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.

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.

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.

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.

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.

VEGF188 promotes corneal reinnervation after injury.

Vascular endothelial growth factor A (VEGF) induces angiogenesis and vascular hyperpermeability in ocular tissues and is therefore a key therapeutic target for eye conditions in which these processes are dysregulated. In contrast, the therapeutic potential of VEGF's neurotrophic roles in the eye has remained unexploited. In particular, it is not known whether modulating levels of any of the 3 major alternatively spliced VEGF isoforms might provide a therapeutic approach to promote neural health in the eye without inducing vascular pathology. Here, we have used a variety of mouse models to demonstrate differences in overall VEGF levels and VEGF isoform ratios across tissues in the healthy eye. We further show that VEGF isoform expression was differentially regulated in retinal versus corneal disease models. Among the 3 major isoforms - termed VEGF120, VEGF164, and VEGF188 - VEGF188 was upregulated to the greatest extent in injured cornea, where it was both necessary and sufficient for corneal nerve regeneration. Moreover, topical VEGF188 application further promoted corneal nerve regeneration without inducing pathological neovascularization. VEGF isoform modulation should therefore be explored further for its potential in promoting neural health in the eye.

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.

Vascular-Derived Vegfa Promotes Cortical Interneuron Migration and Proximity to the Vasculature in the Developing Forebrain.

Vascular endothelial growth factor (Vegfa) is essential for promoting the vascularization of the embryonic murine forebrain. In addition, it directly influences neural development, although its role in the forming forebrain is less well elucidated. It was recently suggested that Vegfa may influence the development of GABAergic interneurons, inhibitory cells with crucial signaling roles in cortical neuronal circuits. However, the mechanism by which it affects interneuron development remains unknown. Here we investigated the developmental processes by which Vegfa may influence cortical interneuron development by analyzing transgenic mice that ubiquitously express the Vegfa120 isoform to perturb its signaling gradient. We found that interneurons reach the dorsal cortex at mid phases of corticogenesis despite an aberrant vascular network. Instead, endothelial ablation of Vegfa alters cortical interneuron numbers, their intracortical distribution and spatial proximity to blood vessels. We show for the first time that vascular-secreted guidance factors promote early-migrating interneurons in the intact forebrain in vivo and identify a novel role for vascular-Vegfa in this process.

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.

VEGF-A and neuropilin 1 (NRP1) shape axon projections in the developing CNS via dual roles in neurons and blood vessels.

Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.

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.

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.

Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

NRP1 function and targeting in neurovascular development and eye disease.

Neuropilin 1 (NRP1) is expressed by neurons, blood vessels, immune cells and many other cell types in the mammalian body and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function. In recent years, several types of mouse knockout models have been developed that have provided useful tools for experimental investigation of NRP1 function, and a multitude of therapeutics targeting NRP1 have been designed, mostly with the view to explore them for cancer treatment. This review provides a general overview of current knowledge of the signalling pathways that are modulated by NRP1, with particular focus on neuronal and vascular roles in the brain and retina. This review will also discuss the potential of NRP1 inhibitors for the treatment for neovascular eye diseases.

Myeloid-Derived Vascular Endothelial Growth Factor and Hypoxia-Inducible Factor Are Dispensable for Ocular Neovascularization--Brief Report.

OBJECTIVE: Ocular neovascularization (ONV) is a pathological feature of sight-threatening human diseases, such as diabetic retinopathy and age-related macular degeneration. Macrophage depletion in mouse models of ONV reduces the formation of pathological blood vessels, and myeloid cells are widely considered an important source of the vascular endothelial growth factor A (VEGF). However, the importance of VEGF or its upstream regulators hypoxia-inducible factor-1α (HIF1α) and hypoxia-inducible factor-2α (HIF2α) as myeloid-derived regulators of ONV remains to be determined. APPROACH AND RESULTS: We used 2 mouse models of ONV, choroidal neovascularization and oxygen-induced retinopathy, to show that Vegfa is highly expressed by several cell types, but not myeloid cells during ONV. Moreover, myeloid-specific VEGF ablation did not reduce total ocular VEGF during choroidal neovascularization or oxygen-induced retinopathy. In agreement, the conditional inactivation of Vegfa, Hif1a, or Epas1 in recruited and resident myeloid cells that accumulated at sites of neovascularization did not significantly reduce choroidal neovascularization or oxygen-induced retinopathy. CONCLUSIONS: The finding that myeloid cells are not a significant local source of VEGF in these rodent models of ONV suggests that myeloid function in neovascular eye disease differs from skin wound healing and other neovascular pathologies.

Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves.

The peripheral nervous system has remarkable regenerative capacities in that it can repair a fully cut nerve. This requires Schwann cells to migrate collectively to guide regrowing axons across a 'bridge' of new tissue, which forms to reconnect a severed nerve. Here we show that blood vessels direct the migrating cords of Schwann cells. This multicellular process is initiated by hypoxia, selectively sensed by macrophages within the bridge, which via VEGF-A secretion induce a polarized vasculature that relieves the hypoxia. Schwann cells then use the blood vessels as "tracks" to cross the bridge taking regrowing axons with them. Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair. This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.