Summary Hematopoietic stem cells (HSCs) originate within the aorta-gonado-mesonephros (AGM) region of the midgestation embryo, but the cell type responsible for their emergence is unknown since critical hematopoietic factors are expressed in both the AGM endothelium and its underlying mesenchyme. Here we employ a temporally restricted genetic tracing strategy to selectively label the endothelium, and separately its underlying mesenchyme, during AGM development. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the endothelium is capable of HSC emergence. The endothelial progeny migrate to the fetal liver, and later to the bone marrow, are capable of expansion, self-renewal, and multi-lineage hematopoietic differentiation. HSC capacity is exclusively endothelial, as ex vivo analyses demonstrate lack of VE-cadherin Cre induction in circulating and fetal liver hematopoietic populations. Moreover, AGM mesenchyme, as selectively traced via a myocardin Cre line, is incapable of hematopoiesis. Our genetic tracing strategy therefore reveals an endothelial origin of HSCs.

Summary Maintenance of single layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of β1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal shaped endothelial cells, dysregulated levels and mis-localization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). β1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in β1 deficient vessels. Combined, our findings demonstrate that β1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation.

The lipolytic processing of triglyceride-rich lipoproteins (e.g., chylomicrons, very low density lipoproteins) by lipoprotein lipase (LPL) is the central event in plasma triglyceride metabolism and plays a crucial role in the delivery of lipid nutrients to parenchymal cells (e.g., adipocytes, myocytes) ( 1-4 ). LPL is synthesized by parenchymal cells and secreted into the interstitial spaces, but it needs to reach the capillary lumen in order to hydrolyze the triglycerides in plasma lipoproteins. Recent studies showed that GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, binds LPL avidly ( 5 ) and is required for the transport of LPL to the capillary lumen ( 6 ). In the absence of GPIHBP1, LPL accumulates in the interstitial spaces around parenchymal cells and is unable to process triglyceride-rich lipoproteins in the bloodstream, resulting in markedly elevated plasma triglyceride levels and interfering with the delivery of lipid nutrients to parenchymal cells ( 5-7 ).Although GPIHBP1 is essential for the delivery of LPL to the luminal face of capillaries, the cellular mechanisms for moving GPIHBP1 and LPL across endothelial cells are poorly defi ned. It is unclear whether GPIHBP1 and LPL move unidirectionally from the basolateral face of endothelial cells to LPL's site of action along the capillary lumen or whether "backwards traffi cking" also occurs (i.e., movement of GPIHBP1 and LPL from the lumen to the basolateral face of cells). A second issue is whether GPIHBP1 Abstract Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni-or bidirectional. We also asked whether GPIHBP1

The vitelline artery is a temporary structure that undergoes extensive remodeling during midgestation to eventually become the superior mesenteric artery (also called the cranial mesenteric artery, in the mouse). Here we show that, during this remodeling process, large clusters of hematopoietic progenitors emerge via extravascular budding and form structures that resemble previously described mesenteric blood islands. We demonstrate through fate mapping of vascular endothelium that these mesenteric blood islands are derived from the endothelium of the vitelline artery. We further show that the vitelline arterial endothelium and subsequent blood island structures originate from a lateral plate mesodermal population. Lineage tracing of the lateral plate mesoderm demonstrates contribution to all hemogenic vascular beds in the embryo, and eventually, all hematopoietic cells in the adult. The intraembryonic hematopoietic cell clusters contain vi- IntroductionThe first wave of embryonic hematopoiesis occurs in the yolk sac from mesodermal precursors at embryonic day 7.0. 1 These early yolk sac mesodermal cells, initially termed hemangioblasts, 2 were thought to have bipotentiality, giving rise to both blood and endothelium. 3 The descendants of hemangioblasts form structures called blood islands, 2 which consist of rounded hematopoietic cells (HCs) circumscribed by an endothelial layer. Although initially thought to propagate the entirety of the adult hematopoietic system, yolk sac hematopoiesis is not fully responsible for definitive hematopoietic stem cell (HSC) emergence but instead provides a transient immature (primitive) hematopoietic population that is later supplanted. [4][5][6] Yet the inability of the yolk sac to produce any definitive adult HCs has recently been questioned. 7,8 In addition, the concept of a mesodermal precursor to HSCs in the early yolk sac has been challenged by recent data suggesting that yolk sac hematopoiesis at the hemangioblast stage occurs through an endothelial intermediate. 9,10 Thus, the yolk sac probably provides both primitive and definitive HCs, but the difference between how the 2 populations emerge and timing of their emergence is still under investigation.Our understanding of intraembryonic definitive hematopoiesis has also been refined by a recent multitude of publications using various systems and model organisms. Previous landmark studies have demonstrated that instead of (or taking into account the more recent data: in addition to) the early yolk sac, later intraembryonic arterial sites give rise to definitive HSCs. [4][5][6][11][12][13] In these sites, large clusters of HSCs are attached to the endothelium, suggesting that a specific subset of endothelial cells (ECs, hemogenic endothelium) 14 are progenitors for HSCs. However, the concept of a mesodermal precursor for HSCs within these intraembryonic sites was also argued. 15,16 The phenomenon of endothelial derivedblood, termed hemogenic endothelium, 9,10,17-21 has become the new paradigm of HSC generation. Vascular...

