The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 877-886 ◽  
Author(s):  
V. Wilson ◽  
L. Manson ◽  
W.C. Skarnes ◽  
R.S. Beddington
Keyword(s):  
T Cells ◽  
Neural Tube ◽  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Distal Portion ◽  
Adhesion Properties ◽  
Cell Clones ◽  
Posterior Neuropore ◽  
Pass Through

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. T/T embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells (Wilson, V., Rashbass, P. and Beddington, R. S. P. (1992) Development 117, 1321–1331). To determine the origin of this abnormal tissue distribution, we have isolated T/T and control T/+ ES cell clones which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. We conclude that prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed. Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak.

Chimeric analysis of T (Brachyury) gene function

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1321-1331 ◽  
Author(s):  
V. Wilson ◽  
P. Rashbass ◽  
R.S. Beddington
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Gene Function ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Morphological Abnormality ◽  
Es Cell ◽  
Wild Type ◽  
Adhesion Properties

To investigate T(Brachyury) gene function, a chimeric analysis of midgestation (9.5-11.5 days post coitum) embryos has been performed. Embryonic stem (ES) cell lines homozygous or heterozygous for the T gene have been introduced into wild-type host embryos by blastocyst injection, and the resulting chimeras scored for morphological abnormality and extent of colonization by T/T cells. As observed previously in earlier stage chimeras (Rashbass, P., Cooke, L. A., Herrmann, B. G. and Beddington, R. S. P. (1991) Nature 353, 348–350), 9.5-11.5 dpc T/T<==>+/+ chimeras exhibit many morphological features of intact T/T mutants. In addition, a dramatic bias of T/T cells towards caudal regions (such as tail and allantois) was observed in all chimeras tested. This is likely to result from accumulation of nascent T/T mesoderm cells with time near the primitive streak, possibly because of altered migration or adhesion properties. T/+ cells colonized rostral regions efficiently, but a slight bias towards the distal end of the tail was still evident. No such bias was observed in control chimeras. The presence of T/T cells in the allantois resulted in its failure to form a correct placental connection and thus arrested later development. In contrast, chimeras in which T/T cells were present predominantly in the tail developed normally but exhibited severe tail abnormalities such as foreshortening, branching and haemorrhagic cavities. Moreover, in these embryos, much higher levels of chimerism were present in the distal end of the tail than in younger (9.5 dpc) embryos. Later in gestation, such abnormal tails probably degenerated, giving rise to neonates with absent or severely abnormal tails but no evidence of chimerism. In situ analysis of T expression in the tail reveals that normally T is expressed highly in the tailbud (the growing portion of the tail) during its elongation between 9.5 and 11.5 dpc. Thus, evidence both from chimeras and from T expression in the tail suggest that T may play a role in the correct deployment of cells emerging from the tailbud.

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

Serum Free Induction of a Lympho-Hematopoietic Precursor Population from Murine Embryonic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3605-3605
Author(s):  
Stefan Irion ◽  
Herve Luche ◽  
Hans J. Fehling ◽  
Gordon Keller
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
B Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Es Cell ◽  
Erythroid Progenitors ◽  
Hematopoietic System ◽  
Serum Free

