Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/-embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/-ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/-ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/-ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.

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Hedgehog is required for murine yolk sac angiogenesis

Development ◽

10.1242/dev.129.2.361 ◽

2002 ◽

Vol 129 (2) ◽

pp. 361-372 ◽

Cited By ~ 1

Author(s):

Noah Byrd ◽

Sandy Becker ◽

Peter Maye ◽

Roopa Narasimhaiah ◽

Benoit St-Jacques ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Vessels ◽

Vascular Remodeling ◽

Hedgehog Signaling ◽

Es Cells ◽

Embryonic Stem ◽

Yolk Sac ◽

Embryoid Bodies ◽

Visceral Endoderm

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh–/– mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and α-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh–/– lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or Smo. Whereas Ihh–/– yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo–/– yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh–/– yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.

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Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro

Development ◽

10.1242/dev.121.11.3877 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3877-3888 ◽

Cited By ~ 22

Author(s):

C. Soudais ◽

M. Bielinska ◽

M. Heikinheimo ◽

C.A. MacArthur ◽

N. Narita ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Embryonic Stem ◽

Yolk Sac ◽

Embryoid Bodies ◽

Targeted Mutagenesis ◽

Clonal Variation ◽

Chimeric Mouse ◽

Visceral Endoderm

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

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Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development

Development ◽

10.1242/dev.114.2.303 ◽

1992 ◽

Vol 114 (2) ◽

pp. 303-316 ◽

Cited By ~ 9

Author(s):

R. Wang ◽

R. Clark ◽

V.L. Bautch

Keyword(s):

Endothelial Cells ◽

Blood Vessel ◽

Light And Electron Microscopy ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Density Lipoprotein ◽

Yolk Sac ◽

Embryoid Bodies ◽

Vascular Channels

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen ‘flowing’ through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.

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Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells

Development ◽

10.1242/dev.124.19.3755 ◽

1997 ◽

Vol 124 (19) ◽

pp. 3755-3764 ◽

Cited By ~ 4

Author(s):

N. Narita ◽

M. Bielinska ◽

D.B. Wilson

Keyword(s):

Transcription Factor ◽

In Situ Hybridization ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Cardiomyocyte Differentiation ◽

Wild Type ◽

Cell Stage

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4−/− embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4−/− ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4−/− embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4−/− embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4−/− ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4−/− ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4−/− ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4−/− ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

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Specific Effect of 5-Fluorouracil on α-Fetoprotein Gene Expression During the In Vitro Mouse Embryonic Stem Cell Differentiation

International Journal of Toxicology ◽

10.1177/1091581810366312 ◽

2010 ◽

Vol 29 (3) ◽

pp. 297-304 ◽

Cited By ~ 8

Author(s):

David Pamies ◽

Néstor Vicente-Salar ◽

Miguel A. Sogorb ◽

Enrique Roche ◽

Juan A. Reig

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryoid Bodies ◽

Screening Methods ◽

Embryonic Stem Cell Differentiation ◽

Visceral Endoderm ◽

Gene Markers ◽

Wide Range

Embryonic stem (ES) cells are considered an important alternative to develop in vitro screening methods for embryotoxicity. Mouse ES cells can be cultured as cell suspension aggregates termed “embryoid bodies” (EBs) in which cells start to differentiate. We have studied the expression of several genes in the presence of a wide range of concentrations of 5-fluorouracil (5-FU). This well-established embryotoxic compound completely inhibited cell viability at 200 nmol/L in monolayer cultures. At lower concentrations, 5-FU led to decrease in the expression of the α-fetoprotein gene, a marker of the visceral endoderm, in the EBs. However, the expression of several mesodermal gene markers was not significantly affected at these concentrations. These results suggest a high sensitivity of the visceral endoderm differentiation to 5-FU. Therefore, the quantification of the α-fetoprotein gene after exposure to potential embryotoxicants should be considered an additional end point in future embryotoxicity assays in vitro with ES cells.

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Endoglin Is Required for Hemangioblast Development.

Blood ◽

10.1182/blood.v104.11.138.138 ◽

2004 ◽

Vol 104 (11) ◽

pp. 138-138 ◽

Cited By ~ 1

Author(s):

