Impaired pancreatic development in Hif2-alpha deficient mice

Accumulating data suggest the existence of a link between hypoxia and maintenance of the undifferentiated cell state, but little is known about the cellular signaling mechanisms underlying this process. Recent reports reveal a direct link between components of the hypoxia signaling pathway and Notch pathway in maintaining precursor cells in an undifferentiated state. Here, we report that in the developing mouse pancreas, Hif2-α is expressed in pancreatic progenitor cells, but its expression is lost in committed endocrine progenitors as well as in differentiated endocrine and exocrine cells. In an attempt to analyze the function of HIF2-α in the developing pancreas, we studied Hif2-α^−/− pancreas. Our analyses revealed that in addition to the decreased size and branching, the Hif2-α deficient pancreas also displayed impaired notch signaling and cell differentiation. Finally, we found that HIF2-α binds directly to Notch-IC and that the responsible site for this interaction is within the RAM domain of Notch protein. These results suggest that HIF2-α is required for normal mouse pancreatic development.     

Redundant and dosage sensitive requirements for Fgf3 and Fgf10 in cardiovascular development

Heart development requires contributions from, and coordinated signaling interactions between, several cell populations, including splanchnic and pharyngeal mesoderm, postotic neural crest and the proepicardium. Here we report that Fgf3 and Fgf10, which are expressed dynamically in and near these cardiovascular progenitors, have redundant and dosage sensitive requirements in multiple aspects of early murine cardiovascular development. Embryos with Fgf3^−/+;Fgf10^−/−, Fgf3^−/−;Fgf10^−/+ and Fgf3^−/−;Fgf10^−/− genotypes formed an allelic series of increasing severity with respect to embryonic survival, with double mutants dead by E11.5. Morphologic analysis of embryos with three mutant alleles at E11.5–E13.5 and double mutants at E9.5–E11.0 revealed multiple cardiovascular defects affecting the outflow tract, ventricular septum, atrioventricular cushions, ventricular myocardium, dorsal mesenchymal protrusion, pulmonary arteries, epicardium and fourth pharyngeal arch artery. Assessment of molecular markers in E8.0–E10.5 double mutants revealed abnormalities in each progenitor population, and suggests that Fgf3 and Fgf10 are not required for specification of cardiovascular progenitors, but rather for their normal developmental coordination. These results imply that coding or regulatory mutations in FGF3 or FGF10 could contribute to human congenital heart defects.     

FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.     

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.     

Endothelial cell migration directs testis cord formation

While the molecular cues initiating testis determination have been identified in mammals, the cellular interactions involved in generating a functional testis with cord and interstitial compartments remain poorly understood. Previous studies have shown that testis cord formation relies on cell migration from the adjacent mesonephros, and have implicated immigrant peritubular myoid cells in this process. Here, we used recombinant organ culture experiments to show that immigrant cells are endothelial, not peritubular myoid or other interstitial cells. Inhibition of endothelial cell migration and vascular organisation using a blocking antibody to VE-cadherin, also disrupted the development of testis cords. Our data reveal that migration of endothelial cells is required for testis cord formation, consistent with increasing evidence of a broader role for endothelial cells in establishing tissue architecture during organogenesis.     

Tbx18 regulates development of the epicardium and coronary vessels

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18^−/− mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α^lacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18^−/− hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.     

Localization of neuropeptide Y and atrial natriuretic peptide in the endothelial cells of human umibilical blood vessels

The localization of neuropeptide Y (NPY) and atrial natriuretic peptide (ANP) in the endothelial cells of human umbilical blood vessels was studied using the pre-embedding peroxidase-antiperoxidase (PAP) technique for electron microscopy and avidin-biotin-complex (ABC) immunostaining for endothelial cells cultured from umbilical vein. Subpopulations of NPY- and ANP-immunoreactive endothelial cells were present in term umbilical vein and artery. The umbilical vein contained more positive cells than the artery. The percentage of NPY- and ANP-immunoreactive umbilical vein cells in culture was 32% and 44%, respectively, out of a total of 3013 cells examined. The possibility that these potent vasoactive substances located in the endothelial cells of the non-innervated umbilical vessels are involved in the local regulation of blood flow is discussed.     