Abstract Hematopoiesis is initiated at several independent sites in the mouse embryo. The earliest site, the yolk sac, supports the development of a restricted hematopoietic program that consists of the production of primitive erythrocytes and macrophages, as well as progenitors of the definitive erythroid, megakaryocytic and mast cell lineages. Lymphoid cells are not generated during the early phase of yolk sac hematopoiesis. Following the onset of yolk sac hematopoiesis, a second hematopoietic program is initiated in a region known as the para-aortic splanchnopleura (P-Sp). The hematopoietic system generated in the P-Sp contains hematopoietic stem cells as well as progenitors of the lymphoid, myeloid and definitive erythroid lineages. The P-Sp does not give rise to primitive erythrocytes. The differentiation of embryonic stem (ES) cells in culture offers an outstanding system for modeling early hematopoietic development and for investigating the mechanisms regulating lineage commitment. While a number of different studies have provided convincing evidence that the ES cell model can recapitulate yolk sac hematopoiesis, it is unclear if the equivalent of the P-Sp hematopoietic system is established in these differentiation cultures. To address this question we induced different hematopoietic populations with a combination of activin A and BMP2 in serum-free media using an ES cell line carrying the GFP cDNA targeted to the mesoderm gene brachyury (GFP-Bry ES cells). When induced with these factors, the GFP-Bry cells generated two distinct populations with respect to expression of GFP-Bry and Flk-1, the receptor for vascular endothelial growth factor. The first expressed GFP-Bry, but no Flk-1 (GFP-Bry+/Flk-1−), whereas the second expressed both markers (GFP-Bry+/Flk-1+). If the GFP-Bry+/Flk-1− cells were allowed to reaggregate and cultured for an additional 24 hours, they generated a second GFP-Bry+/Flk-1+ population. Analysis of these two distinct Flk-1 populations revealed that both contained hematopoietic progenitors, but that their potential differed. The first Flk-1 population contained BL-CFC, the in vitro equivalent of the hemangioblast as well primitive erythroid and macrophage progenitors. It displayed limited potential to generate B and T lymphocytes when cultured on the OP9 and OP9-DL1 cells respectively. In contrast, the second Flk-1 population did generate B cell and T cells following coculture with the OP9 and OP9-DL1 stromal cells. B cell development was monitored by expression of B220, CD19 and surface IgM whereas T cells were identified by expression of CD4, CD8 and CD3. In addition to lymphoid progenitors, the second Flk-1 population contained multipotent, macrophage and definitive erythroid progenitors. It did not, however, contain significant numbers of BL-CFC or primitive erythroid progenitors. Taken together, these findings demonstrate that is it possible to generate two distinct hematopoietic populations in defined culture conditions. The developmental potential of these populations suggests that they could represent the equivalent of the yolk sac and P-Sp hematopoietic programs.

scholarly journals Forced expression of Nanog in hematopoietic stem cells results in a γδT-cell disorder

Blood ◽  
2007 ◽  
Vol 110 (1) ◽  
pp. 107-115 ◽  
Author(s):  
Yosuke Tanaka ◽  
Takumi Era ◽  
Shin-ichi Nishikawa ◽  
Shin Kawamata
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Lymphoid Tissues ◽  
Thymocyte Development ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Γδt Cell

Nanog is a key molecule involved in the maintenance of the self-renewal of undifferentiated embryonic stem (ES) cells. In this work we investigate whether Nanog can enhance self-renewal in hematopoietic stem cells. Contrary to our expectation, no positive effect of Nanog transduction was detected in bone marrow reconstitution assays. However, recipients of Nanog-transduced (Nanog) hematopoietic stem cells (HSCs) invariantly develop a unique disorder typified by an atrophic thymus occupied by Nanog-expressing γδT-cell receptor–positive (TCR+) cells (Nanog T cells). All thymi are eventually occupied by Nanog T cells with CD25+CD44+ surface phenotype that home selectively to the thymus on transfer and suppress normal thymocyte development, which is partly ascribed to destruction of the microenvironment in the thymus cortex. Moreover, this initial disorder invariantly develops to a lymphoproliferative disorder, in which Nanog T cells undergo unlimited proliferation in the peripheral lymphoid tissues and eventually kill the host. This invariable end result suggests that Nanog is a candidate oncogene for γδT-cell malignancy.

scholarly journals Pax3acts cell autonomously in the neural tube and somites by controlling cell surface properties

Development ◽  
2001 ◽  
Vol 128 (11) ◽  
pp. 1995-2005 ◽  
Author(s):  
Ahmed Mansouri ◽  
Patrick Pla ◽  
Lionel Larue ◽  
Peter Gruss
Keyword(s):  
Cell Surface ◽  
Neural Crest ◽  
Surface Properties ◽  
Neural Tube ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Tube Closure ◽  
Paraxial Mesoderm ◽  
Wild Type ◽  
Cell Surface Properties

