A differential display strategy identifies Cryptic, a novel EGF-related gene expressed in the axial and lateral mesoderm during mouse gastrulation

Development ◽  
1997 ◽  
Vol 124 (2) ◽  
pp. 429-442 ◽  
Author(s):  
M.M. Shen ◽  
H. Wang ◽  
P. Leder
Keyword(s):  
Growth Factor ◽  
Differential Display ◽  
Sequence Similarity ◽  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Lateral Plate ◽  
New Family ◽  
Lateral Mesoderm

We have developed a differential display screening approach to identify mesoderm-specific genes, relying upon the differentiation of embryonic stem (ES) cells in vitro. Using this strategy, we have isolated a novel murine gene that encodes a secreted molecule containing a variant epidermal growth factor-like (EGF) motif. We named this gene Cryptic, based on its predicted protein sequence similarity with Cripto, which encodes an EGF-related growth factor. Based on their strong sequence similarities, we propose that Cryptic, Cripto, and the Xenopus FRL-1 gene define a new family of growth factor-like molecules, which we name the ‘CFC’ (Cripto, Frl-1, and Cryptic) family. Analysis of Cryptic expression by in situ hybridization shows that it is expressed during gastrulation in two spatial domains that correspond to the axial and lateral mesoderm. In the first domain of expression, Cryptic expression is progressively localized to the anterior primitive streak, the head process, and the node and notochordal plate. In the second domain, Cryptic expression is initially concentrated in the lateral region of the egg cylinder, and is later found circumferentially in the intermediate and lateral plate mesoderm. Furthermore, Cryptic expression can also be detected at the early head-fold stage in the midline neuroectoderm, and consequently is an early marker for the prospective floor plate of the neural tube. Expression of Cryptic ceases at the end of gastrulation, and has not been observed in later embryonic stages or in adult tissues. Thus, Cryptic encodes a putative signaling molecule whose expression suggests potential roles in mesoderm and/or neural patterning during gastrulation.

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

Basic fibroblast growth factor positively regulates hematopoietic development

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1931-1941 ◽  
Author(s):  
P. Faloon ◽  
E. Arentson ◽  
A. Kazarov ◽  
C.X. Deng ◽  
C. Porcher ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Fibroblast Growth ◽  
Hematopoietic Lineage ◽  
Mesoderm Inducing Factors

Recently identified BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, we show that BL-CFC expresses the receptor tyrosine kinase, Flk1, and thus we took advantage of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1(+) cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, we have analyzed ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, for their potential to generate BL-CFCs and Flk1(+) cells, to further define events leading to hemangioblast development. Our data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast.

scholarly journals Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1253-1263 ◽  
Author(s):  
Masanori Hirashima ◽  
Hiroshi Kataoka ◽  
Satomi Nishikawa ◽  
Norihisa Matsuyoshi ◽  
Shin-Ichi Nishikawa
Keyword(s):  
Cell Adhesion ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Vascular Endothelial ◽  
Cell Cell

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34−to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

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.

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2275-2283 ◽  
Author(s):  
Naoki Nakayama ◽  
Jae Lee ◽  
Laura Chiu
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Morphogenetic Protein ◽  
Endothelial Growth Factor

Abstract The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

scholarly journals Natural Killer and B-Lymphoid Potential in CD34+ Cells Derived From Embryonic Stem Cells Differentiated in the Presence of Vascular Endothelial Growth Factor

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2283-2295 ◽  
Author(s):  
Naoki Nakayama ◽  
Inghwa Fang ◽  
Gary Elliott
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Natural Killer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cytotoxic Lymphocytes ◽  
Vascular Endothelial ◽  
Cd34 Cells ◽  
Endothelial Growth Factor

Abstract Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34−lymphocytes: CD3− cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+intermediate, on which VEGF may play an important role.

225 TERATOMA FORMATION BY BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 229
Author(s):  
M. L. Lim ◽  
I. Vassiliev ◽  
P. J. Verma
Keyword(s):  
Growth Factor ◽  
Es Cells ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Scid Mice ◽  
Bovine Embryos ◽  
Differentiation Potential ◽  
Teratoma Formation

Teratoma formation is commonly used as a model for examining the in vivo differentiation potential of embryonic stem cells. We wanted to investigate the teratoma-forming ability of bovine ES cells; however, there are no reports of teratoma-forming ability of bovine pluripotent cells including pre-implantation embryos. In vivo-produced bovine embryos at stages earlier than Day 14 failed to develop teratomas when transplanted into one of the kidneys of immuno-deficient mice (Anderson et al. 1996 Anim. Reprod. Sci. 45, 231–240), and this prompted questions about the ability of bovine embryos to form teratomas. Bovine oocytes were cultured for 20 to 22 h after aspiration at 39�C (5% CO2/95% air) in TCM-199-bicarbonate medium supplemented with GlutaMax6" (Invitrogen Australia Pty Ltd., Mount Waverley, Victoria, Australia), penicillin/streptomycin, β-mercaptoethanol, 17β-estradiol, fetal calf serum, LH, follicle stimulating hormone, basic fibroblast growth factor, epidermal growth factor, glycine, and l-cysteine. Oocytes were fertilized with IVF media (Cook Australia, Brisbane, Queensland, Australia) and kept for 7 days at 39�C in 5% CO2/95% air to generate blastocysts. The zona pellucida of Day 7 blastocysts was enzymatically removed, and one or two zona-free embryos were injected into each testis of 5-week-old immunodeficient (SCID) mice (CB-17/ICR-Prkdcscid strain; Walter and Eliza Hall Institute, Melbourne, Australia). Eight weeks post-injection, teratomas partially expelled from testes were identified. Histological analysis has confirmed the derivatives of all 3 germ layers in teratomas. In conclusion, we report that Day 7 in vitro-produced embryos can form teratomas when injected into testes of SCID mice.

scholarly journals Transforming growth factor (TGF)beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells

2004 ◽  
Vol 379 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Anouchka SKOUDY ◽  
Meritxell ROVIRA ◽  
Pierre SAVATIER ◽  
Franz MARTIN ◽  
Trinidad LEÓN-QUINTO ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Signalling Pathways ◽  
Fibroblast Growth ◽  
Development And Differentiation

Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor β, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation.

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.

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2275-2283 ◽  
Author(s):  
Naoki Nakayama ◽  
Jae Lee ◽  
Laura Chiu
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Morphogenetic Protein ◽  
Endothelial Growth Factor

The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

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