Aquaporin-1 in blood vessels of rat circumventricular organs

Although the water channel protein aquaporin-1 (AQP1) is widely observed outside the rat brain in continuous, but not fenestrated, vascular endothelia, it has not previously been observed in any endothelia within the normal rat brain and only to a limited extent in the human brain. In this immunohistochemical study of rat brain, AQP1 has also been found in microvessel endothelia, probably of the fenestrated type, in all circumventricular organs (except the subcommissural organ and the vascular organ of the lamina terminalis): in the median eminence, pineal, subfornical organ, area postrema and choroid plexus. The majority of microvessels in the median eminence, pineal and choroid plexus, known to be exclusively fenestrated, are shown to be AQP1-immunoreactive. In the subfornical organ and area postrema in which many, but not all, microvessels are fenestrated, not all microvessels are AQP1-immunoreactive. In the AQP1-immunoreactive microvessels, the AQP1 probably facilitates water movement between blood and interstitium as one component of the normal fluxes that occur in these specialised sensory and secretory areas. AQP1-immunoreactive endothelia have also been seen in a small population of blood vessels in the cerebral parenchyma outside the circumventricular organs, similar to other observations in human brain. The proposed development of AQP1 modulators to treat various brain pathologies in which AQP1 plays a deleterious role will necessitate further work to determine the effect of such modulators on the normal function of the circumventricular organs.     

The homeodomain protein PDX1 is required at mid-pancreatic development for the formation of the exocrine pancreas

The homeoprotein PDX1 is expressed throughout pancreatic development and is thought to play important roles at multiple stages. We describe the properties of a tet-off regulatory scheme to manage the expression of Pdx1 in utero. Cessation of Pdx1 expression at increasingly later gestational times blocked pancreatic development at progressive and morphologically distinct stages and provided the opportunity to assess the requirement for Pdx1 at each stage. Embryonic PDX1 is depleted below effective levels within 1 day of the initiation of doxycycline treatment of pregnant mice. We show that PDX1, which is necessary for early pancreatic development, is also required later for the genesis of acinar tissue, the compartment of the pancreas that produces digestive enzymes. Without PDX1, acini do not form; the precursor epithelium continues to grow and branch, creating a truncated ductal tree comprising immature duct-like cells. The bHLH factor PTF1a, a critical regulator of acinar development, is not expressed and cells producing digestive enzymes are rare. This approach should be generally applicable to study the in vivo functions of other developmental regulators with multiple, temporally distinct roles.     

Intralobular lymphatic vessels and their relationship to blood vessels in the mouse thymus

Summary The spatial distribution and fine structure of the lymphatic vessels within the thymic lobules of normal and hydrocortisone-injected mice were studied by light- and electron microscopy. The lymphatic vessels of the cortex and medulla of normal thymus are irregularly shaped spaces closely associated with branches of the intralobular artery and vein. The overall distribution of these vessels in the greatly involuted thymus of hydrocortisone-treated mice is essentially the same as in the normal thymus. The wall of the lymphatic vessels consists of only a layer of endothelial cells supported by underlying reticular cells. The luminal surface of the endothelial cell is smooth, but trabecular processes are often seen. There are three morphological types of intercellular contacts between contiguous cells, namely, end-to-end, overlapping and interdigitating. The lymphatic vessel has anchoring filaments and collagen fibrils, but a basal lamina is either absent, or if present, is discontinuous. This is in contrast to the continuous basal lamina of the venule. The perivascular space surrounding the postcapillary venule opens into a terminal lymphatic vessel at the cortico-medullary junction and in the medulla. Lymphocytes are seen penetrating the lymphatic endothelium, particularly in acutely involuted thymuses. These findings suggest that the intralobular lymphatic vessels may originate from the vacuities that surround the postcapillary venules, and the lymphatic system may function as a pathway for the migration of lymphocytes into or out of the lymphatic circulation.     

Common origins of blood and blood vessels in adults?