Pax3 is a member of the paired-box-containing transcription factors. It is expressed in the developing somites, dorsal spinal cord, mesencephalon and neural crest derivatives. Several loss-of-function mutations are correlated with the Splotch phenotype in mice and Waardenburg syndrome in humans. Malformations include a lack of muscle in the limb, a failure of neural tube closure and dysgenesis of numerous neural crest derivatives. In this study we have used embryonic stem (ES) cells to generate a lacZ knock-in into the Pax3 locus. The Pax3 knock-in Splotch allele (Sp2G) was used to generate Pax3-deficient ES cells in order to investigate whether, in chimeric embryos, Pax3 is acting cell autonomously in the somites and the neural tube. We found that while Pax3 function is essential for the neuroepithelium and somites, a wild-type environment rescues mutant neural crest cells. In the two affected embryonic tissues, mutant and wild-type cells undergo segregation and do not intermingle.The contribution of mutant cells to the neural tube and the somites displayed temporal differences. All chimeric embryos showed a remarkable contribution of blue cells to the neural tube at all stages analyzed, indicating that the Pax3-deficient cells are not excluded from the neural epithelium while development proceeds. In contrast, this is not true for the paraxial mesoderm. The somite contribution of Pax3−/− ES cells becomes less frequent in older embryos as compared to controls with Pax3+/− ES cells. We propose that although Pax3 function is related to cell surface properties, its role may differ in various tissues. In fact, apoptosis was found in Pax3-deficient cells of the lateral dermomyotome but not in the neural tube.

An analysis of the adaptive immune response towards an embryonic stem cell grafts

2007 ◽  
Vol 30 (4) ◽  
pp. 98
Author(s):  
Douglas Wu ◽  
Kathryn Wood
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Adaptive Immune Response ◽  
Class I ◽  
Es Cell ◽  
Mhc Class I Molecule

Background: Although clinical transplantation has had enormous impact on the treatment of premature organ failure, shortage of donor organs continues to be a crucial limiting factor. Embryonic stem cells represent an attractive potential source of replacement tissue because of their inherent pluripotentiality and ability to self-renew. However, before any ES cell-based cellular replacement strategies can be considered, many issues must be addressed. Among these is an evaluation of the potential immune response elicited by any ES cell graft. Because ES cells express very low levels of MHC class I and no MHC class II, their immunogenicity has been questioned. Here we utilize a BM3 TCR transgenic model to analyze the adaptive immune response against an ES cell graft in vivo. Methods: BM3 CD8 TCR-tg T cells (H2K background) specific for the MHC class I molecule H2Kb were labelled with CFSE and adoptively transferred into CBA rag recipients. The following day, ES cells derived from a CBA, B6, or CBK background were implanted beneath the kidney capsule of adoptively transferred mice. Response of the CD8 T cells was measured via CSFE division profiling and graft infiltration. Results: CFSE division profile of naïve BM3 CD8 T cells was unaltered by the presence of either a syngeneic or an allogeneic ES cell graft. These naïve cells were also unable to recognize and infiltrate either a syngeneic or allogeneic ES cell graft on days 5 and 10 post-implantation, despite strong expression of the MHC class I molecule H2Kb by engrafted allogeneic ES cells. On the other hand, H2Kb+ islets begun to be infiltrated by day 5, and were obliterated by a vigorous allogeneic response by day 10. When H2Kb+ islets were implanted into the same kidney as allogeneic ES cells (opposite poles), islet grafts were rapidly infiltrated by CD4 and CD8 T cells and destroyed, but ES cell grafts exhibited markedly reduced cellular infiltrate. In contrast to naïve BM3 CD8 T cells, however, activated cells recognized and mounted an aggressive cytotoxic response against an allogeneic ES cell graft which could be detected by day 6 and resulted in complete graft destruction by day 10. Conclusions: Under certain circumstances, an ES cell graft may have reduced immunogenicity as compared with other conventional tissue or solid organ allografts. This may be due to their lack of passenger APC, which may in turn cripple their ability to elicit a robust allogeneic response via the direct pathway of allorecognition. However, because of their strong upregulation of allogeneic MHC class I molecules after transplantation, they are still likely to elicit a significant rejection response when transplanted into recipients replete with both CD4 and CD8 T cells.

