Activated Fps/Fes partially rescues the in vivo developmental potential of Flk1-deficient vascular progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 912-920 ◽  
Author(s):  
Jody J. Haigh ◽  
Masatsugu Ema ◽  
Katharina Haigh ◽  
Marina Gertsenstein ◽  
Peter Greer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Es Cells ◽  
Consensus Sequence ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Developmental Potential ◽  
Vascular Progenitor Cells ◽  
Endothelial Growth Factor

AbstractRelatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A–mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A–independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell–derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Roles of vascular endothelial growth factor receptor 3 signaling in differentiation of mouse embryonic stem cell–derived vascular progenitor cells into endothelial cells

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2372-2379 ◽  
Author(s):  
Hiroyuki Suzuki ◽  
Tetsuro Watabe ◽  
Mitsuyasu Kato ◽  
Keiji Miyazawa ◽  
Kohei Miyazono
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Endothelial Differentiation ◽  
Mouse Es Cells ◽  
Vascular Progenitor Cells ◽  
Vegfr2 Signaling ◽  
Endothelial Growth Factor

AbstractVascular endothelial growth factor receptor 2 (VEGFR2/Flk-1)–positive cells derived from embryonic stem (ES) cells serve as vascular progenitors, which differentiate into endothelial cells (ECs) in the presence of VEGF-A. VEGFR3/Flt-4 (fms-like tyrosine kinase 4) signaling is known to be important for the development of lymphatic endothelial cells (LECs). To elucidate the roles of VEGFR3 signaling in the differentiation of vascular progenitor cells into ECs, we introduced various types of VEGFR3 cDNAs into mouse ES cells. VEGF-C, a ligand for VEGFR2 and VEGFR3, stimulated the endothelial differentiation of the VEGFR2+ cells transfected with the VEGFR3 cDNA but not those transfected with kinasenegative mutants of VEGFR3. The VEGFR3-transfected ECs exhibited high expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), one of the markers of LECs, and showed efficient binding of hyaluronan. VEGF-C(C152S), which is able to activate VEGFR3 but not VEGFR2, failed to induce the endothelial differentiation of mock- and VEGFR3-transfected VEGFR2+ cells, suggesting the essential role of VEGFR2 signaling for endothelial differentiation. Furthermore, kinase-negative mutants of VEGFR3 prevented the VEGF-C–mediated endothelial differentiation of the vascular progenitor cells. Thus, VEGFR2 signaling is required for the endothelial differentiation of mouse ES cells induced by VEGF-C, and VEGFR3 signaling may confer lymphatic endothelial-like phenotypes to ECs.

ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1343-1351
Author(s):  
A.M. Muller ◽  
E.A. Dzierzak
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Hematopoietic Stem ◽  
Developmental Potential

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3241-3249 ◽  
Author(s):  
Hiroki Miyashita ◽  
Tohru Yamazaki ◽  
Tetsuya Akada ◽  
Osamu Niizeki ◽  
Minetaro Ogawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Network Formation ◽  
Consensus Sequence ◽  
Embryonic Stem ◽  
Immunohistochemical Analysis ◽  
Subtractive Hybridization ◽  
Amino Acid Sequences ◽  
Antisense Oligodeoxynucleotide

Abstract Using polymerase chain reaction–coupled subtractive hybridization, we have isolated genes expressed during the in vitro differentiation of murine embryonic stem cells into endothelial cells (ECs). Among the genes obtained, we identified one gene that was inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences revealed that the protein was composed of 930 amino acids, including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The recombinant protein hydrolyzed N-terminal leucyl and methionyl residues from synthetic substrates. Immunohistochemical analysis revealed that mPILSAP was expressed in ECs during postnatal angiogenesis. Specific elimination of mPILSAP expression by antisense oligodeoxynucleotide (AS-ODN) attenuated VEGF-stimulated proliferation, migration, and network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP inhibited angiogenesis in vivo. These results suggest a novel function of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.

scholarly journals Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

2021 ◽  
Author(s):  
Natalia López-Anguita ◽  
Seher Ipek Gassaloglu ◽  
Maximilian Stötzel ◽  
Marina Typou ◽  
Iiris Virta ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transcriptional Response ◽  
Primitive Streak ◽  
Transcriptional Networks ◽  
Differentiation Potential

The cellular microenvironment together with intrinsic regulators shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, components of the hypoxia response and how their interplay impacts stem cell transcriptional networks and lineage choices remain poorly understood. Here we investigated the molecular effects of acute and prolonged hypoxia on distinct embryonic and extraembryonic stem cell types as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem (ES) cells. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. Importantly, hypoxia also modulates T levels in conventional gastruloids and enhances representation of endodermal and neural markers. Mechanistically, we identify Hif1α as a central factor that mediates the transcriptional response to hypoxia in balance with epigenetic and metabolic rewiring. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

scholarly journals Generation and Characterization of Smac/DIABLO-Deficient Mice

2002 ◽  
Vol 22 (10) ◽  
pp. 3509-3517 ◽  
Author(s):  
Hitoshi Okada ◽  
Woong-Kyung Suh ◽  
Jianping Jin ◽  
Minna Woo ◽  
Chunying Du ◽  
...  
Keyword(s):  
Physiological Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Caspase Activation ◽  
Proapoptotic Protein ◽  
Apoptosis Proteins ◽  
Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation

2000 ◽  
Vol 113 (1) ◽  
pp. 59-69 ◽  
Author(s):  
M.F. Carlevaro ◽  
S. Cermelli ◽  
R. Cancedda ◽  
F. Descalzi Cancedda
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Hypertrophic Chondrocytes ◽  
Endothelial Cell Migration ◽  
Vegf Receptor ◽  
Hypertrophic Cartilage ◽  
Endothelial Growth Factor

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

Abstract 1457: IGFBP-4-Mediated Wnt Inhibition is Required for Cardiac Myogenesis

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Weidong Zhu ◽  
Ichiro Shiojima ◽  
Li Zhi ◽  
Hiroyuki Ikeda ◽  
Masashi Yoshida ◽  
...  
Keyword(s):  
Growth Factor ◽  
Wnt Signaling ◽  
Heart Diseases ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiomyocyte Differentiation ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

Insulin-like growth factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the effects of IGFBPs appear to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. In this study we demonstrate that IGFBP-4 is a novel cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells and embryonic stem (ES) cells in vitro. Conversely, siRNA-mediated knockdown of IGFBP-4 in P19CL6 cells or ES cells attenuated cardiomyocyte differentiation, and morpholino-mediated knockdown of IGFBP-4 in Xenopus embryos resulted in severe cardiac defects and complete absence of the heart in extreme cases. We also demonstrate that the cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signaling. IGFBP-4 physically interacted with a Wnt receptor Frizzled 8 (Frz8) and a Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Moreover, the cardiogenic defects induced by IGFBP-4 knockdown both in vitro and in vivo was rescued by simultaneous inhibition of canonical Wnt signaling. Thus, IGFBP-4 is an inhibitor of the canonical Wnt signaling, and Wnt inhibition by IGFBP-4 is required for cardiogenesis. The present study provides a molecular link between IGF signaling and Wnt signaling, and suggests that IGFBP-4 may be a novel therapeutic target for heart diseases.

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