Tissue-specific requirements for the proprotein convertase furin/SPC1 during embryonic turning and heart looping

Development ◽  
2000 ◽  
Vol 127 (2) ◽  
pp. 245-254 ◽  
Author(s):  
D.B. Constam ◽  
E.J. Robertson
Keyword(s):  
Serine Proteases ◽  
Es Cells ◽  
Embryonic Stem ◽  
Axial Rotation ◽  
Yolk Sac ◽  
Definitive Endoderm ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Extraembryonic Mesoderm ◽  
Tissue Abnormalities

Furin, the mammalian prototype of a family of serine proteases, is required for ventral closure and axial rotation, and formation of the yolk sac vasculature. Here we show additionally that left-sided expression of pitx2 and lefty-2 are also perturbed in Furin-deficient embryos. These tissue abnormalities are preceded by a marked delay in the expansion of the definitive endoderm during gastrulation. Using a chimera approach, we show that Furin activity is required in epiblast derivatives, including the primitive heart, gut and extraembryonic mesoderm, whereas it is nonessential in the visceral endoderm. Thus, chimeric embryos, derived by injecting wild-type embryonic stem (ES) cells into fur(−/−) blastocysts, develop normally until at least 9.5 d.p.c. In contrast, Furin-deficient chimeras developing in the context of wild-type visceral endoderm fail to undergo ventral closure, axial rotation and yolk sac vascularization. Fur(−/−) cells are recruited into all tissues examined, including the yolk sac vasculature and the midgut, even though these structures fail to form in fur mutants. The presence of wild-type cells in the gut strikingly correlates with the ability of chimeric embryos to undergo turning. Overall, we conclude that Furin activity is essential in both extraembryonic and precardiac mesoderm, and in definitive endoderm derivatives.

scholarly journals The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2433-2445 ◽  
Author(s):  
J.P. Martinez Barbera ◽  
M. Clements ◽  
P. Thomas ◽  
T. Rodriguez ◽  
D. Meloy ◽  
...  
Keyword(s):  
Normal Development ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Definitive Endoderm ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Zona Limitans Intrathalamica ◽  
Ventral Thalamus ◽  
Septum Transversum ◽  
Early Mouse

The homeobox gene Hex is expressed in the anterior visceral endoderm (AVE) and rostral definitive endoderm of early mouse embryos. Later, Hex transcripts are detected in liver, thyroid and endothelial precursor cells. A null mutation was introduced into the Hex locus by homologous recombination in embryonic stem cells. Hex mutant embryos exhibit varying degrees of anterior truncation as well as liver and thyroid dysplasia. The liver diverticulum is formed but migration of hepatocytes into the septum transversum fails to occur. Development of the thyroid is arrested at the thyroid bud stage at 9.5 dpc. Brain defects are restricted to the rostral forebrain and have a caudal limit at the zona limitans intrathalamica, the boundary between dorsal and ventral thalamus. Analysis of Hex(−/−) mutants at early stages shows that the prospective forebrain ectoderm is correctly induced and patterned at 7.5 days post coitum (dpc), but subsequently fails to develop. AVE markers are expressed and correctly positioned but development of rostral definitive endoderm is greatly disturbed in Hex(−/−) embryos. Chimeric embryos composed of Hex(−/−) cells developing within a wild-type visceral endoderm show forebrain defects indicating that Hex is required in the definitive endoderm. All together, these results demonstrate that Hex function is essential in definitive endoderm for normal development of the forebrain, liver and thyroid gland.

Endoglin Is Required for Hemangioblast Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 138-138 ◽  
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.

Hedgehog is required for murine yolk sac angiogenesis

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 361-372 ◽  
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.

scholarly journals Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3720-3730 ◽  
Author(s):  
M Bielinska ◽  
N Narita ◽  
M Heikinheimo ◽  
SB Porter ◽  
DB Wilson
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Cell Stage ◽  
Primitive Hematopoiesis

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.

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

Development ◽  
1995 ◽  
Vol 121 (11) ◽  
pp. 3877-3888 ◽  
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.

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

Blood ◽  
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 &lt; 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.

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

Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1881-1892 ◽  
Author(s):  
Annette Damert ◽  
Lucile Miquerol ◽  
Marina Gertsenstein ◽  
Werner Risau ◽  
Andras Nagy
Keyword(s):  
Yolk Sac ◽  
Endothelial Differentiation ◽  
Vascular Endothelial ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Hypomorphic Allele ◽  
Targeted Mutation ◽  
Blood Formation ◽  
Opposite Situation ◽  
Endothelial Growth Factor

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegflo. Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing ‘Vegf wild-type’ visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the ‘Vegf wild-type’ yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Jie Liu ◽  
Yanmei Qi ◽  
Shu-Chan Hsu ◽  
Siavash Saadat ◽  
Saum Rahimi ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Intercellular Junctions ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Wild Type ◽  
Adhesion Sites ◽  
Cell Cell

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

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