After embryonic development, the vast majority of cells are differentiated and all organs are in place. Growth of the organism then ensues and continues until adulthood, whereupon cell division largely ceases. In some tissues, notably the bone marrow, skin, and gut, cell proliferation continues throughout life to replace cells lost by attrition. This regeneration is fueled by rare, long-lived, and largely quiescent stem cells that give rise to committed progenitors, which in turn generate large numbers of fully differentiated cells. Mounting evidence suggests that such cells can significantly contribute to tissue repair and regeneration in adults and may therefore prove beneficial for autologous cell and gene therapies. This review focuses on the potential of adult stem cells to give rise to hematopoietic and vascular cells. We discuss evidence that a highly purified population of adult stem cells, termed SP cells, serves as a hematopoietic progenitor and can contribute to vascular regeneration after injury. We also discuss the potential relationship of these cells to the embryonic hemangioblast.     

Multilevel complexity of calcium signaling: Modeling angiogenesis

Intracellular calcium signaling is a universal,evolutionary conserved and versatile regulator of cell biochemistry.The complexity of calcium signaling and related cell machinery can be investigated by the use of experimental strategies,as well as by computational approaches.Vascular endothelium is a fascinating model to study the specific properties and roles of calcium signals at multiple biological levels.During the past 20 years,live cell imaging,patch clamp and other techniques have allowed us to detect and interfere with calcium signaling in endothelial cells(ECs),providing a huge amount of information on the regulation of vascularization(angiogenesis) in normal and tumoral tissues.These data range from the spatiotemporal dynamics of calcium within different cell microcompartments to those in entire multicellular and organized EC networks.Beside experimental strategies,in silico endothelial models,specifically designed for simulating calcium signaling,are contributing to our knowledge of vascular physiol-ogy and pathology.They help to investigate and predict the quantitative features of proangiogenic events moving through subcellular,cellular and supracellular levels.This review focuses on some recent developments of computational approaches for proangiogenic endothelial calcium signaling.In particular,we discuss the creation of hybrid simulation environments,which combine and integrate discrete Cellular Potts Models.They are able to capture the phenomenological mechanisms of cell morphological reorganization,migration,and intercellular adhesion,with single-cell spatiotemporal models,based on reaction-diffusion equations that describe the agonist-induced intracellular calcium events.     

Functional symbiosis between endothelium and epithelial cells in glomeruli

In some capillary beds, pericytes regulate endothelial growth. Capillaries with high filtration capacity, such as those in renal glomeruli, lack pericytes. Glomerular endothelium lies adjacent to visceral epithelial cells (podocytes) that are anchored to and cover the anti-luminal surface of the basement membrane. We have tested the hypothesis that podocytes can function as endothelial supporting cells. Endothelial cells were outgrown from circulating endothelial progenitors of normal subjects and were extensively characterized. These blood outgrowth endothelial cells (BOECs) expressed endothelial markers, lacked stem cell markers, and expressed the angiopoietin-1 receptor, Tie-2, and the vascular endothelial growth factor (VEGF) receptor, Flk-1. Differentiated podocytes in culture expressed and secreted VEGF, which was upregulated 4.5-fold by high glucose. In complete medium, BOECs formed thin cell-cell connections and multicellular tubes on Matrigel, the in vitro correlate of angiogenesis. This was impaired in deficient media but rescued by co-incubation with Transwell Anopore inserts containing differentiated podocytes. To assess whether VEGF was the major podocyte-derived signal that rescued BOEC angiogenesis, we examined angiogenesis of control and Flk-1-deficient BOECs. Co-incubation with podocytes or addition of recombinant VEGF each rescued angiogenesis in control BOECs, but both failed to support maintenance and angiogenesis in Flk-1-deficient BOECs. Finally, co-culture with podocytes increased BOEC-proliferation. In concert, these findings suggest a model in which glomerular visceral epithelial cells function as pericyte-like endothelial supporting cells. Podocyte-derived VEGF is a required and sufficient regulator of vascular endothelial maintenance, and its upregulation in podocytes by high glucose may be the mechanism for the increased glomerular angiogenesis that is observed in vivo in early diabetic glomerular injury. These studies were supported by grants from the National Institutes of Health (NIH-NIDDK 63360) and the Juvenile Diabetes Research Foundation (JDRF-1-2004-78).