VWRPY motif–dependent and –independent roles of AML1/Runx1 transcription factor in murine hematopoietic development

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 562-570 ◽  
Author(s):  
Motohiro Nishimura ◽  
Yoko Fukushima-Nakase ◽  
Yasuko Fujita ◽  
Mitsushige Nakao ◽  
Shogo Toda ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Thymocyte Development ◽  
Hematopoietic Development ◽  
Cell Clones ◽  
Definitive Hematopoiesis

Abstract AML1/Runx1 is a frequent target of leukemia-associated gene aberration, and it encodes a transcription factor essential for definitive hematopoiesis. We previously reported that the AML1 molecules with trans-activation subdomains retained can rescue in vitro hematopoietic defects of AML1-deficient mouse embryonic stem (ES) cells when expressed by using a knock-in approach. Extending this notion to in vivo conditions, we found that the knock-in ES cell clones with AML1 mutants, which retain trans-activation subdomains but lack C-terminal repression subdomains including the conserved VWRPY motif, contribute to hematopoietic tissues in chimera mice. We also found that germline mice homozygous for the mutated AML1 allele, which lacks the VWRPY motif, exhibit a minimal effect on hematopoietic development, as was observed in control knock-in mice with full-length AML1. On the other hand, reduced cell numbers and deviant CD4 expression were observed during early T-lymphoid ontogeny in the VWRPY-deficient mice, whereas the contribution to the thymus by the corresponding ES cell clones was inadequate. These findings demonstrate that AML1 with its trans-activating subdomains is essential and sufficient for hematopoietic development in the context of the entire mouse. In addition, its trans-repression activity, depending on the C-terminal VWRPY motif, plays a role in early thymocyte development.

scholarly journals Development of Mouse Embryonic Stem (ES) Cells: IV Differentiation to Mature T and B Lymphocytes after Implantation of Embryoid Bodies Into Nude Mice

1995 ◽  
Vol 4 (2) ◽  
pp. 79-84 ◽  
Author(s):  
Una Chen ◽  
Hoyan Mok
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nude Mice ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Lymphoid Cells

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combinedin vitroandin vivosystem are discussed.

In vitro generation of T lymphocytes from embryonic stem cell–derived prehematopoietic progenitors

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1649-1653 ◽  
Author(s):  
Renée F. de Pooter ◽  
Sarah K. Cho ◽  
James R. Carlyle ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Organ Cultures ◽  
Cell Potential

AbstractEmbryonic stem (ES) cells can differentiate into most blood cells in vitro, providing a powerful model system to study hematopoiesis. However, ES cell–derived T lymphocytes have not been generated in vitro, and it was unresolved whether such potential is absent or merely difficult to isolate. Because the latter case might result from rapid commitment to non–T-cell fates, we isolated ES cell–derived prehematopoietic precursors for reconstitution of fetal thymic organ cultures. We found a transient Flk1+CD45– subset of these precursors generated T lymphocytes in vitro, and the use of reaggregate thymic organ cultures greatly enhanced reconstitution frequency. These findings reveal that ES cells can exhibit in vitro T-cell potential, but this is restricted to early stages of ES cell differentiation. Moreover, the results support the notion that the thymic microenvironment can induce T-cell differentiation from a subset of prehematopoietic progenitors and suggest deficient migration into intact thymi hindered previous attempts to generate T cells in vitro from ES cell–derived progenitors. These findings demonstrate that a defined subset of ES cells has the potential to generate T cells in vitro and could contribute to greater understanding of the molecular events of hematopoietic induction and T-cell lineage commitment.

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.

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