Rita R. Perlingeiro

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Colony Formation ◽

Time Course ◽

Es Cells ◽

Critical Role ◽

Embryonic Stem ◽

Yolk Sac ◽

Embryoid Bodies ◽

Wild Type

Abstract A critical role for endoglin (CD105) in early development has been demonstrated in mice deficient for this gene. Embryos homozygous for the endoglin mutation (eng−/−) fail to progress beyond 10.5 days postcoitum due primarily to vascular and cardiac abnormalities (Bordeau et al, 1999). Analysis of 9.5 dpc eng−/− embryos revealed abnormal vasculature and anemia of the yolk sac, suggesting that endoglin may be required for both blood and endothelial lineages. The hemangioblast, the bipotent precursor for hematopoietic and endothelial cells, can be assessed through the blast colony assay (BL-CFC) using a model system based on the in vitro differentiation of embryonic stem (ES) cells into embryoid bodies (EBs). To evaluate a role for endoglin in this early precursor, we differentiated eng−/−, eng+/−, and eng+/+ (wild-type) ES cells into EBs. At day 3 of EB differentiation, cells were disrupted and plated for blast colony formation in methylcellulose media containing vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO). We found no difference in blast colony formation between heterozygous and wild-type ES cells. However, a significant reduction in the number of BL-CFCs was observed in eng−/− cells when compared to eng+/− or eng+/+ BL-CFCs (p < 0.001). Single eng−/−, eng+/−, and eng+/+ BL-CFCs gave rise to secondary hematopoietic colonies as well as endothelial cells, confirming their nature as hemangioblasts. These results suggest that although endoglin is required for hemangioblast development, its absence does not affect the bipotentiality of formed BL-CFCs. Since anemia was a feature of 9.5 dpc eng−/− yolk sac embryos, we also examined early erythropoiesis using the ES/EB system. For this purpose, eng−/−, eng+/−, and eng+/+ ES cells were differentiated into EBs for 4 days, at which time cells were disrupted and plated for primitive erythroid colonies (EryP) in methylcellulose media containing IL-3, IL-6, SCF, and Epo. We observed a reduction in the number of EryP colonies in eng−/− (p < 0.01) and eng+/− (p < 0.05) EBs when compared to controls (eng+/+). These results corroborate the anemia observed in vivo in the eng−/− embryos. We used RT-PCR and flow cytometry analysis to detect endoglin expression during a time course of EB differentiation. Endoglin is expressed in ES cells and disappears with differentiation. Expression re-appears at day 3 of differentiation, concomitantly with specification of the hemangioblast. Expression thereafter increases, correlating with mature endothelial cells at later time points. We did not find major differences in gene expression for Brachyury, Flk-1, Tie-2, embryonic and adult globins in a time course of EB differentiation for eng−/−, eng+/−, and eng+/+ ES cells. These data point out a role for endoglin, an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, in hemangioblast development.

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Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps

Blood ◽

10.1182/blood.v88.9.3424.bloodjournal8893424 ◽

1996 ◽

Vol 88 (9) ◽

pp. 3424-3431 ◽

Cited By ~ 329

Author(s):

D Vittet ◽

MH Prandini ◽

R Berthier ◽

A Schweitzer ◽

H Martin-Sisteron ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Differentiation ◽

Growth Factor ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Vascular Endothelial ◽

Endothelial Cell Differentiation ◽

Kinetics Of

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

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Deficiency in GATA-4 or GATA-6 Diminishes Definitive Hematopoiesis in Murine Embryonic Stem Cell Derived Embryoid Bodies.

Blood ◽

10.1182/blood.v110.11.2230.2230 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2230-2230

Author(s):

Monique S. Pierre ◽

Mervin Yoder

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Vegf Receptor ◽

Visceral Endoderm ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Blood Island

Abstract Formation of mesoderm derived blood islands in the mouse embryonic yolk sac requires the presence of visceral endoderm (VE) and VE derived factors. Murine embryonic stem (ES) cells can be differentiated into embryoid bodies (EBs) which serve as an in vitro model recapitulating many embryonic developmental processes, including formation of early hematopoietic cells. Previous investigators have reported that differentiation of ES cells deficient in either GATA-4 or GATA-6 results in EBs with disrupted differentiation of visceral endoderm and defective blood island formation. In the current study, we have compared GATA-4 and GATA-6 null ES cell derived EBs to wild-type EBs in their ability to commit to early hematopoietic lineages using hematopoietic progenitor colony assays, and used RT-PCR to assess the expression of endoderm genes. As expected, we observed differences in expression of endoderm genes in wild-type and GATA-4 or GATA-6 null EBs. Blast colony forming cell assays and primitive erythroid progenitor assays revealed no difference in the ability of wild-type and GATA-4 or GATA-6 null EBs to form hemangioblast or primitive erythroid progenitor colonies. In contrast, comparisons of definitive hematopoietic progenitor colonies from day 8, 9 and 10 GATA-4 and GATA-6 null EBs revealed a significant reduction in colony numbers at day 8 (p-values < 0.05) compared to wild-type. Strikingly, definitive progenitor colony numbers are rescued nearly to wild-type levels after the addition of the visceral endoderm derived factor vascular endothelial growth factor (VEGF) during EB differentiation. Furthermore, this rescue response can be blocked by the addition of soluble Flk-1 (VEGF receptor) to EB cultures. These results suggest that GATA-4 and GATA-6 transcription factors and/or visceral endoderm are not necessary for hemangioblast, primitive erythroid, or definitive progenitor emergence from EBs but play a role in definitive progenitor expansion in EBs.

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Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types

Reproduction Fertility and Development ◽

10.1071/rd00079 ◽

2001 ◽

Vol 13 (1) ◽

pp. 15 ◽

Cited By ~ 12

Author(s):

J. Rathjen ◽

S. Dunn ◽

M. D. Bettess ◽

P. D. Rathjen

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Cell Types ◽

Embryoid Bodies ◽

Cell Aggregates ◽

Visceral Endoderm ◽

Es Cell ◽

Pluripotent Cells ◽

Developmental Potential

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

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Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Development ◽

10.1242/dev.102.3.471 ◽

1988 ◽

Vol 102 (3) ◽

pp. 471-478 ◽

Cited By ~ 24

Author(s):

W. Risau ◽

H. Sariola ◽

H.G. Zerwes ◽

J. Sasse ◽

P. Ekblom ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Blood Vessels ◽

Embryonic Stem ◽

Yolk Sac ◽

Embryoid Bodies ◽

Activity Increase ◽

Vasculogenesis And Angiogenesis

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.

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