This article is available online at http://www.jlr.org within cells and tissues is important, but the experimental approaches for creating high-resolution images of lipids within cells and tissues are quite limited. The most commonly used technique for lipid imaging in tissues is fl uorescence microscopy after the delivery of fl uorescently labeled lipids ( 2, 3 ). A drawback of that approach is that some lipids are not readily available as fl uorescent compounds, and one must worry about how a fl uorescent tag affects the biological properties of the lipid. To overcome these issues, label-free lipid imaging techniques have been developed, such as coherent anti-Stokes Raman scattering microscopy ( 4 ) and stimulated Raman scattering microscopy ( 5 ), but the lateral resolution and sensitivity of these approaches are limited ( 6 ).More recently, imaging MS has been utilized to visualize lipids ( 7,8 ). New ionization methods, primary ion beams, and newer instrument designs have made imaging MS more powerful. MALDI techniques, TOF secondary ion MS (SIMS), and magnetic sector SIMS represent complementary techniques for lipid imaging ( 9 ). In that order, these techniques have increasing spatial resolution but decreasing molecular specifi city. MALDI techniques are able to identify and quantify lipids with a resolution of a few micrometers; TOF-SIMS retains the ability to identify specifi c lipids and can achieve 0.5-1 µm lateral resolution. By measuring the elemental composition of lipids, a nanoscale SIMS (NanoSIMS) instrument yields images of lipids with up to 50 nm lateral resolution. We describe the use of NanoSIMS imaging to create high-resolution images of lipids in cells and tissues.Abstract Nanoscale secondary ion MS (NanoSIMS) imaging makes it possible to visualize stable isotope-labeled lipids in cells and tissues at 50 nm lateral resolution. Here we report the use of NanoSIMS imaging to visualize lipids in mouse cells and tissues. After administering stable isotope-labeled fatty acids to mice by gavage, NanoSIMS imaging allowed us to visualize neutral lipids in cytosolic lipid droplets in intestinal enterocytes, chylomicrons at the basolateral surface of enterocytes, and lipid droplets in cardiomyocytes and adipocytes. After an injection of stable isotope-enriched triglyceriderich lipoproteins (TRLs), NanoSIMS imaging documented delivery of lipids to cytosolic lipid droplets in parenchymal cells. Using a combination of backscattered electron (BSE) and NanoSIMS imaging, it was possible to correlate the chemical data provided by NanoSIMS with high-resolution BSE images of cell morphology. This combined imaging approach allowed us to visualize stable isotope-enriched TRLs along the luminal face of heart capillaries and the lipids within heart capillary endothelial cells. We also observed examples of TRLs within the subendothelial spaces of heart capillaries. NanoSIMS imaging provided evidence of defective transport of lipids from the plasma LPs to adipocytes and cardiomyocytes in mice defi cient in glycosyl...

β1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of β1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that β1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of β1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for β1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that β1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of β1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.

Cre/loxP recombination enables cellular specificity and, in the case of inducible systems, temporal control of genomic deletions. Here we used a SM22a tamoxifen-inducible Cre line to inactivate b1 integrin in adult smooth muscle. Interestingly, analysis of two distinct b1 loxP transgenic mice revealed vastly different outcomes after b1 integrin deletion. Lethality occurred 4 weeks postinduction in one Cre/loxP line, while no apparent phenotype was seen in the other line. Genetic analysis revealed appropriate DNA excision in both cases; however, differences were found in the degree of protein loss with absolutely no change in protein levels in the model that lacked a phenotype. Seeking to understand protein persistence despite appropriate recombination, we first validated the flox allele using a constitutive Cre line and demonstrated its ability to mediate effective protein inactivation. We then examined the possibility of heterozygous cell selection, protein turnover, and deletion efficiency with no success for explaining the phenotype. Finally, we documented the presence of the Cre-recombination episomal product, which persisted in tissue samples with no protein loss. The product was only noted in cells with low proliferative capacity. These findings highlight the potential for protein expression from the products of Crerecombinase excised genes, particularly when deletion occurs in low turnover populations.

The S447X polymorphism in lipoprotein lipase (LPL), which shortens LPL by two amino acids, is associated with low plasma triglyceride levels and reduced risk for coronary heart disease. S447X carriers have higher LPL levels in the pre- and post-heparin plasma, raising the possibility that the S447X polymorphism leads to higher LPL levels within capillaries. One potential explanation for increased amounts of LPL in capillaries would be more avid binding of S447X-LPL to GPIHBP1 (the protein that binds LPL dimers and shuttles them to the capillary lumen). This explanation seems plausible because sequences within the carboxyl terminus of LPL are known to mediate LPL binding to GPIHBP1. To assess the impact of the S447X polymorphism on LPL binding to GPIHBP1, we compared the ability of internally tagged versions of wild-type LPL (WT-LPL) and S447X-LPL to bind to GPIHBP1 in both cell-based and cell-free binding assays. In the cell-based assay, we compared the binding of WT-LPL and S447X-LPL to GPIHBP1 on the surface of cultured cells. This assay revealed no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1. In the cell-free assay, we compared the binding of internally tagged WT-LPL and S447X-LPL to soluble GPIHBP1 immobilized on agarose beads. Again, no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1 were observed. We conclude that increased binding of S447X-LPL to GPIHBP1 is unlikely to be the explanation for more efficient lipolysis and lower plasma triglyceride levels in S447X carriers.