of angiogenesis holds the promise to find novel therapeutic targets to stimulate new vessel formation in ischemic tissues or inhibit pathological vessel growth in disease. The embryonic mouse hindbrain provides an excellent model to study sprouting angiogenesis in vivo by histochemical or fluorescent wholemount immunolabeling, thus allowing high-resolution image capture of nascent vasculature and subsequent quantification of relevant angiogenic parameters. This chapter describes how to use the mouse embryonic hindbrain as a model to study physiological angiogenesis, including detailed protocols for hindbrain dissection, wholemount staining, and angiogenic parameters analysis.

Whole-Mount Immunofluorescence Protocol for 3D Imaging, Reconstruction, and Quantification of Fourth Pharyngeal Arch Artery Formation.

Pharyngeal arch arteries (PAA) are formed early during mouse embryogenesis and remodel soon thereafter into the aortic arch arteries. Failure of these vessels to form or remodel results in congenital heart defects. This protocol is designed to study the formation of the PAA using whole-mount immunofluorescence staining, followed by tissue clearing with benzyl alcohol/benzyl benzoate (BAAB) and imaging by confocal microscopy. The fine cellular resolution obtained with this technique allows the embryonic vasculature of the pharyngeal arch artery endothelium to be visualized by surface rendering and quantitatively analyzed by counting the number of endothelial cells in both the PAA and the vascular plexus surrounding them.

Cre toxicity in mouse models of cardiovascular physiology and disease.

The Cre-LoxP system provides a widely used method for studying gene requirements in the mouse as the main mammalian genetic model organism. To define the molecular and cellular mechanisms that underlie cardiovascular development, function and disease, various mouse strains have been engineered that allow Cre-LoxP-mediated gene targeting within specific cell types of the cardiovascular system. Despite the usefulness of this system, evidence is accumulating that Cre activity can have toxic effects in cells, independently of its ability to recombine pairs of engineered LoxP sites in target genes. Here, we have gathered published evidence for Cre toxicity in cells and tissues relevant to cardiovascular biology and provide an overview of mechanisms proposed to underlie Cre toxicity. Based on this knowledge, we propose that each study utilising the Cre-LoxP system to investigate gene function in the cardiovascular system should incorporate appropriate controls to account for Cre toxicity.

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.

Mechanisms and cell lineages in lymphatic vascular development.

Lymphatic vessels have critical roles in both health and disease and their study is a rapidly evolving area of vascular biology. The consensus on how the first lymphatic vessels arise in the developing embryo has recently shifted. Originally, they were thought to solely derive by sprouting from veins. Since then, several studies have uncovered novel cellular mechanisms and a diversity of contributing cell lineages in the formation of organ lymphatic vasculature. Here, we review the key mechanisms and cell lineages contributing to lymphatic development, discuss the advantages and limitations of experimental techniques used for their study and highlight remaining knowledge gaps that require urgent attention. Emerging technologies should accelerate our understanding of how lymphatic vessels develop normally and how they contribute to disease.

First blood: the endothelial origins of hematopoietic progenitors.

Hematopoiesis in vertebrate embryos occurs in temporally and spatially overlapping waves in close proximity to blood vascular endothelial cells. Initially, yolk sac hematopoiesis produces primitive erythrocytes, megakaryocytes, and macrophages. Thereafter, sequential waves of definitive hematopoiesis arise from yolk sac and intraembryonic hemogenic endothelia through an endothelial-to-hematopoietic transition (EHT). During EHT, the endothelial and hematopoietic transcriptional programs are tightly co-regulated to orchestrate a shift in cell identity. In the yolk sac, EHT generates erythro-myeloid progenitors, which upon migration to the liver differentiate into fetal blood cells, including erythrocytes and tissue-resident macrophages. In the dorsal aorta, EHT produces hematopoietic stem cells, which engraft the fetal liver and then the bone marrow to sustain adult hematopoiesis. Recent studies have defined the relationship between the developing vascular and hematopoietic systems in animal models, including molecular mechanisms that drive the hemato-endothelial transcription program for EHT. Moreover, human pluripotent stem cells have enabled modeling of fetal human hematopoiesis and have begun to generate cell types of clinical interest for regenerative medicine.

miR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization.

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183-KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183-KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.

Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges.

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-ß, the toxic driver of Alzheimer's disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.

Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease.

Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis; a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease.