Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

Endocardial cells are a distinct endothelial lineage derived from Flk1+ multipotent cardiovascular progenitors

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal–spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.     

Skeletal muscle stem and progenitor cells: reconciling genetics and lineage

Skeletal muscle provides a unique paradigm for studying stem to differentiated cell transitions, as well as the acquisition of cellular identity. Embryological and genetic studies over the last decades have unveiled key signaling pathways and regulatory genes which are involved in this process. In the adult, regeneration from fiber-associated satellite cells as well as non-muscle cells have opened the perspective for cell therapy studies. Paradoxically, however, the lineage has remained largely elusive. Recent studies have provided clues regarding the cellular organization in this lineage. Furthermore, the complexity of the genetic networks regulating global and local myogenic programs can be correlated with location and lineage. Finally, prenatal and postnatal developmental strategies have similarities and differences which will also be highlighted.     

Proteomic dataset of Sca-1+ progenitor cells

Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com.     

Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding

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Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells

In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRα), c-Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post-gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1⁺ mesodermal cells could be further classified by the level of c-Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1⁺ mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supported in vitro hematopoiesis from ES cells. Flk1⁺ cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1⁺ cells will be required for in vitro ES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos or in vitro cultured ES cells.     

Mechanisms regulating the origins of the vertebrate vascular system

In order to sustain growth, differentiation, and organogenesis, vertebrate embryos must form a functional vascular system early in embryonic development. Intrinsic interest in this process as well as the promise of potential clinical applications has led to significant progress in understanding the mechanisms governing the formation of the vascular system, however the earliest stages of vascular development--the emergence of committed endothelial precursors from the mesoderm--remain unclear. A review of the current literature reveals an unexpected diversity and heterogeneity with respect to where vascular endothelial cells originate in the embryo, when they become committed and the mechanisms governing how endothelial cells acquire their identity. Spatially, a widespread region of the early mesoderm possesses the ability to give rise to vascular endothelial cells; temporally the process is not limited to a small window during embryogenesis, but rather, may continue throughout the lifespan of the organism. On the molecular level, recent findings point to several determinative pathways that regulate, modulate, and extend the scope of the Flk1/VEGF signaling system. An expanding array of novel gene products implicated in endothelial cell type determination appear to act synergistically, with different combinations of factors leading to diverse cellular responses, varying patterns of differentiation, and considerable heterogeneity of endothelial cell types during embryogenesis.     

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.     

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells

Ca²⁺-permeable store-operated channels (SOCs) mediate Ca²⁺ entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1^?/? mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1^?/? VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1^?/? VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1^?/? VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.     

Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells

The pancreas is derived from a pool of multipotent progenitor cells (MPCs) that co-express Pdx-1 and Ptf1a. To more precisely define how the individual and combined loss of Pdx-1 and Ptf1a affects pancreatic MPC specification and differentiation we derived and studied mice bearing a novel Ptf1a^YFP allele. While the expression of Pdx-1 and Ptf1a in pancreatic MPCs coincides between E9.5 and 12.5 the developmental phenotypes of Pdx-1 null and Pdx-1; Ptf1a double null mice are indistinguishable, and an early pancreatic bud is formed in both cases. This finding indicates that Pdx-1 is required in the foregut endoderm prior to Ptf1a for pancreatic MPC specification. We also found that Ptf1a is neither required for specification of Ngn3-positive endocrine progenitors nor differentiation of mature β-cells. In the absence of Pdx-1 Ngn3-positive cells were not observed after E9.5. Thus, in contrast to the deletion of Ptf1a, the loss of Pdx-1 precludes the sustained Ngn3-based derivation of endocrine progenitors from pancreatic MPCs. Taken together, these studies indicate that Pdx-1 and Ptf1a have distinct but interdependent functions during pancreatic MPC specification.     

FOXO1 delays skeletal muscle regeneration and suppresses myoblast proliferation

Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.     

Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.     

The hallmarks of myotonic dystrophy type 1 muscle dysfunction

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3′ untranslated region (3′UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin–proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.     

Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Blood islands' of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper. These lineages arise from a layer of extraembryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules (Indian hedgehog) is produced by visceral endoderm, can induce formation of blood and endothelial cells in explant cultures and can reprogram prospective neurectoderm along hematopoietic and endothelial cell lineages. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. These findings may have important implications for regulating hematopoiesis and vascular development for therapeutic purposes in humans and for the development of new sources of stem cells for transplantation and gene therapy.     

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF‐ß, and Ang‐1,2. Among these, the role of VEGF‐A signaling in vessel morphogenesis is best understood. The biological activity of VEGF‐A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF‐A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1‐tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1‐tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development. genesis 50:561–571, 2012. © 2012 Wiley Periodicals, Inc.     

Cell lineage of flight muscle fibers in Drosophila: a fate map of the induced shibire phenotype in mosaics

Summary A blastoderm fate map has been prepared for Drosophila, using mosaics of a temperature-sensitive mutation, shibire (shi). The mutation can cause abnormal flight muscle morphology, inducible only by a short heat pulse in early metamorphosis. Thus muscle lineage and development are unperturbed until the heat pulse in the early pupa. The developmental focus of the shi muscle phenotype maps to the ventral thorax at the expected site of thoracic mesoderm, and probably indicates the blastoderm progenitors of the adult flight muscle. The fate map provides greater detail than previously available for the dorsolongitudinal fibers (DLM) of flight muscle, showing wide separation of the fibers of flight muscle. DLM fibers a and b map close together, and far anterior to fibers e and f, which also map together. On a fate map, common developmental focus indicates a common blastoderm origin. Thus, the observed pattern for DLM fibers suggests that the blastoderm progenitors for each of these syncytial fiber pairs (a, b; e, f) include only one or two cells. It follows that there is usually a single genotype within each fiber pair (a, b; e, f), and that this genotype is directly reflected in the fiber phenotype. In a large number of cases, DLM fibers a and b differ in phenotype from other DLM fibers, in parallel with their other differences (e.g., timing of development in pupa, innervation, motor activity). The separation of fate map locations of the developmental focus for DLM fibers within mesoderm suggests that specific fibers of flight muscle may, in normal development, originate in all three thoracic mesodermal parasegments.